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Sample records for epithelial raft culture

  1. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  2. Generation of organotypic raft cultures from primary human keratinocytes.

    Science.gov (United States)

    Anacker, Daniel; Moody, Cary

    2012-02-22

    The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)(1). The life cycle of HPV is tightly linked to the differentiation of squamous epithelium(2). Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production(3,4,5). In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras(6) and modified by Kopan et al.(7), the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies(8). Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as

  3. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  4. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

    Science.gov (United States)

    Maza, Paloma K; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

  5. Lipid raft facilitated ligation of K-α1-tubulin by specific antibodies on epithelial cells: Role in pathogenesis of chronic rejection following human lung transplantation

    International Nuclear Information System (INIS)

    Tiriveedhi, Venkataswarup; Angaswamy, Nataraju; Weber, Joseph; Mohanakumar, T.

    2010-01-01

    Research highlights: → Addition of KAT Abs (+) sera to NHBE culture causes upregulation of growth factors. → Cholesterol depletion causes down regulation of growth factor expression. → Cholesterol depletion is accompanied by loss of membrane bound caveolin. → Thus, we demonstrate lipid raft are critical for efficient ligation of the KAT Abs. -- Abstract: Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-α1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft-domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF (6.4 ± 1.1-, 3.2 ± 0.9-, and 3.4 ± 1.1-fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of β-methyl cyclodextran (βMCD) had significantly reduced growth factor expression (1.3 ± 0.3, vs βMCD untreated being 6.4 ± 1.1-fold increase) upon stimulation with KAT Abs. Depletion

  6. Lipid raft facilitated ligation of K-{alpha}1-tubulin by specific antibodies on epithelial cells: Role in pathogenesis of chronic rejection following human lung transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Tiriveedhi, Venkataswarup; Angaswamy, Nataraju [Department of Surgery, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO (United States); Weber, Joseph [Department of Medicine, Washington University School of Medicine, St. Louis, MO (United States); Mohanakumar, T., E-mail: kumart@wustl.edu [Department of Surgery, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO (United States)

    2010-08-20

    Research highlights: {yields} Addition of KAT Abs (+) sera to NHBE culture causes upregulation of growth factors. {yields} Cholesterol depletion causes down regulation of growth factor expression. {yields} Cholesterol depletion is accompanied by loss of membrane bound caveolin. {yields} Thus, we demonstrate lipid raft are critical for efficient ligation of the KAT Abs. -- Abstract: Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-{alpha}1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft-domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF (6.4 {+-} 1.1-, 3.2 {+-} 0.9-, and 3.4 {+-} 1.1-fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of {beta}-methyl cyclodextran ({beta}MCD) had significantly reduced growth factor expression (1.3 {+-} 0.3, vs {beta}MCD untreated being 6.4 {+-} 1.1-fold

  7. Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts.

    Science.gov (United States)

    Watad, A A; Kochba, M; Nissim, A; Gaba, V

    1995-03-01

    An efficient method was developed using floating membrane rafts (Liferaft(™)) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.

  8. Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

    Science.gov (United States)

    Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

    2018-02-01

    Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Response of human limbal epithelial cells to wounding on 3D RAFT tissue equivalents: effect of airlifting and human limbal fibroblasts.

    Science.gov (United States)

    Massie, Isobel; Levis, Hannah J; Daniels, Julie T

    2014-10-01

    Limbal epithelial stem cell deficiency can cause blindness but may be treated by human limbal epithelial cell (hLE) transplantation, normally on human amniotic membrane. Clinical outcomes using amnion can be unreliable and so we have developed an alternative tissue equivalent (TE), RAFT (Real Architecture for 3D Tissue), which supports hLE expansion, and stratification when airlifted. Human limbal fibroblasts (hLF) may be incorporated into RAFT TEs, where they support overlying hLE and improve phenotype. However, the impact of neither airlifting nor hLF on hLE function has been investigated. hLE on RAFT TEs (±hLF and airlifting) were wounded using heptanol and re-epithelialisation (fluorescein diacetate staining), and percentage putative stem cell marker p63α and proliferative marker Ki67 expression (wholemount immunohistochemistry), measured. Airlifted, hLF- RAFT TEs were unable to close the wound and p63α expression was 7 ± 0.2% after wounding. Conversely, non-airlifted, hLF- RAFT TEs closed the wound within 9 days and p63α expression was higher at 22 ± 5% (p < 0.01). hLE on both hLF- and hLF+ RAFT TEs (non-airlifted) closed the wound and p63α expression was 26 ± 8% and 36 ± 3% respectively (ns). Ki67 expression by hLE increased from 1.3 ± 0.5% before wounding to 7.89 ± 2.53% post-wounding for hLF- RAFT TEs (p < 0.01), and 0.8 ± 0.08% to 17.68 ± 10.88% for hLF+ RAFT TEs (p < 0.05), suggesting that re-epithelialisation was a result of proliferation. These data suggest that neither airlifting nor hLF are necessarily required to maintain a functional epithelium on RAFT TEs, thus simplifying and shortening the production process. This is important when working towards clinical application of regenerative medicine products. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

  11. Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

    Science.gov (United States)

    Hahn, Soojung; Yoo, Jongman

    2017-08-17

    An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

  12. The autologus graft of epithelial tissue culture

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    Minaee B

    1999-08-01

    Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

  13. Depletion of plankton in a raft culture of Mytilus galloprovincialis in Ría de Vigo, NW Spain. I. Phytoplankton

    NARCIS (Netherlands)

    Petersen, J.K.; Nielsen, T.G.; Van Duren, L.A.; Maar, M.

    2008-01-01

    Food depletion in mussel cultivation has been rarely studied and seldom demonstrated. In this study, concentrations of phytoplankton in and around a blue mussel Mytilus galloprovincialis raft culture unit in the Ría de Vigo were measured during a 2 wk study period in July 2004. Flow direction and

  14. Turbulent Dynamics of Epithelial Cell Cultures

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    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  15. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

  16. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  17. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

    NARCIS (Netherlands)

    Huang, L.; van Loveren, C.; Ling, J.; Wei, X.; Crielaard, W.; Deng, D.M.

    2016-01-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated

  18. Lingual Epithelial Stem Cells and Organoid Culture of Them

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    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  19. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  20. Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.

    Science.gov (United States)

    da Luz, Camila Macedo; Boyles, Matthew Samuel Powys; Falagan-Lotsch, Priscila; Pereira, Mariana Rodrigues; Tutumi, Henrique Rudolf; de Oliveira Santos, Eidy; Martins, Nathalia Balthazar; Himly, Martin; Sommer, Aniela; Foissner, Ilse; Duschl, Albert; Granjeiro, José Mauro; Leite, Paulo Emílio Corrêa

    2017-01-31

    Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover

  1. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    International Nuclear Information System (INIS)

    Norgaard, J.O.

    1987-01-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

  2. Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells

    Science.gov (United States)

    Emam, Aufaugh; Carter, William G; Lingwood, Clifford

    2010-01-01

    Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed PMID:21270937

  3. A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

    Science.gov (United States)

    Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

    2014-02-10

    Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally

  4. In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

    Directory of Open Access Journals (Sweden)

    Han Hu

    Full Text Available BACKGROUND: The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. METHODOLOGY: Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. PRINCIPAL FINDINGS: The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n=60. Furthermore, they were capable of synthesizing beta-casein (CSN2, acetyl-CoA carboxylase-alpha (ACACA and butyrophilin (BTN1A1. An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. CONCLUSIONS: The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs.

  5. Regulation of AMPA receptor localization in lipid rafts

    OpenAIRE

    Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

    2008-01-01

    Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the...

  6. Regulation of AMPA receptor localization in lipid rafts

    Science.gov (United States)

    Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

    2009-01-01

    Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

  7. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  8. A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

    International Nuclear Information System (INIS)

    Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

    2005-01-01

    Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

  9. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  10. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  11. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  12. Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure.

    Science.gov (United States)

    Massie, Isobel; Dale, Sarah B; Daniels, Julie T

    2015-06-01

    Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP.

  13. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    OpenAIRE

    Liu, Z.; Zhang, P.; Zhou, Y.; Qin, H.; Shen, T.

    2010-01-01

    Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithel...

  14. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Raquel Rodrigues-Diez

    2014-01-01

    Full Text Available Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β. Epithelial mesenchymal transition (EMT is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2 with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription. The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β.

  15. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  16. Raft River Geothermal Aquaculture Experiment. Phase II

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, D.K.; Rose, F.L.; Kent, J.C.; Watson, L.R.; Sullivan, J.F.

    1979-08-01

    Channel catfish, tilapia and Malaysian prawns were cultured directly in geothermal water for approximately seven months at the Department of Energy, Raft River Geothermal Site, to evaluate the organisms throughout a grow-out cycle. Parameters evaluated included survival, growth, bioaccumulation of metals and fluoride, collagen synthesis, and bone calcium levels. Growth at Raft River was slightly lower than at a companion commercial facility at Buhl, Idaho, but was attributed to facility differences rather than an adverse impact of geothermal water. No significant differences were recorded between Raft River and Buhl fish for bone calcium or collagen concentrations. No significant accumulation of heavy metals by fish or prawns was recorded.

  17. Polarized Airway Epithelial Models for Immunological Co-Culture Studies

    DEFF Research Database (Denmark)

    Papazian, Dick; Würtzen, Peter A; Hansen, Søren Werner Karlskov

    2016-01-01

    Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation...... of the epithelial response towards environmental antigens and genetic susceptibility, resulting in inflammation and T cell-derived immune responses. In vivo animal models have long been used to study immune homeostasis of the airways but are limited by species restriction and lack of exposure to a natural...

  18. Life raft stabilizer

    Science.gov (United States)

    Radnofsky, M. I.; Barnett, J. H., Jr.; Harrison, F. L.; Marak, R. J. (Inventor)

    1973-01-01

    An improved life raft stabilizer for reducing rocking and substantially precluding capsizing is discussed. The stabilizer may be removably attached to the raft and is defined by flexible side walls which extend a considerable depth downwardly to one another in the water. The side walls, in conjunction with the floor of the raft, form a ballast enclosure. A weight is placed in the bottom of the enclosure and water port means are provided in the walls. Placement of the stabilizer in the water allows the weighted bottom to sink, producing submerged deployment thereof and permitting water to enter the enclosure through the port means, thus forming a ballast for the raft.

  19. Response of cultured normal human mammary epithelial cells to X rays

    International Nuclear Information System (INIS)

    Yang, T.C.; Stampfer, M.R.; Smith, H.S.

    1983-01-01

    The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D 0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique

  20. Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

    NARCIS (Netherlands)

    Kabgani, N.; Grigoleit, T.; Schulte, K.; Sechi, A.; Sauer-Lehnen, S.; Tag, C.; Boor, P.; Kuppe, C.; Warsow, G.; Schordan, S.; Mostertz, J.; Chilukoti, R.K.; Homuth, G.; Endlich, N.; Tacke, F.; Weiskirchen, R.; Fuellen, G.; Endlich, K.; Floege, J.; Smeets, B.; Moeller, M.J.

    2012-01-01

    Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or

  1. Interactions of virulent and avirulent leptospires with primary cultures of renal epithelial cells

    DEFF Research Database (Denmark)

    Ballard, S A; Williamson, M; Adler, B

    1986-01-01

    A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar cop...

  2. Dose response of tracheal epithelial cells to ionizing radiation in air-liquid interface cultures

    International Nuclear Information System (INIS)

    Fukutsu, K.; Yamada, Y.; Shimo, M.

    2002-01-01

    The dose-response relationships of tracheal epithelial cells to ionizing radiation was examined in air-liquid interface cultures, which were developed for the purpose of simulating in vivo conditions. The cultures investigated in this study were expected to be advantageous for the performance of irradiation experiments using short-range α rays. The level of dose response of air-liquid interface cultures to ionizing radiation proved to be the same as that for in vivo conditions. This result indicates that air-liquid interface cultures will prove most useful, to facilitate future studies for the investigation of the biological effects induced in tracheal epithelial cells by ionizing radiation, especially by α-rays. (orig.)

  3. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  4. Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

    International Nuclear Information System (INIS)

    Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

    2005-01-01

    Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

  5. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

    Directory of Open Access Journals (Sweden)

    Ricardo Chaves Vilela

    2013-02-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  6. Self Righting Life Raft

    Science.gov (United States)

    1982-01-01

    The Givens Buoy Raft was designed and manufactured for inventor Jim Givens of Givens Marine Survival Co. Inc., by RPR Industries, Inc. The Raft consists of a canopied topside and an underwater hemispheric ballast chamber. It has a heavy ballast stabilization system, adopted from NASA technology, which negates the capsizing problem. A "flapper valve" admits large amounts of water to the hemisphere chamber providing ballast to keep the center of gravity constant; stabilization system compensates for changes in wave angle and weight shifting of raft occupants. Mr. Givens has an exclusive patent license for use of the NASA technology. Produced in various sizes, capacities range from six to 20 persons. Raft is housed in a canister, available in several configurations. A pull on a line triggers the automatic inflation process, which takes 12 seconds. The raft has been credited with saving 230 lives in the last five years. It has found wide acceptance with operators of fishing boats, pleasure craft and other vessels. The Coast Guard is purchasing the rafts for use on its rescue helicopters and the Navy has a development program to adapt the system. The Coast Guard last year announced a proposed amendment of its regulations that would require large ballast chambers on inflatable life rafts.

  7. Organoid culture systems for prostate epithelial and cancer tissue

    NARCIS (Netherlands)

    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans

    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material

  8. Sinking a Granular Raft

    Science.gov (United States)

    Protière, Suzie; Josserand, Christophe; Aristoff, Jeffrey M.; Stone, Howard A.; Abkarian, Manouk

    2017-03-01

    We report experiments that yield new insights on the behavior of granular rafts at an oil-water interface. We show that these particle aggregates can float or sink depending on dimensionless parameters taking into account the particle densities and size and the densities of the two fluids. We characterize the raft shape and stability and propose a model to predict its shape and maximum length to remain afloat. Finally we find that wrinkles and folds appear along the raft due to compression by its own weight, which can trigger destabilization. These features are characteristics of an elastic instability, which we discuss, including the limitations of our model.

  9. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

    Directory of Open Access Journals (Sweden)

    Miessen Katrin

    2012-03-01

    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  10. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri.

    Science.gov (United States)

    Miessen, Katrin; Einspanier, Ralf; Schoen, Jennifer

    2012-03-19

    Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  11. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    Directory of Open Access Journals (Sweden)

    Aaron H Fronk

    2016-07-01

    Full Text Available The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

  12. Cultured alveolar epithelial cells from septic rats mimic in vivo septic lung.

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    2010-06-01

    Full Text Available Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200-300 g were sacrificed 24 hours after cecal ligation and double puncture (2CLP or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A and MAPk (JNK, ERK, an p38 signaling activation, or barrier function was examined by measuring transepithelial resistance (TER or the flux of two molecular tracers (5 and 20 A. Inhibitors of JNK (SP600125, 20 microM and ERK (U0126, 10 microM were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm(2, respectively, however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique

  13. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    International Nuclear Information System (INIS)

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G.

    2007-01-01

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells

  14. Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

    Science.gov (United States)

    Murase, Hiromi; Tsuruma, Kazuhiro; Kuse, Yoshiki; Shimazawa, Masamitsu; Hara, Hideaki

    2017-12-01

    Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  16. Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique.

    Science.gov (United States)

    Noruddin, Nur Adelina Ahmad; Saim, Aminuddin B; Chua, Kien Hui; Idrus, Ruszymah

    2007-12-01

    To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application. Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells. Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques. Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.

  17. Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

    Science.gov (United States)

    2013-01-01

    Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

  18. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  19. Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

    Science.gov (United States)

    Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

    2018-06-26

    Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

  20. Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model.

    Science.gov (United States)

    Speroni, Lucia; Whitt, Gregory S; Xylas, Joanna; Quinn, Kyle P; Jondeau-Cabaton, Adeline; Barnes, Clifford; Georgakoudi, Irene; Sonnenschein, Carlos; Soto, Ana M

    2014-01-01

    The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone-sensitive 3D culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose-dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

  1. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) accelerates expression of differentiation markers in cultures of rat palatal epithelial cells

    DEFF Research Database (Denmark)

    Arenholt, D; Dabelsteen, Erik

    1987-01-01

    Cultures of rat palatal epithelium grown on collagen rafts were treated with different doses of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Sections from biopsies taken 1, 6, 24, and 48 hr after the addition of TPA were examined for the localization of staining by blood ...

  2. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    International Nuclear Information System (INIS)

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-01-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

  3. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  4. Advanced three-dimensional culture of equine intestinal epithelial stem cells.

    Science.gov (United States)

    Stewart, A Stieler; Freund, J M; Gonzalez, L M

    2018-03-01

    Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Descriptive study. Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease

  5. Response of cultured human airway epithelial cells to X-rays and energetic α-particles

    International Nuclear Information System (INIS)

    Yang, T.C.; Holley, W.R.; Curtis, S.B.; Gruenert, D.C.; California Univ., San Francisco, CA

    1990-01-01

    Radon and its progeny, which emit α-particles during decay, may play an important role in inducing human lung cancer. To gain a better understanding of the biological effects of α-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions. Experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus. The RBE for cell inactivation determined by the ratio of D 0 for X-rays to that for 8 MeV helium ions was 1.8-2.2. The cross-section for helium ions, calculated from the D 0 value, was about 24 μm 2 for cells of the primary culture. This cross-section is significantly smaller than the average geometric nuclear area (∼ 180 μm 2 ), suggesting that an average of 7.5 α-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion. (author)

  6. Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

    OpenAIRE

    Klinkspoor, J H; Yoshida, T; Lee, S P

    1998-01-01

    1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoprot...

  7. Nacre formation by epithelial cell cultures from mantle of the black-lip pearl oyster, Pinctada margaritifera.

    Science.gov (United States)

    Jayasankar, Vidya; Vasudevan, Srinivasa Raghavan; Poulose, Suja C; Divipala, Indira

    2018-06-12

    Mantle tissue from the black-lip pearl oyster, Pinctada margaritifera, was cultured in vitro using sterilized seawater supplemented with 0.1% yeast extract as the culture medium. Granular and agranular epithelial cells, hyalinocytes, and fibroblast-like cells were observed in the initial stages of culture. Epithelial cells later formed pseudopodial cell networks containing clusters of granulated cells, which upon maturation released their colored granules. These granules induced formation of nacre crystal deposits on the bottom of the culture plate. Cultures comprised of only granulated epithelial cells were established through periodic sub-culturing of mantle cells and maintained for over 18 mo in a viable condition. Reverse transcriptase PCR of cultured cells demonstrated gene expression of the shell matrix protein, nacrein. To further evaluate the functional ability of cultured granulated epithelial cells, nuclear shell beads were incubated in culture medium containing these cells to induce nacre formation on the beads. Observation of the bead surface under a stereomicroscope at periodic intervals showed the gradual formation of blackish yellow colored nacre deposits. Examination of the bead surface by scanning electron microscopy and energy dispersive X-ray analysis at periodic intervals revealed a distinct brick and mortar formation characteristic of nacre, comprised of aragonite platelets and matrix proteins. Calcium, carbon, and oxygen were the major elements in all stages examined. Our study shows that mantle epithelial cells in culture retain the ability to secrete nacre and can therefore form the basis for future studies on the biomineralization process and its application in development of sustainable pearl culture.

  8. Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

    Science.gov (United States)

    Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

    2015-01-01

    PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

  9. Apoptosis related genes expressed in cultured Fallopian tube epithelial cells infected in vitro with Neisseria gonorrhoeae

    Directory of Open Access Journals (Sweden)

    PAZ A REYES

    2007-01-01

    Full Text Available Background: Infection of the Fallopian tubes (FT by Neisseria gonorrhoeae (Ngo can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. Aim: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. Materials and Methods: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Results: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1 without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I and TNFRSF1B (TNFR-II. Conclusion: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. Results strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed

  10. Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Kopecky, K.J.; Nakamura, Nori; Jones, M.P.; Ito, Toshio; Clifton, K.H.

    1986-09-01

    Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and X-irradiated to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5 %, the average mean lethal dose D 0 was 97.6 cGy for HY cells, and 96.7 cGy and 94.3 cGy, respectively, for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average D 0 of 85.0 cGy and PC cells a significantly (p = .001) lower average D 0 of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (p = .82) or sex (p = .10). These results suggest that malignant thyroid cells may be especially radiosensitive. (author)

  11. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

    Science.gov (United States)

    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...

  13. Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

    Directory of Open Access Journals (Sweden)

    E Michael Danielsen

    Full Text Available The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs, was absent from detergent resistant membranes (DRMs, implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

  14. Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

    Science.gov (United States)

    Danielsen, E Michael; Hansen, Gert H

    2013-01-01

    The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

  15. A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    2018-01-01

    Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

  16. RaftProt: mammalian lipid raft proteome database.

    Science.gov (United States)

    Shah, Anup; Chen, David; Boda, Akash R; Foster, Leonard J; Davis, Melissa J; Hill, Michelle M

    2015-01-01

    RaftProt (http://lipid-raft-database.di.uq.edu.au/) is a database of mammalian lipid raft-associated proteins as reported in high-throughput mass spectrometry studies. Lipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids thought to act as dynamic signalling and sorting platforms. Given their fundamental roles in cellular regulation, there is a plethora of information on the size, composition and regulation of these membrane microdomains, including a large number of proteomics studies. To facilitate the mining and analysis of published lipid raft proteomics studies, we have developed a searchable database RaftProt. In addition to browsing the studies, performing basic queries by protein and gene names, searching experiments by cell, tissue and organisms; we have implemented several advanced features to facilitate data mining. To address the issue of potential bias due to biochemical preparation procedures used, we have captured the lipid raft preparation methods and implemented advanced search option for methodology and sample treatment conditions, such as cholesterol depletion. Furthermore, we have identified a list of high confidence proteins, and enabled searching only from this list of likely bona fide lipid raft proteins. Given the apparent biological importance of lipid raft and their associated proteins, this database would constitute a key resource for the scientific community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

    Directory of Open Access Journals (Sweden)

    Chengpeng Zhu

    Full Text Available We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM. The cultured pterygial epithelial cells (PECs were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4, which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(PH dehydrogenase (quinone 1 (NQO1, NF-E2-related factor-2 (NRF2 and superoxide dismutases (SOD2. Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: β-catenin, nonphospho-β-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6. We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

  18. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    Science.gov (United States)

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  19. Culture promotes transfer of thyroid epithelial cell hyperplasia and proliferation by reducing regulatory T cell numbers.

    Science.gov (United States)

    Kayes, Timothy D; Braley-Mullen, Helen

    2013-01-01

    IFN-γ(-/-) NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ(-/-) SCID mice by splenocytes from mice with severe (4-5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ(-/-) NOD.H-2h4 mice have elevated numbers of FoxP3(+) T reg cells, up to 2-fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Sildenafil Effect on Nitric Oxide Secretion by Normal Human Endometrial Epithelial Cells Cultured In vitro

    Directory of Open Access Journals (Sweden)

    Farzaneh Chobsaz

    2011-01-01

    Full Text Available Background: Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specificphosphodiesterase type 5. It increases intracellular nitric oxide (NO production in some cells.There are reports on its positive effect on uterine circulation, endometrial thickness, and infertilityimprovement. Endometrial epithelial cells (EEC play an important role in embryo attachment andimplantation. The present work investigates the effect of sildenafil on human EEC and their NOsecretion in vitro.Materials and Methods: In this experimental in vitro study, endometrial biopsies (n=10 werewashed in a phosphate buffered solution (PBS and digested with collagenase I (2 mg/ml in DMEM/F12 medium at 37°C for 90 minutes. Epithelial glands were collected by sequential filtrationthrough nylon meshes (70 and 40 μm pores, respectively. Epithelial glands were then treated withtrypsin to obtain individual cells. The cells were counted and divided into four groups: control and1, 10, and 20 μM sildenafil concentrations. Cells were cultured for 15 days at 37ºC and 5% CO2; themedia were changed every 3 days, and their supernatants were collected for the NO assay. NO wasmeasured by standard Greiss methods. Data were analyzed by one way ANOVA.Results: There was no significant difference between groups in cell count and NO secretion, but thelevel of NO increased slightly in the experimental groups. The 10 μM dose showed the highest cellcount. EEC morphology changed into long spindle cells in the case groups.Conclusion: Sildenafil (1, 10, and 20 μM showed a mild proliferative effect on human EECnumbers, but no significant change was seen in NO production.

  1. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-01-01

    Human thyroid epithelial tissues from 23 individuals were obtained from surgical tissue, and cultured in vitro. Dose response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X-rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (D q ) values and extrapolation number (n) values greater than 1) at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X-rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  2. Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells.

    Directory of Open Access Journals (Sweden)

    Vincent M Bruno

    2009-08-01

    Full Text Available Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP kinase and NF-kappaB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.

  3. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-09-01

    Human thyroid epithelial tissue from 23 individuals was obtained from surgical tissue, and cultured in vitro. Dose-response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (Dsub(q)) values and extrapolation number (n) values greater than 1)* at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  4. Treatment of chronic desquamative gingivitis using tissue-engineered human cultured gingival epithelial sheets: a case report.

    Science.gov (United States)

    Okuda, Kazuhiro; Momose, Manabu; Murata, Masashi; Saito, Yoshinori; lnoie, Masukazu; Shinohara, Chikara; Wolff, Larry F; Yoshie, Hiromasa

    2004-04-01

    Human cultured gingival epithelial sheets were used as an autologous grafting material for regenerating gingival tissue in the maxillary left and mandibular right quadrants of a patient with chronic desquamative gingivitis. Six months post-surgery in both treated areas, there were gains in keratinized gingiva and no signs of gingival inflammation compared to presurgery. In the maxillary left quadrant, preoperative histopathologic findings revealed the epithelium was separated from the connective tissue and inflammatory cells were extensive. After grafting with the gingival epithelial sheets, inflammatory cells were decreased and separation between epithelium and connective tissue was not observed. The human cultured gingival epithelial sheets fabricated using tissue engineering technology showed significant promise for gingival augmentation in periodontal therapy.

  5. Alpha-particles induce preneoplastic transformation of rat tracheal epithelial cells in culture

    International Nuclear Information System (INIS)

    Thomassen, D.G.; Seiler, F.A.; Shyr, L.-J.; Griffith, W.C.

    1990-01-01

    To characterize the potential role of high-l.e.t. radiation in respiratory carcinogenesis, the cytotoxic and transforming potency of 5.5 MeV α-particles from electroplated sources of 238 Pu were determined using primary cultures of rat tracheal epithelial cells. RBE for cell killing by α-particles versus X-rays varied with dose, and ranged between 4 and 1.5 for α doses in the range 0.2-4 Gy. At equally toxic doses (relative survival 0.18-0.2), all three agents induced similar frequencies of preneoplastic transformation. For preneoplastic transformation induced by doses of α- and X-radiations giving 80 per cent toxicity, an α RBE of 2.4 was derived. The similar RBEs for cell killing and for preneoplastic transformation suggest an association between the type or degree of radiation-induced damage responsible for both cell killing and cell transformation. (author)

  6. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE

    Science.gov (United States)

    Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in CultureE.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.Previous studies have ...

  7. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19...

  8. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  9. RAFT polymerization mediated bioconjugation strategies

    OpenAIRE

    Bulmuş, Volga

    2011-01-01

    This review aims to highlight the use of RAFT polymerization in the synthesis of polymer bioconjugates. It covers two main bioconjugation strategies using the RAFT process: (i) post-polymerization bioconjugations using pre-synthesized reactive polymers, and (ii) bioconjugations via in situ polymerization using biomolecule-modified monomers or chain transfer agents. © 2011 The Royal Society of Chemistry.

  10. Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

    Science.gov (United States)

    Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

    2013-11-01

    Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

  11. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  12. Blue-light filtering alters angiogenic signaling in human retinal pigmented epithelial cells culture model.

    Science.gov (United States)

    Vila, Natalia; Siblini, Aya; Esposito, Evangelina; Bravo-Filho, Vasco; Zoroquiain, Pablo; Aldrees, Sultan; Logan, Patrick; Arias, Lluis; Burnier, Miguel N

    2017-11-02

    Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein

  13. Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

    Science.gov (United States)

    Klinkspoor, J H; Yoshida, T; Lee, S P

    1998-05-15

    1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with beta-cyclodextrin (2.5-15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (>100 microM Triton X-100, >0.8 mM Tween-20), caused increased LDH release. Incubation with beta-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.

  14. Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

    Directory of Open Access Journals (Sweden)

    Beales Ian LP

    2002-04-01

    Full Text Available Abstract Background Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study. Methods AGS cells were cultured with IL-1β. DNA synthesis was assed by [3H]thymidine incorporation and total viable cell numbers by MTT assay. Results IL-1β dose dependently increased DNA synthesis and cell numbers. The enhanced proliferation was blocked by interleukin-1 receptor antagonist. Addition of neutralising antibody to GM-CSF reduced IL-1β-stimulated proliferation by 31 ± 4 %. GM-CSF alone significantly stimulated proliferation. Addition or neutralisation of IL-8 had no effect on basal or IL-1β-stimulated proliferation. The tyrosine kinase inhibitor genistein completely blocked IL-1β-stimulated proliferation and inhibition of the extracellular signal related kinase pathway with PD 98059 inhibited IL-1β stimulated proliferation by 58 ± 5 %. Conclusions IL-1β stimulates proliferation in gastric epithelial cells. Autocrine stimulation by GM-CSF contributes to this proliferative response. Signalling via tyrosine kinase activity is essential to the mitogenic response to IL-1β. The extracellular signal related kinase pathway is involved in, but not essential to downstream signalling. IL-1β may contribute to the hyperproliferation seen in H. pylori- infected gastric mucosa, and be involved in the carcinogenic process.

  15. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    Energy Technology Data Exchange (ETDEWEB)

    Aslanova, Afag [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Takagi, Ryo; Yamato, Masayuki; Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yamamoto, Masakazu, E-mail: yamamoto.ige@twmu.ac.jp [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  16. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    International Nuclear Information System (INIS)

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-01-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  17. Study of the effects of low-dose radiation and rhEGF on growth of cultured human epithelial cells

    International Nuclear Information System (INIS)

    Yang Jicheng; Zhao Xiaoyu; Sheng Weihua; Tang Zhongyi

    1998-01-01

    In authors' study, the method of taking skin sample, mincing and trypsinizing the sample are presented. The cells were inoculated on adherent membrane or, for sublethally injured 3T3 cells, in culture dish fed with Eargles' medium supplemented with fetal calf serum and various growth-stimulating factors. The cultures were incubated at 37 degree C in an atmosphere containing 5% CO 2 . The medium was changed every three days. The cultured cells became confluent in about two weeks. At the same time, low-dose-radiation and rhEGF were used to influence the growth of the epithelial cells and to test the effects of dosage and concentration. The results showed that low-dose-radiation in the conditions like authors' study could enhance the growth of human epithelial cells just like rhEGF, and it has synergetic effects with rhEGF. The mechanism is discussed

  18. A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

    Science.gov (United States)

    Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

    2017-01-01

    Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

  19. Cultivated vaginal microbiomes alter HIV-1 infection and antiretroviral efficacy in colonized epithelial multilayer cultures.

    Science.gov (United States)

    Pyles, Richard B; Vincent, Kathleen L; Baum, Marc M; Elsom, Barry; Miller, Aaron L; Maxwell, Carrie; Eaves-Pyles, Tonyia D; Li, Guangyu; Popov, Vsevolod L; Nusbaum, Rebecca J; Ferguson, Monique R

    2014-01-01

    There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

  20. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    Science.gov (United States)

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Identification of differences in gene expression in primary cell cultures of human endometrial epithelial cells and trophoblast cells following their interaction

    DEFF Research Database (Denmark)

    Høgh, Mette; Islin, Henrik; Møller, Charlotte

    2006-01-01

    The interaction between the cell types was simulated in vitro by growing primary cell cultures of human endometrial epithelial cells and trophoblast cells together (co-culture) and separately (control cultures). Gene expression in the cell cultures was compared using the Differential Display method and confirmed...

  2. Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo

    International Nuclear Information System (INIS)

    Cuadras, Mariela A.; Greenberg, Harry B.

    2003-01-01

    The pathway by which rotavirus is released from the cell is poorly understood but recent work has shown that, prior to cell lysis, rotavirus is released almost exclusively from the apical surface of the infected cell. By virtue of their unique biochemical and physical properties, viruses have exploited lipid rafts for host cell entry and/or assembly. Here we characterized the association of rhesus rotavirus (RRV) with lipid rafts during the rotavirus replication cycle. We found that newly synthesized infectious virus associates with rafts in vitro and in vivo. RRV proteins cosegregated with rafts on density gradients. Viral infectivity and genomic dsRNA also cosegregated with the raft fractions. Confocal microscopic analysis of raft and RRV virion proteins demonstrated colocalization within the cell. In addition, cholesterol depletion interfered with the association of RRV particles with rafts and reduced the release of infectious particles from the cell. Furthermore, murine rotavirus associates with lipid rafts in intestinal epithelial cells during a natural infection in vivo. Our results confirm the association of rotavirus infectious particles with rafts during replication in vitro and in vivo and strongly support the conclusion that this virus uses these microdomains for transport to the cell surface during replication

  3. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Liara M Gonzalez

    Full Text Available Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA, Minichromosome Maintenance Complex 2 (MCM2, Bromodeoxyuridine (BrdU and phosphorylated Histone H3 (pH3 distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2; enteroendocrine cells by Chromogranin A (CGA, Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII and sucrase isomaltase (SIM. Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

  4. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    Science.gov (United States)

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

  5. A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

    Science.gov (United States)

    Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

    2017-06-01

    Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  6. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  7. Human airway epithelial cell cultures for modeling respiratory syncytial virus infection.

    Science.gov (United States)

    Pickles, Raymond J

    2013-01-01

    Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.

  8. Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Alice L Yu

    Full Text Available The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE induces cell loss, cellular senescence, and extracellular matrix (ECM synthesis in primary human retinal pigment epithelial (RPE cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J, connective tissue growth factor (CTGF, fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

  9. Myo-inositol uptake by cultured calf retinal pigment epithelial cells: regulation by glucose

    International Nuclear Information System (INIS)

    Khatami, M.; Rockey, J.H.

    1986-01-01

    Confluent primary (P-1) or subcultured passage 2 or 3 (P-2, P-3) calf retinal pigment epithelial cells (RPE) were incubated with [ 3 H]-myo-inositol (MI, 100-200 μM) in balanced salt solution (BSS), for 5 to 60 min at 37 0 C. MI uptake into RPE (P-2, 5 days old) was saturable with K/sub m/ of 147 μM and V/sub max/ of 5.5 pmole/min/μg DNA. P-1 or P-2 incubated with 10 μM MI for 40 min accumulated MI against a concentration gradient ([MI]in/[MI]out > 20). Replacement of 150 mM NaCl in BSS by 150 mM choline-Cl reduced the uptake of MI by 87%. MI uptake was inhibited (39%) when cells were incubated in BSS in the absence of Ca Cl 2 . Transport of MI into RPE incubated in the presence of phloridzin, ouabain or 2,4-dinitrophenol (1 mM each) for 10 min was inhibited by 65, 37 and 21%, respectively. α-D-Glucose (20 mM) in the incubation media inhibited MI uptake into primary (or P-2) cultured RPE by 30 or 43% when cells were incubated for 10 or 60 min, respectively. The ability of RPE cells, grown in the presence of 50 mM glucose for 15-25 days, to concentrate MI (40 μM) was reduced up to 41%. Cultured RPE cells accumulated myo-inositol by an active transport system, sensitive to ouabain, DNP and phloridzin. High glucose in the incubation media or in the growth media inhibited the uptake of MI into calf RPE cells

  10. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

    Science.gov (United States)

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  11. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    Directory of Open Access Journals (Sweden)

    Babak Qasemi-Panahi

    2013-02-01

    Full Text Available Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1 on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 μL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  12. Esophageal 3D Culture Systems as Modeling Tools in Esophageal Epithelial Pathobiology and Personalized MedicineSummary

    Directory of Open Access Journals (Sweden)

    Kelly A. Whelan

    Full Text Available The stratified squamous epithelium of the esophagus shows a proliferative basal layer of keratinocytes that undergo terminal differentiation in overlying suprabasal layers. Esophageal pathologies, including eosinophilic esophagitis, gastroesophageal reflux disease, Barrett's esophagus, squamous cell carcinoma, and adenocarcinoma, cause perturbations in the esophageal epithelial proliferation-differentiation gradient. Three-dimensional (3D culture platforms mimicking in vivo esophageal epithelial tissue architecture ex vivo have emerged as powerful experimental tools for the investigation of esophageal biology in the context of homeostasis and pathology. Herein, we describe types of 3D culture that are used to model the esophagus, including organotypic, organoid, and spheroid culture systems. We discuss the development and optimization of various esophageal 3D culture models; highlight the applications, strengths, and limitations of each method; and summarize how these models have been used to evaluate the esophagus under homeostatic conditions as well as under the duress of inflammation and precancerous/cancerous conditions. Finally, we present future perspectives regarding the use of esophageal 3D models in basic science research as well as translational studies with the potential for personalized medicine. Keywords: Organotypic Culture, Organoid, Spheroid Culture, Esophageal Disease

  13. A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

    Science.gov (United States)

    Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

    2011-05-01

    A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  14. Changes in responsiveness of rat tracheal epithelial cells to growth factors during preneoplastic transformation in cell culture

    International Nuclear Information System (INIS)

    Thomassen, D.G.

    1988-01-01

    Preneoplastic rat tracheal epithelial (RTE) cell lines require fewer growth factors for clonal proliferation in culture than normal cells. Serum-free media missing various combinations of growth factors (e.g., cholera toxin, serum albumin, epidermal growth factor, hydrocortisone) required for proliferation of normal, but not preneoplastic, RTE cells can be used to select for carcinogen-induced preneoplastic variants having an increased proliferative potential in culture. These results suggest that reductions in growth factor requirements are primary events in the carcinogenic process. (author)

  15. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

    Science.gov (United States)

    Lai, Jui-Yang; Ma, David Hui-Kang

    2013-01-01

    Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

  16. The effects of platelet gel on cultured human retinal pigment epithelial (hRPE cells

    Directory of Open Access Journals (Sweden)

    Sahar Balagholi

    2017-11-01

    Full Text Available The positive role of platelet gel (PG in tissue regeneration is well known, however, other characteristics of PG still remain to be determined. We investigated cellular and molecular changes in cultured human retinal pigment epithelial (hRPE cells when treated with different concentrations of PG named PG1, PG2, and PG3. hRPE cells were isolated from donor eyes of two newborn children, within 24 hours after their death. The cells were treated with three concentrations of PG for 7 days: 3 × 104/ml (PG1, 6 × 104/ml (PG2, and 9 × 104/ml (PG3. Fetal bovine serum was used as a control. Immunocytochemistry was performed with anti-RPE65 (H-85, anti-Cytokeratin 8/18 (NCL-5D3, and anti-PAX6 antibody. We used MTT assay to determine cell viability. Gene expressions of PAX6, MMP2, RPE65, ACTA2, MKI67, MMP9, and KDR were analyzed using real-time PCR. A significant increase in viability was observed for PG3-treated cells compared to control (p = 0.044 and compared to PG1 group (p = 0.027, on day 7. Cellular elongation together with dendritiform extensions were observed in PG-treated cells on days 1 and 3, while epithelioid morphology was observed on day 7. All cells were immunoreactive for RPE65, cytokeratin 8/18, and PAX6. No significant change was observed in the expression of MKI67 and PAX6, but the expressions of MMP2, MMP9, ACTA2, and KDR were significantly higher in PG2-treated cells compared to controls (p < 0.05. Our results indicate that increased concentration of PG and extended exposure time have positive effects on viability of hRPE cells. PG may be useful for hRPE cell encapsulation in retinal cell replacement therapy.

  17. Tissue Engineering the Cornea: The Evolution of RAFT

    Science.gov (United States)

    Levis, Hannah J.; Kureshi, Alvena K.; Massie, Isobel; Morgan, Louise; Vernon, Amanda J.; Daniels, Julie T.

    2015-01-01

    Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro. PMID:25809689

  18. Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

    DEFF Research Database (Denmark)

    Jantzen, Kim; Roursgaard, Martin; Madsen, Claus Desler

    2012-01-01

    Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung...... treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine...... relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells....

  19. Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

    International Nuclear Information System (INIS)

    Wan, X. Steven; Ware, Jeffrey H.; Zhou, Zhaozong; Donahue, Jeremiah J.; Guan, Jun; Kennedy, Ann R.

    2006-01-01

    Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, α-lipoic acid, L-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, γ-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects

  20. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

    Directory of Open Access Journals (Sweden)

    Lai JY

    2013-10-01

    Full Text Available Jui-Yang Lai,1–3 David Hui-Kang Ma4,5 1Institute of Biochemical and Biomedical Engineering, 2Biomedical Engineering Research Center, 3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 4Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 5Department of Chinese Medicine, Chang Gung University, Taoyuan, Taiwan Abstract: Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA cross-linked amniotic membrane (AM on limbal epithelial cell (LEC cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous

  1. Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    International Nuclear Information System (INIS)

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

  2. Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio

    Directory of Open Access Journals (Sweden)

    Wang YB

    2013-10-01

    Full Text Available Yanbo Wang, Xuxia Yan, Linglin Fu Marine Resources and Nutrition Biology Research Center, Food Quality and Safety Department, Zhejiang Gongshang University, Hangzhou, People's Republic of China Abstract: Nano-selenium (Se, with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp. Keywords: selenium nanoparticle, intestinal epithelial cell, crucian carp, primary culture

  3. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Mytych, Jennifer, E-mail: jennifermytych@gmail.com [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland)

    2017-01-15

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  4. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    International Nuclear Information System (INIS)

    Mytych, Jennifer; Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek

    2017-01-01

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  5. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  6. Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells: the protective effects of lactoferrin

    International Nuclear Information System (INIS)

    Shimmura, Shigeto; Suematsu, Makoto; Shimoyama, Masaru; Oguchi, Yoshihisa; Ishimura, Yuzuru

    1996-01-01

    Acute exposure to suprathreshold ultraviolet B radiation (UV-B) is known to cause photokeratitis resulting from the necrosis and shedding of corneal epithelial cells. However, the corneal effects of low dose UV-B in the environmental range is less clear. In this study, subthreshold UV-B was demonstrated to cause non-necrotic peroxide formation in cultured corneal epithelial cells, which was attenuated by the major tear protein lactoferrin. Intracellular oxidative insults and cell viability of rabbit corneal epithelial cells (RCEC) were assessed by dual-color digital microfluorography using carboxydichlorofluorescin (CDCFH) diacetate bis (acetoxymethyl) ester, a hydroperoxide-sensitive fluoroprobe, and propidium iodode (PI) respectively. The magnitude of UV-induced oxidative insults was calibrated by concentrations of exogenously applied H 2 O 2 which evoke compatible levels of CDCFH oxidation. Exposure of RCEC to low-dose UV-B (2.0 mJ cm -2 at 313 nm, 10.0 mJ cm -2 total UV-B) caused intracellular oxidative changes which were equivalent to those elicited by 240 μM hydrogen peroxide under the conditions of the study. The changes were dose dependent, non-necrotic, and were partially inhibited by lactoferrin ( 1 mg ml -1 ) but not by iron-saturated lactoferrin. Pretreatment with deferoxamine (2 mΜ) or catalase (100 U ml -1 ) also attenuated the UV-induced oxidative stress. The results indicate that UV-B comparable to solar irradiation levels causes significant intracellular peroxide formation in corneal epithelial cells, and that lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation. (Author)

  7. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.

  8. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    International Nuclear Information System (INIS)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs

  9. Culture of primary epithelial adenoma cells from familial adenomatous polyposis patients

    Czech Academy of Sciences Publication Activity Database

    Fostira, F.; Apessos, A.; Oikonomou, E.; Kouklis, P.; Baratsis, S.; Manifikos, G.; Anděra, Ladislav; Yannoukakos, D.; Pintzas, A.; Nasioulas, G.

    2008-01-01

    Roč. 28, 2A (2008), s. 843-846 ISSN 0250-7005 Institutional research plan: CEZ:AV0Z50520514 Keywords : colorectal neoplasia * adenomatous polyposis coli * epithelial cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.390, year: 2008

  10. Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

    Science.gov (United States)

    Ciesiółka, S; Bryja, A; Budna, J; Kranc, W; Chachuła, A; Bukowska, D; Piotrowska, H; Porowski, L; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation

  11. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  12. Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Mee-Hae Kim

    2017-10-01

    Full Text Available In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL, which can be set to 37 °C and 5% CO2, and uses a gas-permeable resin (poly- dimethylsiloxane as the vessel wall. RPE cells were seeded at 5.0 × 104 cells/cm2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

  13. Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

    Science.gov (United States)

    Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

    2016-02-01

    To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

  14. Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

    OpenAIRE

    Beales, Ian LP

    2002-01-01

    Abstract Background Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study. Methods ...

  15. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    International Nuclear Information System (INIS)

    Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

    2013-01-01

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment

  16. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    Energy Technology Data Exchange (ETDEWEB)

    Katano, Takahito [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Ootani, Akifumi [Department of Gastroenterology and GI Endoscopy Center, Shin-Kokura Hospital, Federation of National Public Service Personnel Mutual Aid Associations, 1-3-1 Kanada, Kokurakita-ku, Kitakyushu 803-0816 (Japan); Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Mizoshita, Tsutomu, E-mail: tmizoshi@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Toda, Shuji [Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    2013-03-22

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

  17. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from [U- 14 C]glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state

  18. Correlation of Hypoxia and Pro-senescence Protein Expression in Green Sea Turtle (Chelonia mydas Lung Epithelial and Dermal Fibroblast Cell Culture

    Directory of Open Access Journals (Sweden)

    Anggraini Barlian

    2018-03-01

    Full Text Available Recent studies have shown hypoxia-induced gene expression correlated with cellular senescence. HIF-1α (hypoxia-inducible factor 1-alpha, p53, and pRB were induced under hypoxia and correlated with cellular senescence. The localization and expression of HIF-1α, p53, and pRB in Chelonia mydas lung epithelial and dermal fibroblast cell cultures were analyzed under normoxic and hypoxic conditions (at 4 and 24 hours. Human dermal fibroblast was used for comparison purposes. Protein localization was analyzed with immunocytochemistry, while protein expression was analyzed with the Western blot and enhanced chemiluminescence (ECL method. HIF-1α, p53, and pRB were localized in the nuclei of the C. mydas cell cultures treated with hypoxia. The C. mydas lung epithelial cell cultures had a higher increase of HIF-1α expression than the human dermal fibroblast cell culture. The hypoxic conditions did not affect p53 expression significantly in C. mydas lung epithelial and dermal fibroblast cell cultures. Meanwhile, pRB expression changed significantly under hypoxia in the C. mydas dermal fibroblast cells. Expression of p53 and pRB in the human cell cultures was higher than in the C. mydas cell cultures. This research suggests that C. mydas and human cell cultures have different pro-senescence protein expression responses under hypoxic conditions.

  19. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

    Science.gov (United States)

    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

  20. Dose Optimization of Calcusol™ and Calcium Oxalate Monohydrate (COM on Primary Renal Epithelial Cells Cultures of Mice ( Mus musculus

    Directory of Open Access Journals (Sweden)

    Ahmad Soni

    2014-05-01

    Full Text Available Kidney stones are one of the urologic diseases that have plagued mankind for centuries. The main constituents of stones in the kidney are calcium oxalate monohydrate (COM crystals. Nowadays, there are varieties of drugs and treatments that can be made to minimize the grievances due to kidney stone disease. The treatment can be done either by using chemicals or traditional medicine. Calcusol™ is one of the popular herbal products that have been used by Indonesian people in curing the kidney stone disease. The main constituent that was contained in Calcusol™ is an extract of the tempuyung leaves (Sonchus arvensis L., which is expected could cure the kidney stone disease. This study used primary cultured renal epithelial cells of mice to determine the optimal dose of Calcusol™ and the optimal dose of COM. The primary Kidney epithelial cell were treated with Calcusol™ and COM at various doses. The analysis of the cell death either by necrosis or apoptosis pathways was analyzed by flow cytometric analysis. The results that were obtained is the percentage of cell death that is then analyzed by using a complete randomized design (CRD One Way Anova. Based on the results that were obtained, it is known that the optimal dose of Calcusol™ in vitro were ranging from 75 ppm to 100 ppm, whereas the optimal dose of COM suggested for 500 ppm.

  1. Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

    Science.gov (United States)

    Labilloy, Anatália; Youker, Robert T; Bruns, Jennifer R; Kukic, Ira; Kiselyov, Kirill; Halfter, Willi; Finegold, David; do Monte, Semiramis Jamil Hadad; Weisz, Ora A

    2014-02-01

    Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed. © 2013 Elsevier Inc. All rights reserved.

  2. Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

    NARCIS (Netherlands)

    Pyrc, Krzysztof; Sims, Amy C.; Dijkman, Ronald; Jebbink, Maarten; Long, Casey; Deming, Damon; Donaldson, Eric; Vabret, Astrid; Baric, Ralph; van der Hoek, Lia; Pickles, Raymond

    2010-01-01

    Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is

  3. Clonal differences in generation times of GPK epithelial cells in monolayer culture.

    Science.gov (United States)

    Riley, P A; Hola, M

    1980-01-01

    Pedigrees of cells in eight clones of guinea pig keratocyte (GPK) cells in monolayer culture were analyzed from a time-lapse film. The generation times and the position in the field of observation were recorded up to the sixth generation when the cultures were still subconfluent. Statistical analysis of the results indicates that the position in the culture has less significance than the clonal origin of the cell in determining the interval between successive mitoses.

  4. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts

    Science.gov (United States)

    Iskandar, Anita R.; Xiang, Yang; Frentzel, Stefan; Talikka, Marja; Leroy, Patrice; Kuehn, Diana; Guedj, Emmanuel; Martin, Florian; Mathis, Carole; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

    2015-01-01

    Organotypic 3D cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model. PMID:26085348

  5. Effect of Zebularine loaded MePEG-PCL nanoparticles on viability, attachment of in vitro cultured lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Si-Wei Liu

    2015-01-01

    Full Text Available AIM: To investigate the effect of zebularine(Zebloaded Poly(ethylene glycol-block-poly(ε-caprolactonemethyl ether(MePEG-PCLnanoparticles(NPson the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells(LECs. METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L(ZebF1 group, 100μmol/L(ZebF2 group, Zeb -loaded MePEG-PCL NPs 50μmol/L(ZebNP1 group, Zeb -loaded MePEG-PCL NPs 100μmol/L(ZebNP2 group, MePEG-PCL empty NPs(NPs groupor blank medium(group Crespectively. A tetrazolium dye assay(MTTtest and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis. RESULTS: Determined by MTT colorimetric method: Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner(PPP ZebNP1>ZebF2(PCONCLUSION: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups, as well as enhancing the apoptosis.

  6. Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

    International Nuclear Information System (INIS)

    Choudhary, S.; Xiao, T.; Srivastava, S.; Zhang, W.; Chan, L.L.; Vergara, L.A.; Van Kuijk, F.J.G.M.; Ansari, N.H.

    2005-01-01

    Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H 2 O 2 , 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H 2 O 2 -, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H 2 O 2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3 H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST) (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD

  7. A comparative study on the raft chemical properties of various alginate antacid raft-forming products.

    Science.gov (United States)

    Dettmar, Peter W; Gil-Gonzalez, Diana; Fisher, Jeanine; Flint, Lucy; Rainforth, Daniel; Moreno-Herrera, Antonio; Potts, Mark

    2018-01-01

    Research to measure the chemical characterization of alginate rafts for good raft performance and ascertain how formulation can affect chemical parameters. A selection of alginate formulations was investigated all claiming to be proficient raft formers with significance between products established and ranked. Procedures were selected which demonstrated the chemical characterization allowing rafts to effectively impede the reflux into the esophagus or in severe cases to be refluxed preferentially into the esophagus and exert a demulcent effect, with focus of current research on methods which complement previous studies centered on physical properties. The alginate content was analyzed by a newly developed HPLC method. Methods were used to determine the neutralization profile and the acid neutralization within the raft determined along with how raft structure affects neutralization. Alginate content of Gaviscon Double Action (GDA) within the raft was significantly superior (p raft acid neutralization capacity were GDA and Rennie Duo, the latter product not being a raft former. Raft structure was key and GDA had the right level of porosity to allow for longer duration of neutralization. Alginate formulations require three chemical reactions to take place simultaneously: transformation to alginic acid, sodium carbonate reacting to form carbon dioxide, calcium releasing free calcium ions to bind with alginic acid providing strength to raft formation. GDA was significantly superior (p <.0001) to all other comparators.

  8. An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

    Directory of Open Access Journals (Sweden)

    Kajohnkiart Janebodin

    2013-01-01

    Full Text Available Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10, decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

  9. Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses.

    Science.gov (United States)

    Punyadarsaniya, Darsaniya; Winter, Christine; Mork, Ann-Kathrin; Amiri, Mahdi; Naim, Hassan Y; Rautenschlein, Silke; Herrler, Georg

    2015-02-01

    Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica

    2008-01-01

    (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures....... In BME-UV1, spermine significantly inhibited cell proliferation (Pcultured in the presence of all amines...

  11. Chemical driving force for rafting in superalloys

    CSIR Research Space (South Africa)

    Nabarro, FRN

    1997-08-15

    Full Text Available The author provides a brief overview of the chemical driving forces for rafting in superalloys. Until recently, all theories of the driving force for rafting have considered the compositions of the two phases to be fixed, although accepting...

  12. Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

    Science.gov (United States)

    Ye, P; Yu, H; Simonian, M; Hunter, N

    2014-04-01

    Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin

  13. RAFT polymerization and some of its applications.

    Science.gov (United States)

    Moad, Graeme; Rizzardo, Ezio; Thang, San H

    2013-08-01

    Reversible addition-fragmentation chain transfer (RAFT) is one of the most robust and versatile methods for controlling radical polymerization. With appropriate selection of the RAFT agent for the monomers and reaction conditions, it is applicable to the majority of monomers subject to radical polymerization. The process can be used in the synthesis of well-defined homo-, gradient, diblock, triblock, and star polymers and more complex architectures, which include microgels and polymer brushes. In this Focus Review we describe how the development of RAFT and RAFT application has been facilitated by the adoption of continuous flow techniques using tubular reactors and through the use of high-throughput methodology. Applications described include the use of RAFT in the preparation of polymers for optoelectronics, block copolymer therapeutics, and star polymer rheology control agents. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  15. Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid.

    Directory of Open Access Journals (Sweden)

    Robert A Hirst

    Full Text Available The diagnosis of primary ciliary dyskinesia (PCD requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n  111 was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture.Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

  16. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

    Science.gov (United States)

    Pilgrim, Matthew G; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C; Messinger, Jeffrey D; Read, Russell W; Guidry, Clyde; Curcio, Christine A

    2017-02-01

    Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

  17. Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions

    NARCIS (Netherlands)

    Hiemstra, P.S.; Grootaers, G.G.; Does, A.M. van der; Krul, C.A.M.; Kooter, I.M.

    2018-01-01

    The epithelium that covers the conducting airways and alveoli is a primary target for inhaled toxic substances, and therefore a focus in inhalation toxicology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the

  18. Specificity in calcium oxalate adherence to papillary epithelial cells in culture

    International Nuclear Information System (INIS)

    Riese, R.J.; Riese, J.W.; Kleinman, J.G.; Wiessner, J.H.; Mandel, G.S.; Mandel, N.S.

    1988-01-01

    Attachment of microcystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with [ 14 C]CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a β value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/α ox value of 287 μg/cm 2 . On incubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/α AP value of 93 μg/cm 2 . Microcystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding

  19. Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

    Science.gov (United States)

    Gajewska, Malgorzata; McNally, Sara

    2017-01-01

    Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

  20. Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

    Science.gov (United States)

    Ammar, David A; Kahook, Malik Y

    2011-10-01

    We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6). BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p < 0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p < 0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p < 0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%). These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

  1. Retinal Pigment Epithelial Cell Culture and Cooperation of L-carnitine in Reducing Stress Induced Cellular Damage

    International Nuclear Information System (INIS)

    Shamsi, Farrukh A.; Al-Rajhi, Ali A.; Athmanathan, S.; Boulton, M.; Chaudhry, Imtiaz A.

    2006-01-01

    Purpose was to show that L-carnitine (LC) is capable of reducing non-oxidative stress in the retinal pigment epithelial cells (RPE) of the human eye. The RPE cells were cultured from donor eyes, obtained immediately after post-mortem. The interaction between bovine serum albumin (BSA) and non-oxidative (sodium hydroxide and methyl methane sulphonate) stress-inducers was observed by recording the change in the absorption profiles of the interacting molecules after incubation in light for 5 hours and after treatment with LC. The isolated and cultured RPE cells from the human eyes were treated with sodium hydroxide or methyl methane sulphonate and/or LC for 5 hours under light, and the qualitative effect on cell morphology after treatment was analyzed by staining cells with Giemsa and visualization by light microscopy. The cell morphology was also qualitatively analyzed by scanning electron microscopy (SEM). L-carnitine and stress-inducers interact with BSA and bring about changes in the spectral profile of the interacted molecules. Light microscopy as well as SEM show that the changes in the cellular morphology, induced by 100 uM concentrations of non-oxidative stress-inducers, are considerably reduced in the presence of 100 uM LC. However, L-carnitine alone does not cause any qualitative damage to the cell morphology during incubation under similar conditions. The results give a preliminary indication that LC has ability to reduce the changes brought about by the non-oxidative stress-inducers in the RPF cells in culture. (author)

  2. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    Science.gov (United States)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  3. The use of cultured epithelial autograft in the treatment of major burn injuries: a critical review of the literature.

    Science.gov (United States)

    Wood, F M; Kolybaba, M L; Allen, P

    2006-06-01

    The need to achieve rapid wound closure in patients with massive burns and limited skin donor sites led to the investigation of in vitro cellular expansion of keratinocytes. The use of cultured epithelial autografts (CEA) was first reported in the treatment of major burns in 1981. Since that time, support for the use of CEA has varied, ranging from 'a useful agent' to having 'no demonstrable effect on the outcome of extensively burned patients'. This critical review of the literature examines issues associated with the use of CEA and the introduction of the technology into clinical practice. The factors potentially limiting the use of cultured CEA are the time necessary to culture CEA sheets, the reliability of 'take', vulnerability of grafts on the newly healed surface, long-term durability and the cost implications of such treatment. The available literature was located and critically evaluated using the Australian National Health and Medical Research Council Guidelines. In the identified literature, the level of evidence to support the use of CEA in major burn injures is limited and often restricted to case studies and case series with no Level 1 evidence currently available. The main question arising 'Does CEA have a role in the treatment of major burns?' has proven difficult to answer due to the wide variation in both the quality of study design and the findings. At best, the literature review has highlighted areas of concern that have hindered the successful use of CEA. Our review critically evaluates the use of CEA and explores the advances in techniques towards attempting to improve reliable clinical implementation of CEA. The need for higher level research into the use of CEA is emphasised by this review.

  4. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  5. Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains

    DEFF Research Database (Denmark)

    Riemann, D; Hansen, Gert Helge; Niels-Christiansen, L

    2001-01-01

    in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural...... from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T......-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes...

  6. The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

    Science.gov (United States)

    Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

    2006-06-01

    Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

  7. HC-130 Wing Life Raft Replacement Study

    National Research Council Canada - National Science Library

    Scher, Bob

    1997-01-01

    The U.S. Coast Guard (USCG) uses HC-130 aircraft for search and rescue (SAR) and other missions. The aircraft are presently equipped with two to four 20 person inflatable life rafts, stowed in cells in the wings...

  8. Assessing the nature of lipid raft membranes

    DEFF Research Database (Denmark)

    Niemelä, Perttu S; Ollila, Samuli; Hyvönen, Marja T

    2007-01-01

    of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins......-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide...... heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid-lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads...

  9. Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells

    Science.gov (United States)

    You, Minghai; Chen, Jianling; Wang, Shaobing; Dong, Shiqing; Wang, Yuhua; Xie, Shusen; Wang, Zhengchao; Yang, Hongqin

    2018-04-01

    Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells.

  10. Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.

    Science.gov (United States)

    Hall, M O; Prieto, A L; Obin, M S; Abrams, T A; Burgess, B L; Heeb, M J; Agnew, B J

    2001-10-01

    The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye. Copyright 2001 Academic Press.

  11. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  12. Lipid Raft, Regulator of Plasmodesmal Callose Homeostasis.

    Science.gov (United States)

    Iswanto, Arya Bagus Boedi; Kim, Jae-Yean

    2017-04-03

    A bstract: The specialized plasma membrane microdomains known as lipid rafts are enriched by sterols and sphingolipids. Lipid rafts facilitate cellular signal transduction by controlling the assembly of signaling molecules and membrane protein trafficking. Another specialized compartment of plant cells, the plasmodesmata (PD), which regulates the symplasmic intercellular movement of certain molecules between adjacent cells, also contains a phospholipid bilayer membrane. The dynamic permeability of plasmodesmata (PDs) is highly controlled by plasmodesmata callose (PDC), which is synthesized by callose synthases (CalS) and degraded by β-1,3-glucanases (BGs). In recent studies, remarkable observations regarding the correlation between lipid raft formation and symplasmic intracellular trafficking have been reported, and the PDC has been suggested to be the regulator of the size exclusion limit of PDs. It has been suggested that the alteration of lipid raft substances impairs PDC homeostasis, subsequently affecting PD functions. In this review, we discuss the substantial role of membrane lipid rafts in PDC homeostasis and provide avenues for understanding the fundamental behavior of the lipid raft-processed PDC.

  13. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pearlman, Andrew Leonard [Univ. of California, Berkeley, CA (United States)

    1978-08-01

    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G2M by about 50%. When added to G1 cells, DE delayed recruitment of apparently quiescent (G0) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  14. Proximate biochemical composition and caloric potential in the raft-grown green mussel Perna viridis

    Digital Repository Service at National Institute of Oceanography (India)

    Rivonker, C.U.; Parulekar, A.H.

    The biochemical components of the mussel tissue, cultured on the raft were estimated. The range of variation in the content of moisture, protein, carbohydrates, lipid, organic carbon and ash were 66.8-90.1; 46.2-67.4; 11.7-18.6; 2.9-7.4; 19...

  15. The Settlement Behavior of Piled Raft Interaction in Undrained Soil

    DEFF Research Database (Denmark)

    Ghalesari, Abbasali Taghavi; Barari, Amin; Amini, Pedram Fardad

    2013-01-01

    Offshore piled raft foundations are one of the most commonly used foundations in offshore structures. When a raft foundation alone does not satisfy the design requirements, the addition of piles may improve both the ultimate load capacity and the settlement performance of the raft. In this paper......, the behavior of a piled raft on undrained soil is studied based on a series of parametric studies on the average and differential settlement of piled raft using three-dimensional finite element analysis. The settlement behavior is found to be dependent on the number of piles and raft thickness....

  16. Sphingolipid trafficking and protein sorting in epithelial cells

    NARCIS (Netherlands)

    Slimane, TA; Hoekstra, D

    2002-01-01

    Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an

  17. Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine

    Directory of Open Access Journals (Sweden)

    Eva Maier

    2017-12-01

    Full Text Available Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2 activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2. The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER, of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

  18. Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine.

    Science.gov (United States)

    Maier, Eva; Anderson, Rachel C; Roy, Nicole C

    2017-12-12

    Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

  19. Studies of Bystander Effect and Intercellular Communication in Human Epithelial Cell Cultures Irradiated with X-rays

    International Nuclear Information System (INIS)

    Romppanen, E.; Trott, K. R.; Musatonen, R.; Leszcznski, D.; Belyakov, O.

    2004-01-01

    The bystander effect is a phenomenon whereby biological consequences of irradiation are expressed in nonexposed cells in the vicinity of exposed cells. Two main pathways have been proposed to mediate the bystander effect: Gap Junction Intercellular Communication (GJIC) and medium borne soluble factors dependent mechanisms. The present study was designed to evaluate the relative contributions of gap junction intercellular communication and of soluble extracellular factors on the bystander effects of low dose X-ray irradiation. HaCaT human epithelial cell monolayers were exposed to X-ray using specially constructed shield, which cover 95% or 56% or 0% of the cells from the radiation. To evaluate whether the GJIC is involved in transmission of the bystander signal from irradiated to nonirradiated cells, irradiations were performed in presence or absence of GJIC inhibitor lindane. The cytochalasin B block technique was used to quantify fractions of micronucleated cells 48 hours after the irradiation. Our results suggest that more micronucleated cells are induced in partially shielded monolayers than expected according to back extrapolation of the data from open field irradiation. Treatment with lindane considerably reduced amount of the bystander damage. We demonstrated that fraction of micronucleated cells after X-rays irradiation of 5% of cells with 1 Gy was 0.07±0.08 (without lindane) and 0.05±0.004 (in presence of lindane). Irradiation of 100% of cells with the same dose resulted in 0.023±0.04 /without lindane) and 0.013±0.02 (in presence of lindane) fractions of micronucleated cells. Comparison with open field data showed that the fraction of micronucleated cells after irradiation of 5% of the cell culture was 5-10 times greater than the estimated fraction assuming no bystander effect. Irradiation of 44% of cells ded not demonstrate a pronounced bystander effect. (Author) 20 refs

  20. Culture of Iris Pigment Epithelial Cells on Expanded-Polytetrafluroethylene (ePTFE Substrates for the Treatment of Age-Related Macular Degeneration

    Directory of Open Access Journals (Sweden)

    S Nian

    2011-05-01

    Full Text Available Introduction: Transplantation of an intact differentiated retinal pigment epithelial (RPE cell layer may provide a means to treat Age-Related Macular Degeneration (AMD. However, harvesting RPE cells can be a technically complicated procedure. Our current work aimed to prepare intact differentiated iris pigment epithelial (IPE cell layers, which are easy to obtain and have the same embryonic origin and similar properties as RPE cells, on ePTFE substrates for transplantation purposes to rescue deteriorated photoreceptors in AMD. Methods: IPE cells isolated from rat eyes were seeded on different substrates, including fibronectin n-heptylamine (HA ePTFE substrates, HA ePTFE substrates, ePTFE substrates and fibronectin tissue culture polystyrene (TCPS as control. Cell number and morphology were assessed at each time interval. The formation of tight junction was examined by immunostaining of junction proteins. Results: An obvious increasing trend of cell number was observed in IPE cells on fibronectin n-heptylamine (HA ePTFE substrate, exhibiting heavy pigmentation and epithelial morphology. At Day 28, tight junction formation was indicated by cell-cell junctional proteins along cell borders. Conclusion: Harvested IPE cells cultured on fibronectin HA-ePTFE substrates can differentiate and form a cell monolayer that may be suitable for transplantation.

  1. Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells: Induction of assembly on fibrillar type I collagen substrata

    International Nuclear Information System (INIS)

    David, G.; van der Schueren, B.; van den Berghe, H.; Nusgens, B.; Van Cauwenberge, D.; Lapiere, C.

    1987-01-01

    Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable [ 3 H]hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the allover rates of collagen synthesis and degradation. The proportion of the [ 3 H]collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibers. Transformed cells on collagen produce and accumulate more [ 3 H]collage, yet are less effective in basement membrane formation than normal cells, indicting that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface

  2. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    Science.gov (United States)

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  3. Lipid Raft: A Floating Island Of Death or Survival

    Science.gov (United States)

    George, Kimberly S.; Wu, Shiyong

    2012-01-01

    Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid rafts microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid rafts disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity. PMID:22289360

  4. Multiblock copolymers synthesized in aqueous dispersions using multifunctional RAFT agents

    NARCIS (Netherlands)

    Bussels, R.; Bergman-Göttgens, C.M.; Meuldijk, J.; Koning, C.E.

    2005-01-01

    Triblock copolymers were synthesized in aqueous dispersions in two polymerization steps using a low molar mass difunctional dithiocarbamate-based RAFT agent, and in merely one polymerization step using a macromolecular difunctional dithiocarbamate-based RAFT agent. Segmented block copolymers

  5. Oceanic rafting by a coastal community.

    Science.gov (United States)

    Fraser, Ceridwen I; Nikula, Raisa; Waters, Jonathan M

    2011-03-07

    Oceanic rafting is thought to play a fundamental role in assembling the biological communities of isolated coastal ecosystems. Direct observations of this key ecological and evolutionary process are, however, critically lacking. The importance of macroalgal rafting as a dispersal mechanism has remained uncertain, largely owing to lack of knowledge about the capacity of fauna to survive long voyages at sea and successfully make landfall and establish. Here, we directly document the rafting of a diverse assemblage of intertidal organisms across several hundred kilometres of open ocean, from the subantarctic to mainland New Zealand. Multispecies analyses using phylogeographic and ecological data indicate that 10 epifaunal invertebrate species rafted on six large bull kelp specimens for several weeks from the subantarctic Auckland and/or Snares Islands to the Otago coast of New Zealand, a minimum distance of some 400-600 km. These genetic data are the first to demonstrate that passive rafting can enable simultaneous trans-oceanic transport and landfall of numerous coastal taxa.

  6. Erythropoietin Receptor Signaling Is Membrane Raft Dependent

    Science.gov (United States)

    McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

    2012-01-01

    Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

  7. Lipid Raft, Regulator of Plasmodesmal Callose Homeostasis

    Directory of Open Access Journals (Sweden)

    Arya Bagus Boedi Iswanto

    2017-04-01

    Full Text Available Abstract: The specialized plasma membrane microdomains known as lipid rafts are enriched by sterols and sphingolipids. Lipid rafts facilitate cellular signal transduction by controlling the assembly of signaling molecules and membrane protein trafficking. Another specialized compartment of plant cells, the plasmodesmata (PD, which regulates the symplasmic intercellular movement of certain molecules between adjacent cells, also contains a phospholipid bilayer membrane. The dynamic permeability of plasmodesmata (PDs is highly controlled by plasmodesmata callose (PDC, which is synthesized by callose synthases (CalS and degraded by β-1,3-glucanases (BGs. In recent studies, remarkable observations regarding the correlation between lipid raft formation and symplasmic intracellular trafficking have been reported, and the PDC has been suggested to be the regulator of the size exclusion limit of PDs. It has been suggested that the alteration of lipid raft substances impairs PDC homeostasis, subsequently affecting PD functions. In this review, we discuss the substantial role of membrane lipid rafts in PDC homeostasis and provide avenues for understanding the fundamental behavior of the lipid raft–processed PDC.

  8. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

    Science.gov (United States)

    Schlage, Walter K; Iskandar, Anita R; Kostadinova, Radina; Xiang, Yang; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Talikka, Marja; Geertz, Marcel; Mathis, Carole; Ivanov, Nikolai; Hoeng, Julia; Peitsch, Manuel C

    2014-10-01

    Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

  9. Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

    Science.gov (United States)

    Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

    2017-11-01

    Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation. © 2017 Wiley Periodicals, Inc.

  10. Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface.

    Science.gov (United States)

    Chang-Lin, Joan-En; Kim, Kwang-Jin; Lee, Vincent H L

    2005-06-01

    Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear-Snapwells precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na(+) and Cl(-) transport to overall short-circuit current (I(sc)) was evaluated by removing Na(+) and Cl(-), respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na(+) and Cl(-) using (22)Na and (36)Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na(+)-channel activities to net Na(+) fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl(2) and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl(-)-channel, and basolateral K(+)-channel and Na(+)(K(+))2Cl(-)-cotransporter activities, respectively, in active ion transport across RCrECL. I(sc) of RCrECL derived from pigmented rabbits was comprised of 64+/-2% and 44+/-5% for active Na(+) and Cl(-) transport, respectively, consistent with net Na(+) absorption and Cl(-) secretion of 0.062+/-0.006 and 0.046+/-0.008 muEq/cm(2)/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I(sc) by up to approximately 50% with an IC(50) of 1 and 0.1 microm, respectively, consistent with participation of apical epithelial Na(+)-channels to net Na(+) absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl(2) to the basolateral and bumetanide to basolateral fluid decreased I(sc) by 86+/-1.5%, 53+/-3%, 18+/-1.8% and 13+/-1.9% in RCr

  11. Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size – implication for FasR-associated apoptosis

    Science.gov (United States)

    Gilbert, Stéphane; Loranger, Anne; Omary, M. Bishr

    2016-01-01

    ABSTRACT Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. PMID:27422101

  12. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  13. Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures.

    Science.gov (United States)

    Almada, Evangelina; Tonucci, Facundo M; Hidalgo, Florencia; Ferretti, Anabela; Ibarra, Solange; Pariani, Alejandro; Vena, Rodrigo; Favre, Cristián; Girardini, Javier; Kierbel, Arlinet; Larocca, M Cecilia

    2017-11-02

    The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

  14. Diesel exhaust alters the response of cultured primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD) to non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Zarcone, Maria C; van Schadewijk, Annemarie; Duistermaat, Evert; Hiemstra, Pieter S; Kooter, Ingeborg M

    2017-01-28

    Exacerbations constitute a major cause of morbidity and mortality in patients suffering from chronic obstructive pulmonary disease (COPD). Both bacterial infections, such as those with non-typeable Haemophilus influenzae (NTHi), and exposures to diesel engine emissions are known to contribute to exacerbations in COPD patients. However, the effect of diesel exhaust (DE) exposure on the epithelial response to microbial stimulation is incompletely understood, and possible differences in the response to DE of epithelial cells from COPD patients and controls have not been studied. Primary bronchial epithelial cells (PBEC) were obtained from age-matched COPD patients (n = 7) and controls (n = 5). PBEC were cultured at the air-liquid interface (ALI) to achieve mucociliary differentiation. ALI-PBECs were apically exposed for 1 h to a stream of freshly generated whole DE or air. Exposure was followed by 3 h incubation in presence or absence of UV-inactivated NTHi before analysis of epithelial gene expression. DE alone induced an increase in markers of oxidative stress (HMOX1, 50-100-fold) and of the integrated stress response (CHOP, 1.5-2-fold and GADD34, 1.5-fold) in cells from both COPD patients and controls. Exposure of COPD cultures to DE followed by NTHi caused an additive increase in GADD34 expression (up to 3-fold). Importantly, DE caused an inhibition of the NTHi-induced expression of the antimicrobial peptide S100A7, and of the chaperone protein HSP5A/BiP. Our findings show that DE exposure of differentiated primary airway epithelial cells causes activation of the gene expression of HMOX1 and markers of integrated stress response to a similar extent in cells from COPD donors and controls. Furthermore, DE further increased the NTHi-induced expression of GADD34, indicating a possible enhancement of the integrated stress response. DE reduced the NTHi-induced expression of S100A7. These data suggest that DE exposure may cause adverse health effects in part by

  15. Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model.

    Science.gov (United States)

    Tanaka, Yohei; Nakayama, Jun

    2016-01-01

    Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000-1,800 nm wavelengths and excluded 1,400-1,500 nm wavelengths. A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm(2) irradiation (Psolar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage.

  16. Replication and interaction of herpes simplex virus and human papillomavirus in differentiating host epithelial tissue

    International Nuclear Information System (INIS)

    Meyers, Craig; Andreansky, Samita S.; Courtney, Richard J.

    2003-01-01

    We have investigated the interactions and consequences of superinfecting and coreplication of human papillomavirus (HPV) and herpes simplex virus (HSV) in human epithelial organotypic (raft) culture tissues. In HPV-positive tissues, HSV infection and replication induced significant cytopathic effects (CPE), but the tissues were able to recover and maintain a certain degree of tissue integrity and architecture. HPV31b not only maintained the episomal state of its genomic DNA but also maintained its genomic copy number even during times of extensive HSV-induced CPE. E2 transcripts encoded by HPV31b were undetectable even though HPV31b replication was maintained in HSV- infected raft tissues. Expression of HPV31b oncogenes (E6 and E7) was also repressed but to a lesser degree than was E2 expression. The extent of CPE induced by HSV is dependent on the magnitude of HPV replication and gene expression at the time of HSV infection. During active HSV infection, HPV maintains its genomic copy number even though genes required for its replication were repressed. These studies provide new insight into the complex interaction between two common human sexually transmitted viruses in an in vitro system, modeling their natural host tissue in vivo

  17. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Wrinkles, folds, and plasticity in granular rafts

    Science.gov (United States)

    Jambon-Puillet, Etienne; Josserand, Christophe; Protière, Suzie

    2017-09-01

    We investigate the mechanical response of a compressed monolayer of large and dense particles at a liquid-fluid interface: a granular raft. Upon compression, rafts first wrinkle; then, as the confinement increases, the deformation localizes in a unique fold. This characteristic buckling pattern is usually associated with floating elastic sheets, and as a result, particle laden interfaces are often modeled as such. Here, we push this analogy to its limits by comparing quantitative measurements of the raft morphology to a theoretical continuous elastic model of the interface. We show that, although powerful to describe the wrinkle wavelength, the wrinkle-to-fold transition, and the fold shape, this elastic description does not capture the finer details of the experiment. We describe an unpredicted secondary wavelength, a compression discrepancy with the model, and a hysteretic behavior during compression cycles, all of which are a signature of the intrinsic discrete and frictional nature of granular rafts. It suggests also that these composite materials exhibit both plastic transition and jamming dynamics.

  19. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    International Nuclear Information System (INIS)

    Zaccone, Eric J.; Goldsmith, W. Travis; Shimko, Michael J.; Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A.; Fedan, Jeffrey S.

    2015-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (R t ) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na + transport, without affecting Cl − transport or Na + ,K + -pump activity. R t was unaffected. Na + transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. - Highlights: • Butter flavoring vapor effects on human cultured airway epithelium were studied. • Na transport was reduced by a 6-h exposure to 25 ppm diacetyl and 2,3-pentanedione. • Na transport recovered 18 h after exposure. • > 60 ppm transepithelial voltage and resistance were abolished; cells were damaged. • Cells metabolized diacetyl and 2,3-pentanedione into acetoin and 2-OH-3-pentanone.

  20. Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

    Directory of Open Access Journals (Sweden)

    Elhaseen Elamin

    Full Text Available BACKGROUND: Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. METHODS AND FINDINGS: Caco-2 cells were grown in a basement membrane matrix (Matrigel™ to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM or acetaldehyde (25-200 µM for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. CONCLUSIONS: These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in

  1. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    Science.gov (United States)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  2. Differential effect of TGFβ on the proteome of cancer associated fibroblasts and cancer epithelial cells in a co-culture approach - a short report.

    Science.gov (United States)

    Koczorowska, Maria Magdalena; Friedemann, Charlotte; Geiger, Klaus; Follo, Marie; Biniossek, Martin Lothar; Schilling, Oliver

    2017-12-01

    Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor β (TGFβ), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFβ on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFβ on CAFs and ETCs at the proteomic level. We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFβ. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. We found that TGFβ treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFβ. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFβ treated CAFs. In addition, we found that the TGFβ treated CAFs exhibited increased N-cadherin levels. From our data we conclude that CAFs respond to TGFβ treatment by changing their proteome composition, while ETCs appear to be rather resilient.

  3. Mycotoxin production by Fusarium avenaceum strains isolated from Norwegian grain and the cytotoxicity of rice culture extracts to porcine kidney epithelial cells.

    Science.gov (United States)

    Morrison, Ellen; Kosiak, Barbara; Ritieni, Alberto; Aastveit, Are H; Uhlig, Silvio; Bernhoft, Aksel

    2002-05-08

    The secondary metabolites of 24 isolates of Fusarium avenaceum from Norwegian cereals and grown on rice have been characterized. Moniliformin (MON), enniatins (ENNs), and beauvericin (BEA) were analyzed by high-performance liquid chromatography. Porcine kidney epithelial cells (PK15, American Type Culture Collection) were used to study the cytotoxicity of MON in the extracts. The following metabolites were produced by all isolates, ranked by concentration in rice cultures: ENN-B, MON, ENN-B1, and ENN-A. BEA was produced by eight isolates. The productions of BEA and ENN-A were significantly correlated, as was the case with ENN-B and ENN-B1. MON production was correlated neither to any of the other toxins nor to toxicity.

  4. Polarity in Mammalian Epithelial Morphogenesis

    OpenAIRE

    Roignot, Julie; Peng, Xiao; Mostov, Keith

    2013-01-01

    Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture model...

  5. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    International Nuclear Information System (INIS)

    Mulari, Mika T.K.; Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-01-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-β-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption

  6. The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

    Science.gov (United States)

    De Chatterjee, Atasi; Mendez, Tavis L; Roychowdhury, Sukla; Das, Siddhartha

    2015-05-01

    Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.

    Science.gov (United States)

    Alawin, Osama A; Ahmed, Rayan A; Ibrahim, Baher A; Briski, Karen P; Sylvester, Paul W

    2016-01-01

    A large percentage of human breast cancers are characterized by excessive or aberrant HER2 activity. Lipid rafts are specialized microdomains within the plasma membrane that are required for HER2 activation and signal transduction. Since the anticancer activity of γ-tocotrienol is associated with suppression in HER2 signaling, studies were conducted to examine the effects of γ-tocotrienol on HER2 activation within the lipid raft microdomain in HER2-positive SKBR3 and BT474 human breast cancer cells. Treatment with 0-5μM γ-tocotrienol induced a significant dose-dependent inhibition in cancer cell growth after a 5-day culture period, and these growth inhibitory effects were associated with a reduction in HER2 dimerization and phosphorylation (activation). Phosphorylated HER2 was found to be primarily located in the lipid raft microdomain of the plasma membrane in vehicle-treated control groups, whereas γ-tocotrienol treatment significantly inhibited this effect. Assay of plasma membrane subcellular fractions showed that γ-tocotrienol also accumulates exclusively within the lipid raft microdomain. Hydroxypropyl-β-cyclodextrin (HPβCD) is an agent that disrupts lipid raft integrity. Acute exposure to 3mM HPβCD alone had no effect, whereas an acute 24-h exposure to 20μM γ-tocotrienol alone significantly decreased SKBR3 and BT474 cell viability. However, combined treatment with these agents greatly reduced γ-tocotrienol accumulation in the lipid raft microdomain and cytotoxicity. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are associated with its accumulation in the lipid raft microdomain and subsequent interference with HER2 dimerization and activation in SKBR3 and BT474 human breast cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

    Science.gov (United States)

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

    2014-02-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

  9. mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

    Science.gov (United States)

    Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

    2016-08-15

    The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation

  10. Investigating physiological characteristics of mint in the Raft aquaponic system and perlite medium

    OpenAIRE

    H. R. Roosta; A. R. Sajjadinia

    2010-01-01

    Aquaponic is a combination of fish and plant cultivated in recirculating systems. Fish culture in closed recirculating systems causes organic waste accumulation in the system. If these metabolites are used for plant nutrition, they are not mere waste, but have economical value and however benefit the fish production systems. Most aquaponic systems are designed as perlite-filled media, thin layer of nutrient solution, or Raft system. Photosynthesis parameters and water relations are good index...

  11. Lipid rafts and B cell signaling.

    Science.gov (United States)

    Gupta, Neetu; DeFranco, Anthony L

    2007-10-01

    B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secrete pathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process them for presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasma membrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation including B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we review the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.

  12. Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

    Science.gov (United States)

    Stájer, Anette; Ungvári, Krisztina; Pelsoczi, István K; Polyánka, Hilda; Oszkó, Albert; Mihalik, Erzsébet; Rakonczay, Zoltán; Radnai, Márta; Kemény, Lajos; Fazekas, András; Turzó, Kinga

    2008-11-01

    High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

  13. Lipid raft involvement in yeast cell growth and death

    Energy Technology Data Exchange (ETDEWEB)

    Mollinedo, Faustino, E-mail: fmollin@usal.es [Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas - Universidad de Salamanca, Salamanca (Spain)

    2012-10-10

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na{sup +}, K{sup +}, and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  14. Lipid raft involvement in yeast cell growth and death

    International Nuclear Information System (INIS)

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na + , K + , and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  15. The effects of gas humidification with high-flow nasal cannula on cultured human airway epithelial cells.

    Science.gov (United States)

    Chidekel, Aaron; Zhu, Yan; Wang, Jordan; Mosko, John J; Rodriguez, Elena; Shaffer, Thomas H

    2012-01-01

    Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC) effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH) 90% (HFNC) for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n = 6/condition). Transepithelial resistance and cell viability decreased over time (P < 0.001) between HFNC and dry groups (P < 0.001). Total protein secretion increased at 8 hours in the dry group (P < 0.001). Secretion of interleukin (IL)-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P < 0.001). Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation.

  16. The Effects of Gas Humidification with High-Flow Nasal Cannula on Cultured Human Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Aaron Chidekel

    2012-01-01

    Full Text Available Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH 90% (HFNC for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n=6/condition. Transepithelial resistance and cell viability decreased over time (P<0.001 between HFNC and dry groups (P<0.001. Total protein secretion increased at 8 hours in the dry group (P<0.001. Secretion of interleukin (IL-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P<0.001. Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation.

  17. Molecularly Imprinted Polymer Synthesis Using RAFT Polymerisation

    International Nuclear Information System (INIS)

    Cormack, P.A.G.; Faizatul Shimal Mehamod; Faizatul Shimal Mehamod

    2013-01-01

    In this paper, the synthesis and characterisation of caffeine-imprinted polymers are described. The polymers were prepared in monolithic form via both reversible addition-fragmentation chain-transfer (RAFT) polymerisation and conventional free radical polymerisation, using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and crosslinking agent, respectively. The potential benefits in applying RAFT polymerisation techniques towards the synthesis of molecularly imprinted polymers (MIPs) are explored and elucidated. The pore structures of the polymers produced were characterised by nitrogen sorption porosimetry and the molecular recognition properties of representative products were evaluated in high-performance liquid chromatography (HPLC) mode. Molecular imprinting effects were confirmed by analysing the relative retentions of analytes on imprinted and non-imprinted HPLC stationary phases. It was found that a caffeine-imprinted polymer synthesised by RAFT polymerisation was superior to a polymer prepared using a conventional synthetic approach; the imprinting factor and column efficiency were found to be higher for the former material. (author)

  18. Raft river geoscience case study, volume 1

    Science.gov (United States)

    Dolenc, M. R.; Hull, L. C.; Mizell, S. A.; Russell, B. F.; Skiba, P. A.; Strawn, J. A.; Tullis, J. A.; Garber, R.

    1981-11-01

    The Raft River Geothermal Site has been evaluated over the past eight years by the United States Geological Survey and the Idaho National Engineering Laboratory as a moderate-temperature geothermal resource. The geoscience data gathered in the drilling and testing of seven geothermal wells suggest that the Raft River thermal reservoir is: (1) produced from fractures found at the contact metamorphic zone apparently the base of detached normal faulting from the Bridge and Horse Well Fault zones of the Jim Sage Mountains; (2) anisotropic, with the major axis of hydraulic conductivity coincident to the Bridge Fault Zone; (3) hydraulically connected to the shallow thermal fluid of the Crook and BLM wells based upon both geochemistry and pressure response; (4) controlled by a mixture of diluted meteoric water recharging from the northwest and a saline sodium chloride water entering from the southwest. Although the hydrogeologic environment of the Raft River geothermal area is very complex and unique, it is typical of many Basin and Range systems.

  19. Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

    Directory of Open Access Journals (Sweden)

    Tanaka Y

    2016-08-01

    Full Text Available Yohei Tanaka,1,2 Jun Nakayama2 1Department of Plastic Surgery, Clinica Tanaka Plastic, Reconstructive Surgery and Anti-aging Center, 2Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan Background and objective: Humans are increasingly exposed to near-infrared (NIR radiation from both natural (eg, solar and artificial (eg, electrical appliances sources. Although the biological effects of sun and ultraviolet (UV exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues.Materials and methods: DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C. The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths.Results: A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05.Conclusion: We found that NIR irradiation induced the

  20. Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

    Science.gov (United States)

    Zonta, Barbara; Minichiello, Liliana

    2013-10-18

    Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75(NTR)) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

  1. Synaptic membrane rafts: traffic lights for local neurotrophin signalling?

    Directory of Open Access Journals (Sweden)

    Barbara eZonta

    2013-10-01

    Full Text Available Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signalling, plasticity and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signalling. The tyrosine kinase neurotrophin receptors (Trk and the low-affinity p75 neurotrophin receptor (p75NTR are enriched in neuronal lipid rafts together with the intermediates of downstream signalling pathways, suggesting a possible role of rafts in neurotrophin signalling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

  2. Membrane raft association is a determinant of plasma membrane localization.

    Science.gov (United States)

    Diaz-Rohrer, Blanca B; Levental, Kandice R; Simons, Kai; Levental, Ilya

    2014-06-10

    The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

  3. Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    Science.gov (United States)

    Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

    2014-01-01

    Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

  4. Two-dimensional Forster resonance energy transfer (2-D FRET) and the membrane raft hypothesis

    OpenAIRE

    Acasandrei, Maria; Dale, Robert; VAN DE VEN, Martin; AMELOOT, Marcel

    2006-01-01

    A model for analyzing Forster resonance energy transfer (FRET) data in relation to the cell plasma membrane raft hypothesis is developed to take into account: (a) the distribution of FRET donors and acceptors at the surface of probing antibody fragments specific for a putative raft component; (b) partitioning of the raft component between raft and non-raft areas of the membrane; and (c) the dependence of the raft partition on the expression level of the considered component. Analysis of relev...

  5. Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

    Science.gov (United States)

    Zeng, Hui; Goldsmith, Cynthia S.; Maines, Taronna R.; Belser, Jessica A.; Gustin, Kortney M.; Pekosz, Andrew; Zaki, Sherif R.; Katz, Jacqueline M.

    2013-01-01

    Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses. PMID:23255802

  6. Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

    Science.gov (United States)

    Zhang, Yuanyuan; George, Jasmine; Li, Yun; Olufade, Rebecca; Zhao, Xueying

    2015-01-01

    As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy

  7. Functional interactions between 17 β -estradiol and progesterone regulate autophagy during acini formation by bovine mammary epithelial cells in 3D cultures.

    Science.gov (United States)

    Zielniok, Katarzyna; Motyl, Tomasz; Gajewska, Malgorzata

    2014-01-01

    Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2) and progesterone (P4). Mammary epithelial cells (MECs) cultured on reconstituted basement membrane (rBM) form three-dimensional (3D) acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR) involved in autophagy induction.

  8. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    Energy Technology Data Exchange (ETDEWEB)

    Zaccone, Eric J. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Goldsmith, W. Travis [Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States); Shimko, Michael J. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A. [Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States); Fedan, Jeffrey S. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States)

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (R{sub t}) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na{sup +} transport, without affecting Cl{sup −} transport or Na{sup +},K{sup +}-pump activity. R{sub t} was unaffected. Na{sup +} transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. - Highlights: • Butter flavoring vapor effects on human cultured airway epithelium were studied. • Na transport was reduced by a 6-h exposure to 25 ppm diacetyl and 2,3-pentanedione. • Na transport recovered 18 h after exposure. • > 60 ppm transepithelial voltage and resistance were abolished; cells were damaged. • Cells metabolized diacetyl and 2,3-pentanedione

  9. Uncoupling of attenuated myo-[3H]inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    International Nuclear Information System (INIS)

    Cammarata, P.R.; Tse, D.; Yorio, T.

    1991-01-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-[3H]inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-[3H]inositol uptake. No significant difference in the rates of passive efflux of myo-[3H]inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia

  10. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Cammarata, P.R.; Tse, D.; Yorio, T. (Department of Anatomy, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth (USA))

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  11. Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.

    Science.gov (United States)

    Yi, Jin-Kyu; Mehrazarin, Shebli; Oh, Ju-Eun; Bhalla, Anu; Oo, Jenessa; Chen, Wei; Lee, Min; Kim, Reuben H; Shin, Ki-Hyuk; Park, No-Hee; Kang, Mo K

    2014-11-01

    Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  12. Ganoderma extract prevents albumin-induced oxidative damage and chemokines synthesis in cultured human proximal tubular epithelial cells.

    Science.gov (United States)

    Lai, Kar Neng; Chan, Loretta Y Y; Tang, Sydney C W; Leung, Joseph C K

    2006-05-01

    Ganoderma lucidum (Ganoderma or lingzhi) is widely used as an alternative medicine remedy to promote health and longevity. Recent studies have indicated that components extracted from Ganoderma have a wide range of pharmacological actions including suppressing inflammation and scavenging free radicals. We recently reported that tubular secretion of interleukin-8 (IL-8) induced by albumin is important in the pathogenesis of tubulointerstitial injury in the proteinuric state. In this study, we explored the protective effect of Ganoderma extract (LZ) on albumin-induced kidney epithelial injury. Growth arrested human proximal tubular epithelial cells (PTECs) were incubated with 0.625 to 10 mg/ml human serum albumin (HSA) for up to 72 h. HSA induced DNA damage and apoptosis in PTEC in a dose- and time-dependent manner. Co-incubation of PTEC with 4-64 microg/ml LZ significantly reduced the oxidative damage and cytotoxic effect of HSA in a dose-dependent manner (PGanoderma (16 microg/ml). To explore the components of LZ that exhibited most protective effect in HSA-induced PTEC damages, LZ was further separated into two sub-fractions, LZF1 (MW effective in reducing sICAM-1 released from HSA-activated PTEC whereas the high molecular weight LZ (unfractionated LZ) was more effective in diminishing IL-8 production. Our results suggest that Ganoderma significantly reduces oxidative damages and apoptosis in PTEC induced by HSA. The differential reduction of IL-8 or sICAM-1 released from HSA-activated PTEC by different components of the LZ implicates that components of Ganoderma with different molecular weights could play different roles and operate different mechanisms in preventing HSA-induced PTEC damage.

  13. Comparison of gene expression profiles of normal human bronchial epithelial cells in 2D and 3D cultural conditions

    Data.gov (United States)

    National Aeronautics and Space Administration — The experiment is part of a project to study DNA repair process after ionizing radiation in organotypic 3-dimentional human bronchial epithlial cell culture. Human...

  14. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

    International Nuclear Information System (INIS)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

    2012-01-01

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

  15. The effect of isolation and culture methods on epithelial stem cell populations and their progeny-toward an improved cell expansion protocol for clinical application.

    Science.gov (United States)

    Lenihan, Catherine; Rogers, Caroline; Metcalfe, Anthony D; Martin, Yella H

    2014-12-01

    The use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. We compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. During the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Synthesis and characterization of telechelic polymethacrylates via RAFT polymerization

    NARCIS (Netherlands)

    Lima, V.G.R.; Jiang, X.; Brokken-Zijp, J.C.M.; Schoenmakers, P.J.; Klumperman, B.; Linde, van der R.

    2005-01-01

    The reversible addn.-fragmentation chain transfer (RAFT) polymn. technique has been employed to synthesize linear a,w -telechelic polymers with either hydroxyl or carboxyl end groups. Me methacrylate, Bu methacrylate, and Bu acrylate were polymd. with RAFT polymn. The polymns. exhibited the usual

  17. Synthesis and characterization of telechelic polymers prepared by RAFT

    NARCIS (Netherlands)

    Lima, V.G.R.; Brokken-Zijp, J.C.M.; Klumperman, B.; Benthem - van Duuren, van A.M.G.; Linde, van der R.

    2003-01-01

    The reversible addn.-fragmentation chain transfer (RAFT) polymn. technique was employed to synthesize telechelic polymers. Me methacrylate, Bu methacrylate were polymd. using RAFT polymn. The polymns. exhibit the usual characteristics of living processes, and were followed by a two-step chain-end

  18. Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Thayanithy, Venugopal [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States); Babatunde, Victor [Moore Laboratory, Department of Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Dickson, Elizabeth L. [Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Minnesota, Minneapolis, MN 55455 (United States); Wong, Phillip [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States); Oh, Sanghoon; Ke, Xu; Barlas, Afsar; Fujisawa, Sho; Romin, Yevgeniy [Molecular Cytology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Moreira, André L. [Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Downey, Robert J. [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Steer, Clifford J. [Departments of Medicine and Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States); Subramanian, Subbaya [Department of Surgery, University of Minnesota, Minneapolis, MN 55455 (United States); Manova-Todorova, Katia [Molecular Cytology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Moore, Malcolm A.S. [Moore Laboratory, Department of Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Lou, Emil, E-mail: emil-lou@umn.edu [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States)

    2014-04-15

    Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes and

  19. Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

    International Nuclear Information System (INIS)

    Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.; Wong, Phillip; Oh, Sanghoon; Ke, Xu; Barlas, Afsar; Fujisawa, Sho; Romin, Yevgeniy; Moreira, André L.; Downey, Robert J.; Steer, Clifford J.; Subramanian, Subbaya; Manova-Todorova, Katia; Moore, Malcolm A.S.; Lou, Emil

    2014-01-01

    Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes and

  20. Helicobacter pylori antigen HP0986 (TieA) interacts with cultured gastric epithelial cells and induces IL8 secretion via NF-κB mediated pathway.

    Science.gov (United States)

    Devi, Savita; Ansari, Suhail A; Vadivelu, Jamuna; Mégraud, Francis; Tenguria, Shivendra; Ahmed, Niyaz

    2014-02-01

    The envisaged roles and partly understood functional properties of Helicobacter pylori protein HP0986 are significant in the context of proinflammatory and or proapoptotic activities, the two important facilitators of pathogen survival and persistence. In addition, sequence analysis of this gene predicts a restriction endonuclease function which remained unknown thus far. To evaluate the role of HP0986 in gastric inflammation, we studied its expression profile using a large number of clinical isolates but a limited number of biopsies and patient sera. Also, we studied antigenic role of HP0986 in altering cytokine responses of human gastric epithelial (AGS) cells including its interaction with and localization within the AGS cells. For in vitro expression study of HP0986, 110 H. pylori clinical isolates were cultured from patients with functional dyspepsia. For expression analysis by qRT PCR of HP0986, 10 gastric biopsy specimens were studied. HP0986 was also used to detect antibodies in patient sera. AGS cells were incubated with recombinant HP0986 to determine cytokine response and NF-κB activation. Transient transfection with HP0986 cloned in pEGFPN1 was used to study its subcellular localization or homing in AGS cells. Out of 110 cultured H. pylori strains, 34 (31%) were positive for HP0986 and this observation was correlated with in vitro expression profiles. HP0986 mRNA was detected in 7 of the 10 biopsy specimens. Further, HP0986 induced IL-8 secretion in gastric epithelial cells in a dose and time-dependent manner via NF-κB pathway. Serum antibodies against HP0986 were positively associated with H. pylori positive patients. Transient transfection of AGS cells revealed both cytoplasmic and nuclear localization of HP0986. HP0986 was moderately prevalent in clinical isolates and its expression profile in cultures and gastric biopsies points to its being naturally expressed. Collective observations including the induction of IL-8 via TNFR1 and NF

  1. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification.

    Directory of Open Access Journals (Sweden)

    Nina Recek

    Full Text Available Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO due to remaining lens epithelial cells (LECs by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation at a highly targeted cell level compared to treatment on top of the medium (indirect treatment. Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity to the posterior lens capsule and PCO formation.

  2. Preserved and unpreserved 12 anti-allergic ophthalmic solutions and ocular surface toxicity: in vitro assessment in four cultured corneal and conjunctival epithelial cell lines.

    Science.gov (United States)

    Ayaki, Masahiko; Iwasawa, Atsuo; Yaguchi, Shigeo; Koide, Ryohei

    2010-12-01

    In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.

  3. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Hitchener, W.R.

    1986-01-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either 3 H 2 O or 1- 14 C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of 3 H 2 O or 1- 14 C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10 6 cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with 3 H 2 O and 4.0 +/- 1.1 times greater when measured with 14 C-acetate. Thus, 3 H 2 O and 14 C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid

  4. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  5. Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

    Science.gov (United States)

    Le-Bel, Gaëtan; Ghio, Sergio Cortez; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

    2018-05-27

    Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

  6. Monomethylfumarate induces γ-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina.

    Science.gov (United States)

    Promsote, Wanwisa; Makala, Levi; Li, Biaoru; Smith, Sylvia B; Singh, Nagendra; Ganapathy, Vadivel; Pace, Betty S; Martin, Pamela M

    2014-05-13

    Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina. Human globin gene expression was evaluated by RT-quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. γ-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 μM dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase-qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate γ-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous β(s) mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 μM) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1β, and VEGF expression were also analyzed. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated γ-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  7. Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

    Science.gov (United States)

    Sawazaki, Ryoichi; Ishihara, Tomoaki; Usui, Shinya; Hayashi, Erika; Tahara, Kayoko; Hoshino, Tatsuya; Higuchi, Akihiro; Nakamura, Shigeru; Tsubota, Kazuo; Mizushima, Tohru

    2014-04-21

    Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

  8. Raft tectonics in northern Campos Basin; Tectonica de jangada (raft tectonics) na area norte da Bacia de Campos

    Energy Technology Data Exchange (ETDEWEB)

    Castro, Marilia R. de [Universidade Estadual do Norte Fluminense (UENF), Campos dos Goytacases, RJ (Brazil)]|[PETROBRAS, Macae, RJ (Brazil). Unidade de Negocio da Bacia de Campos; Fugita, Adhemar M. [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Programa de Recursos Humanos da ANP

    2004-07-01

    In the northern area of Campos Basin salt gliding/spreading processes promoted the break-up and transport of Cretaceous and Tertiary rocks overlying the evaporites. This process is known as raft tectonics, and it represents the most extreme form of thin-skinned extension above the salt decollement surface. Three distinct geotectonic domains were recognized that formed in response to the raft tectonics. The first one, confined to the shallower shelf portion of the basin, is characterized by minor extension (pre-raft domain), probably because of small salt thickness and low gradient. In the second domain (or disorganized rafts domain), located in distal platformal and slope areas, seismic sections show the occurrence of blocks or rafts with angular shapes, sometimes imbricated and frequently discontinuous. In the third domain, or domain of organized rafts, located in bacinal region, seismic sections show a more continuous raft pattern, often folded because of salt compression in the distal portions of the basin. The main purposes of this work is to characterize these three tectonic domains distinguished by raft tectonics, as well as their importance in hydrocarbon accumulations in calcarenites. (author)

  9. Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

    Science.gov (United States)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

  10. Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H

    2013-01-01

    The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces...... functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither...... major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present...

  11. Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model.

    Science.gov (United States)

    Matsumoto, Reiko; Sugimoto, Masazumi

    2007-02-01

    We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.

  12. Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Zhang

    Full Text Available As a subfamily of matrix metalloproteinases (MMPs, gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive

  13. 8-prenylnaringenin and tamoxifen inhibit the shedding of irradiated epithelial cells and increase the latency period of radiation-induced oral mucositis. Cell culture and murine model

    Energy Technology Data Exchange (ETDEWEB)

    Ryck, Tine de; Impe, Annouchka van; Bracke, Marc E. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Vanhoecke, Barbara W. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Ghent University, Laboratory of Microbial Ecology and Technology (LabMET), Ghent (Belgium); Heyerick, Arne [Ghent University, Laboratory of Pharmacognosy and Phytochemistry, Ghent (Belgium); Vakaet, Luc; Neve, Wilfried de [Ghent University Hospital, Department of Radiation Oncology, Ghent (Belgium); Mueller, Doreen [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Schmidt, Margret [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); German Cancer Consortium (DKTK) partner site Dresden and German Cancer Center (DKFZ), Heidelberg (Germany); Doerr, Wolfgang [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Medical University, Department of Radiation Oncology, CCC, and CD-Laboratory RadOnc, Vienna (Austria)

    2015-05-01

    The major component in the pathogenesis of oral radiation-induced mucositis is progressive epithelial hypoplasia and eventual ulceration. Irradiation inhibits cell proliferation, while cell loss at the surface continues. We conceived to slow down this desquamation by increasing intercellular adhesion, regulated by the E-cadherin/catenin complex. We investigated if 8-prenylnaringenin (8-PN) or tamoxifen (TAM) decrease the shedding of irradiated human buccal epithelial cells in vitro and thus delay the ulcerative phase of radiation-induced mucositis in vivo. In vitro, aggregates of buccal epithelial cells were irradiated and cultured in suspension for 11 days. 8-PN or TAM were investigated regarding their effect on cell shedding. In vivo, the lower tongue surface of mice was irradiated with graded single doses of 25 kV X-rays. The incidence, latency, and duration of the resulting mucosal ulcerations were analyzed after topical treatment with 8-PN, TAM or solvent. 8-PN or TAM prevented the volume reduction of the irradiated cell aggregates during the incubation period. This was the result of a higher residual cell number in the treated versus the untreated irradiated aggregates. In vivo, topical treatment with 8-PN or TAM significantly increased the latency of mucositis from 10.9 to 12.1 and 12.4 days respectively, while the ulcer incidence was unchanged. 8-PN and TAM prevent volume reduction of irradiated cell aggregates in suspension culture. In the tongues of mice, these compounds increase the latency period. This suggests a role for these compounds for the amelioration of radiation-induced mucositis in the treatment of head and neck tumors. (orig.) [German] Die wesentliche Komponente in der Pathogenese der radiogenen Mukositis ist eine progressive epitheliale Hypoplasie und letztendlich Ulzeration. Die Bestrahlung hemmt die Zellproliferation, waehrend der Zellverlust an der Oberflaeche fortbesteht. Wir versuchten, diese Desquamation durch eine Stimulation der

  14. Analysis of lipid raft molecules in the living brain slices.

    Science.gov (United States)

    Kotani, Norihiro; Nakano, Takanari; Ida, Yui; Ito, Rina; Hashizume, Miki; Yamaguchi, Arisa; Seo, Makoto; Araki, Tomoyuki; Hojo, Yasushi; Honke, Koichi; Murakoshi, Takayuki

    2017-08-24

    Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Effects of RAFT Agent on the Selective Approach of Molecularly Imprinted Polymers

    OpenAIRE

    Asman, Saliza; Mohamad, Sharifah; Sarih, Norazilawati

    2015-01-01

    Two types of reversible addition-fragmentation chain transfer molecularly imprinted polymers (RAFT-MIPs) were synthesized using different monomers, which were methacrylic acid functionalized β-cyclodextrin (MAA-β-CD) and 2-hydroxyethyl methacrylate functionalized β-cyclodextrin (HEMA-β-CD), via reversible addition-fragmentation chain transfer (RAFT) polymerization, and were represented as RAFT-MIP(MAA-β-CD) and RAFT-MIP(HEMA-β-CD), respectively. Both RAFT-MIPs were systematically characterize...

  16. Cell kinetics of differentiation of Na+-dependent hexose transport in a cultured renal epithelial cell line

    International Nuclear Information System (INIS)

    Cook, J.S.; Weiss, E.R.

    1985-01-01

    Fully differentiated cells of the renal proximal tubule have the capability of taking up hexoses across their apical borders by transport coupled to the Na + -electrochemical gradient. This property is also found in postconfluent cultures of the cloned cell line LLC-PK 1 , a morphologically polarized line of renal cells. Postconfluent cells develop the Na + -dependent capacity to transport hexoses at their apical surface. This function is not observable during the growth phase of the cultures. To analyze the developmental process at the cellular level a method has been derived to separate transporting cells, expressing the differentiated function, from nontransporting cells. The method is based on the swelling of the cells accompanying the uptake of the nonmetabolizable glucose analog alpha methylglucoside. The swollen cells have a lower buoyant density than the undifferentiated cells and may be separated from them on density gradients. Analysis of the distribution of cells on such gradients shows that after the cells reach confluence the undifferentiated subpopulation is recruited onto the differentiation pathway with a rate constant of 0.2 per day, that 5 to 7 days are required for a cell to traverse this pathway to the fully differentiated state, and that once the maximum uptake capacity is achieved the cells do not develop further

  17. Functional Proteomic Analysis of Lipid Raft Kinase Complexes

    Science.gov (United States)

    2009-08-01

    0.0 623 + + + + + IPI00219425 PVR Isoform Beta of Poliovirus receptor precursor Non-raft 9 86/372 + + + + + 19.0 1.5 12.7 17 3 5.7 21 0 70.0 1st EXP...Counts No. Prob. Both Acc.Number Gene Symbol Description Fraction Uni. Pep. IPI00219425 PVR Isoform Beta of Poliovirus receptor precursor Raft 2 25...372 + + 1.0 0.0 3.3 1 0 3.3 1 0 3.3 624 + + + + + IPI00022661 PVRL2 Isoform Delta of Poliovirus receptor-related protein 2 precursor Non-raft 7 92/538

  18. Apparatus for measuring the finite load-deformation behavior of a sheet of epithelial cells cultured on a mesoscopic freestanding elastomer membrane

    International Nuclear Information System (INIS)

    Selby, John C.; Shannon, Mark A.

    2007-01-01

    Details are given for the design, calibration, and operation of an apparatus for measuring the finite load-deformation behavior of a sheet of living epithelial cells cultured on a mesoscopic freestanding elastomer membrane, 10 μm thick and 5 mm in diameter. Although similar in concept to bulge tests used to investigate the mechanical properties of micromachined thin films, cell-elastomer composite diaphragm inflation tests pose a unique set of experimental challenges. Composite diaphragm (CD) specimens are extremely compliant (E MIN =0 μl, V MAX ≤40 μl) while simultaneously recording the inflation pressure acting at the fixed boundary of the specimen, p(r=a). Using a carefully prescribed six-cycle inflation test protocol, the apparatus is shown to be capable of measuring the [V,p(r=a)] inflation response of a cell-elastomer CD with random uncertainties estimated at ±0.45 μl and ±2.5 Pa, respectively

  19. Effects of Lutein on Hyperosmoticity-Induced Upregulation of IL-6 in Cultured Corneal Epithelial Cells and Its Relevant Signal Pathways

    Directory of Open Access Journals (Sweden)

    Shih-Chun Chao

    2016-01-01

    Full Text Available Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye.

  20. TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    International Nuclear Information System (INIS)

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah; Cho, Jin Won; Lee, Joon H.

    2011-01-01

    Highlights: ► TGF-β induces aberrant expression of βIII in RPE cells via the ERK pathway. ► TGF-β increases O-GlcNAc modification of βIII in RPE cells. ► Mature RPE cells have the capacity to express a neuron-associated gene by TGF-β. -- Abstract: The class III β-tubulin isotype (β III ) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III β-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-β (TGF-β) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.

  1. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

    Science.gov (United States)

    Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na + , K + -ATPase, or the Na + pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β 1 subunit of Na + , K + -ATPase plays an important role in this mechanism because homotypic β 1 -β 1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na + pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β 2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na + , K + -ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na + pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K + -ATPase in RPE cells depends on the expression of the β 2 subunit.

  2. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Jee [Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyeonggi-do (Korea, Republic of); Chun, Ji Na; Jung, Sun-Ah [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of); Cho, Jin Won [Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lee, Joon H., E-mail: joonhlee@konyang.ac.kr [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  3. Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Kevin M. Dorney

    2013-12-01

    Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

  4. Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

    Science.gov (United States)

    Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

    2013-01-01

    The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

  5. optimizing conventional des concrete raft ng conventional design

    African Journals Online (AJOL)

    eobe

    concrete cross section area of raft slab foundation ... accurately modeling boundary cond never be ... the design of reinforced concrete flat ... undation and soil layer configuration adopted for the finite element analy .... After the application of.

  6. Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

    Science.gov (United States)

    Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

    2018-01-01

    Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

  7. Different apoptotic effects of [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin on normal and cancerous human epithelial breast cells in primary culture.

    Science.gov (United States)

    Vetrugno, Carla; Muscella, Antonella; Fanizzi, Francesco Paolo; Cossa, Luca Giulio; Migoni, Danilo; De Pascali, Sandra Angelica; Marsigliante, Santo

    2014-11-01

    The aim of this study was to determine whether [platinum (Pt)(O,O'-acetylacetonate (acac))(γ-acac)(dimethylsulphide (DMS))] is differentially cytotoxic in normal and cancer cells, and to measure comparative levels of cytotoxicity compared with cisplatin in the same cells. We performed experiments on cancerous and normal epithelial breast cells in primary culture obtained from the same patients. The apoptotic effects [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin in cancerous and normal breast cells were compared. Cancer cells were more sensitive to [Pt(O,O'-acac)(γ-acac)(DMS)] (IC50 = 5.22 ± 1.2 μmol·L(-1)) than normal cells (IC50 = 116.9 ± 8.8 μmol·L(-1)). However, the difference was less strong when cisplatin was used (IC50 = 96.0 ± 6.9 and 61.9 ± 6.1 μmol·L(-1) for cancer and normal cells respectively). Both compounds caused reactive oxygen species (ROS) production with different mechanisms: [Pt(O,O'-acac)(γ-acac)(DMS)] quickly activated NAD(P)H oxidase while cisplatin caused a slower formation of mitochondrial ROS. Cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)] caused activation of caspases, proteolysis of PARP and modulation of Bcl-2, Bax and Bid. [Pt(O,O'-acac)(γ-acac)(DMS)] also caused leakage of cytochrome c from the mitochondria. Overall, these processes proceeded more quickly in cells treated with [Pt(O,O'-acac)(γ-acac)(DMS)] compared with cisplatin. [Pt(O,O'-acac)(γ-acac)(DMS)] effects were faster and quantitatively greater in cancer than in normal cells. [Pt(O,O'-acac)(γ-acac)(DMS)] caused a fast decrease of mitochondrial membrane potential, especially in cancer cells. [Pt(O,O'-acac)(γ-acac)(DMS)] was specific to breast cancer cells in primary culture, and this observation makes this compound potentially more interesting than cisplatin. © 2014 The British Pharmacological Society.

  8. Different apoptotic effects of [Pt(O,O′-acac)(γ-acac)(DMS)] and cisplatin on normal and cancerous human epithelial breast cells in primary culture

    Science.gov (United States)

    Vetrugno, Carla; Muscella, Antonella; Fanizzi, Francesco Paolo; Cossa, Luca Giulio; Migoni, Danilo; De Pascali, Sandra Angelica; Marsigliante, Santo

    2014-01-01

    Background and Purpose The aim of this study was to determine whether [platinum (Pt)(O,O′-acetylacetonate (acac))(γ-acac)(dimethylsulphide (DMS))] is differentially cytotoxic in normal and cancer cells, and to measure comparative levels of cytotoxicity compared with cisplatin in the same cells. Experimental Approach We performed experiments on cancerous and normal epithelial breast cells in primary culture obtained from the same patients. The apoptotic effects [Pt(O,O′-acac)(γ-acac)(DMS)] and cisplatin in cancerous and normal breast cells were compared. Key Results Cancer cells were more sensitive to [Pt(O,O′-acac)(γ-acac)(DMS)] (IC50 = 5.22 ± 1.2 μmol·L−1) than normal cells (IC50 = 116.9 ± 8.8 μmol·L−1). However, the difference was less strong when cisplatin was used (IC50 = 96.0 ± 6.9 and 61.9 ± 6.1 μmol·L−1 for cancer and normal cells respectively). Both compounds caused reactive oxygen species (ROS) production with different mechanisms: [Pt(O,O′-acac)(γ-acac)(DMS)] quickly activated NAD(P)H oxidase while cisplatin caused a slower formation of mitochondrial ROS. Cisplatin and [Pt(O,O′-acac)(γ-acac)(DMS)] caused activation of caspases, proteolysis of PARP and modulation of Bcl-2, Bax and Bid. [Pt(O,O′-acac)(γ-acac)(DMS)] also caused leakage of cytochrome c from the mitochondria. Overall, these processes proceeded more quickly in cells treated with [Pt(O,O′-acac)(γ-acac)(DMS)] compared with cisplatin. [Pt(O,O′-acac)(γ-acac)(DMS)] effects were faster and quantitatively greater in cancer than in normal cells. [Pt(O,O′-acac)(γ-acac)(DMS)] caused a fast decrease of mitochondrial membrane potential, especially in cancer cells. Conclusions and Implications [Pt(O,O′-acac)(γ-acac)(DMS)] was specific to breast cancer cells in primary culture, and this observation makes this compound potentially more interesting than cisplatin. PMID:24990093

  9. Sorafenib suppresses TGF-β responses by inducing caveolae/lipid raft-mediated internalization/degradation of cell-surface type II TGF-β receptors: Implications in development of effective adjunctive therapy for hepatocellular carcinoma.

    Science.gov (United States)

    Chung, Chih-Ling; Wang, Shih-Wei; Sun, Wei-Chih; Shu, Chih-Wen; Kao, Yu-Chen; Shiao, Meng-Shin; Chen, Chun-Lin

    2018-04-18

    Sorafenib is the only FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other malignancies. Studies indicate that TGF-β signalling is associated with tumour progression in HCC. Autocrine and paracrine TGF-β promotes tumour growth and malignancy by inducing epithelial-mesenchymal transition (EMT). Sorafenib is believed to antagonize tumour progression by inhibiting TGF-β-induced EMT. It improves survival of patients but HCC later develops resistance and relapses. The underlying mechanism of resistance is unknown. Understanding of the molecular mechanism of sorafenib inhibition of TGF-β-induced signalling or responses in HCC may lead to development of adjunctive effective therapy for HCC. In this study, we demonstrate that sorafenib suppresses TGF-β responsiveness in hepatoma cells, hepatocytes, and animal liver, mainly by downregulating cell-surface type II TGF-β receptors (TβRII) localized in caveolae/lipid rafts and non-lipid raft microdomains via caveolae/lipid rafts-mediated internalization and degradation. Furthermore, sorafenib-induced downregulation and degradation of cell-surface TβRII is prevented by simultaneous treatment with a caveolae disruptor or lysosomal inhibitors. On the other hand, sorafenib only downregulates cell-surface TβRII localized in caveolae/lipid rafts but not localized in non-lipid raft microdomains in hepatic stellate cells. These results suggest that sorafenib inhibits TGF-β signalling mainly by inducing caveolae/lipid raft-mediated internalization and degradation of cell-surface TβR-II in target cells. They may also imply that treatment with agents which promote formation of caveolae/lipid rafts, TGF-β receptor kinase inhibitors (e.g., LY2157299) or TGF-β peptide antagonists (by liver-targeting delivery) may be considered as effective adjunct therapy with sorafenib for HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

    Science.gov (United States)

    Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

    2014-01-01

    Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

  11. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity...... subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum...

  12. High temperature initiator-free RAFT polymerization of methyl methacrylate in a microwave reactor

    NARCIS (Netherlands)

    Paulus, R.M.; Becer, C.R.; Hoogenboom, R.; Schubert, U.S.

    2009-01-01

    The reversible additionfragmentation chain transfer (RAFT) polymerization of methyl methacrylate (MMA) was investigated under microwave irradiation. At first, a comparison was made between microwave and thermal heating for the RAFT polymerization of MMA with azobis(isobutyronitrile) (AIBN) as

  13. Involvement of glycosphingolipid-enriched lipid rafts in inflammatory responses.

    Science.gov (United States)

    Iwabuchi, Kazuhisa

    2015-01-01

    Glycosphingolipids (GSLs) are membrane components consisting of hydrophobic ceramide and hydrophilic sugar moieties. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid rafts. Biochemical analyses have demonstrated that GSL-enriched lipid rafts contain several kinds of transducer molecules, including Src family kinases. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1), as well as being associated with several key cellular processes. LacCer recruits PCKα/ε and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and their adhesion to endothelial cells, as well as regulating β1-integrin clustering and endocytosis on cell surfaces. This review describes the organizational and inflammation-related functions of LacCer-enriched lipid rafts.

  14. RAFT: a computer program for fault tree risk calculations

    International Nuclear Information System (INIS)

    Seybold, G.D.

    1977-11-01

    A description and user instructions are presented for RAFT, a FORTRAN computer code for calculation of a risk measure for fault tree cut sets. RAFT calculates release quantities and a risk measure based on the product of probability and release quantity for cut sets of fault trees modeling the accidental release of radioactive material from a nuclear fuel cycle facility. Cut sets and their probabilities are supplied as input to RAFT from an external fault tree analysis code. Using the total inventory available of radioactive material, along with release fractions for each event in a cut set, the release terms are calculated for each cut set. Each release term is multiplied by the cut set probability to yield the cut set risk measure. RAFT orders the dominant cut sets on the risk measure. The total risk measure of processed cut sets and their fractional contributions are supplied as output. Input options are available to eliminate redundant cut sets, apply threshold values on cut set probability and risk, and control the total number of cut sets output. Hash addressing is used to remove redundant cut sets from the analysis. Computer hardware and software restrictions are given along with a sample problem and cross-reference table of the code. Except for the use of file management utilities, RAFT is written exclusively in FORTRAN language and is operational on a Control Data, CYBER 74-18--series computer system. 4 figures

  15. Tunable, Quantitative Fenton-RAFT Polymerization via Metered Reagent Addition.

    Science.gov (United States)

    Nothling, Mitchell D; McKenzie, Thomas G; Reyhani, Amin; Qiao, Greg G

    2018-05-10

    A continuous supply of radical species is a key requirement for activating chain growth and accessing quantitative monomer conversions in reversible addition-fragmentation chain transfer (RAFT) polymerization. In Fenton-RAFT, activation is provided by hydroxyl radicals, whose indiscriminate reactivity and short-lived nature poses a challenge to accessing extended polymerization times and quantitative monomer conversions. Here, an alternative Fenton-RAFT procedure is presented, whereby radical generation can be finely controlled via metered dosing of a component of the Fenton redox reaction (H 2 O 2 ) using an external pumping system. By limiting the instantaneous flux of radicals and ensuring sustained radical generation over tunable time periods, metered reagent addition reduces unwanted radical "wasting" reactions and provides access to consistent quantitative monomer conversions with high chain-end fidelity. Fine tuning of radical concentration during polymerization is achieved simply via adjustment of reagent dose rate, offering significant potential for automation. This modular strategy holds promise for extending traditional RAFT initiation toward more tightly regulated radical concentration profiles and affords excellent prospects for the automation of Fenton-RAFT polymerization. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Research status of wave energy conversion (WEC) device of raft structure

    Science.gov (United States)

    Dong, Jianguo; Gao, Jingwei; Tao, Liang; Zheng, Peng

    2017-10-01

    This paper has briefly described the concept of wave energy generation and six typical conversion devices. As for raft structure, detailed analysis is provided from its development process to typical devices. Taking the design process and working principle of Plamis as an example, the general principle of raft structure is briefly described. After that, a variety of raft structure models are introduced. Finally, the advantages and disadvantages, and development trend of raft structure are pointed out.

  17. Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells.

    Science.gov (United States)

    Liu, Xunxian; Allen, Jeffrey D; Arnold, Julia T; Blackman, Marc R

    2008-04-01

    Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

  18. Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes

    DEFF Research Database (Denmark)

    Calay, Damien; Vind-Kezunovic, Dina; Frankart, Aurelie

    2010-01-01

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...

  19. 78 FR 17087 - Special Local Regulation; New River Raft Race, New River; Fort Lauderdale, FL

    Science.gov (United States)

    2013-03-20

    ...-AA08 Special Local Regulation; New River Raft Race, New River; Fort Lauderdale, FL AGENCY: Coast Guard... on the New River in Fort Lauderdale, Florida during the Rotary Club of Fort Lauderdale New River Raft... States during the Rotary Club of Fort Lauderdale New River Raft Race. On March 23, 2013, Fort Lauderdale...

  20. Radiation-induced controlled polymerization of acrylic acid by RAFT and RAFT-MADIX methods in protic solvents

    Science.gov (United States)

    Sütekin, S. Duygu; Güven, Olgun

    2018-01-01

    The kinetic investigation of one-pot synthesis of poly(acrylic acid) (PAA) prepared via gamma radiation induced controlled polymerization was reported. PAA homopolymers were prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization in the presence of trithiocarbonate-based chain transfer agent (CTA) 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and also by Reversible Addition-Fragmentation/Macromolecular Design by Inter-change of Xanthates (RAFT/MADIX) polymerization in the presence of a xanthate based CTA O-ethyl-S-(1-methoxycarbonyl) ethyl dithiocarbonate (RA1). The polymerizations were performed at room temperature by the virtue of ionizing radiation. Protic solvents were used for the RAFT polymerization of AA considering environmental profits. The linear first-order kinetic plot, close control of molecular weight by the monomer/CTA molar ratio supported that the polymerization proceeds in a living fashion. The linear increase in molecular weight with conversion monitored by Size Exclusion Chromatography (SEC) is another proof of controlling of polymerization. [Monomer]/[RAFT] ratio and conversion was controlled to obtain PAA in the molecular weight range of 6900-35,800 with narrow molecular weight distributions. Reaction kinetics and effect of the amount of RAFT agent were investigated in detail. Between two different types of CTA, trithiocarbonate based DDMAT was found to be more efficient in terms of low dispersity (Đ) and linear first-order kinetic behavior for the radiation induced controlled synthesis of PAA homopolymers.

  1. Raft-like membrane domains in pathogenic microorganisms.

    Science.gov (United States)

    Farnoud, Amir M; Toledo, Alvaro M; Konopka, James B; Del Poeta, Maurizio; London, Erwin

    2015-01-01

    The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Rapid, long-distance dispersal by pumice rafting.

    Directory of Open Access Journals (Sweden)

    Scott E Bryan

    Full Text Available Pumice is an extremely effective rafting agent that can dramatically increase the dispersal range of a variety of marine organisms and connect isolated shallow marine and coastal ecosystems. Here we report on a significant recent pumice rafting and long-distance dispersal event that occurred across the southwest Pacific following the 2006 explosive eruption of Home Reef Volcano in Tonga. We have constrained the trajectory, and rate, biomass and biodiversity of transfer, discovering more than 80 species and a substantial biomass underwent a >5000 km journey in 7-8 months. Differing microenvironmental conditions on the pumice, caused by relative stability of clasts at the sea surface, promoted diversity in biotic recruitment. Our findings emphasise pumice rafting as an important process facilitating the distribution of marine life, which have implications for colonisation processes and success, the management of sensitive marine environments, and invasive pest species.

  3. With or without rafts? Alternative views on cell membranes.

    Science.gov (United States)

    Sevcsik, Eva; Schütz, Gerhard J

    2016-02-01

    The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity. © 2015 WILEY Periodicals, Inc.

  4. RAFT technology for the production of advanced photoresist polymers

    Science.gov (United States)

    Sheehan, Michael T.; Farnham, William B.; Okazaki, Hiroshi; Sounik, James R.; Clark, George

    2008-03-01

    Reversible Addition Fragmentation Chain Transfer (RAFT) technology has been developed for use in producing high yield low polydispersity (PD) polymers for many applications. RAFT technology is being used to produce low PD polymers and to allow control of the polymer architecture. A variety of polymers are being synthesized for use in advanced photoresists using this technique. By varying the RAFT reagent used we can modulate the system reactivity of the RAFT reagent and optimize it for use in acrylate or methacrylate monomer systems (193 and 193i photoresist polymers) or for use in styrenic monomer systems (248 nm photoresist polymers) to achieve PD as low as 1.05. RAFT polymerization technology also allows us to produce block copolymers using a wide variety of monomers. These block copolymers have been shown to be useful in self assembly polymer applications to produce unique and very small feature sizes. The mutual compatibilities of all the components within a single layer 193 photoresist are very important in order to achieve low LWR and low defect count. The advent of immersion imaging demands an additional element of protection at the solid/liquid interface. We have used RAFT technology to produce block copolymers comprising a random "resist" block with composition and size based on conventional dry photoresist materials, and a "low surface energy" block for use in 193i lithography. The relative block lengths and compositions may be varied to tune solution behavior, surface energy, contact angles, and solubility in developer. The use of this technique will be explored to produce polymers used in hydrophobic single layer resists as well as additives compatible with the main photoresist polymer.

  5. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  6. Generic sorting of raft lipids into secretory vesicles in yeast

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W

    2011-01-01

    Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma...... a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...

  7. Detecting Temporal and Spatial Effects of Epithelial Cancers with Raman Spectroscopy

    Directory of Open Access Journals (Sweden)

    Matthew D. Keller

    2008-01-01

    Full Text Available Epithelial cancers, including those of the skin and cervix, are the most common type of cancers in humans. Many recent studies have attempted to use Raman spectroscopy to diagnose these cancers. In this paper, Raman spectral markers related to the temporal and spatial effects of cervical and skin cancers are examined through four separate but related studies. Results from a clinical cervix study show that previous disease has a significant effect on the Raman signatures of the cervix, which allow for near 100% classification for discriminating previous disease versus a true normal. A Raman microspectroscopy study showed that Raman can detect changes due to adjacent regions of dysplasia or HPV that cannot be detected histologically, while a clinical skin study showed that Raman spectra may be detecting malignancy associated changes in tissues surrounding nonmelanoma skin cancers. Finally, results of an organotypic raft culture study provided support for both the skin and the in vitro cervix results. These studies add to the growing body of evidence that optical spectroscopy, in this case Raman spectral markers, can be used to detect subtle temporal and spatial effects in tissue near cancerous sites that go otherwise undetected by conventional histology.

  8. Development of optimum design from static response of pile–raft interaction

    DEFF Research Database (Denmark)

    Taghavi Ghalesari, A; Barari, Amin; Fardad Amini, P

    2015-01-01

    Piled raft foundations are among the most commonly used support structures for offshore projects. When a raft foundation alone does not satisfy the design requirements, piles may be added to improve the ultimate load capacity and the settlement performance of the raft. In this study, design...... for the piled raft were highly influenced by the number of piles and the raft thickness. Optimal design configurations of piles for cohesive soils are discussed. Increasing the pile spacing decreased the pile butt load ratio by allowing for a more uniform load distribution between the piles....

  9. Biomedical applications of polymers derived by reversible addition - fragmentation chain-transfer (RAFT).

    Science.gov (United States)

    Fairbanks, Benjamin D; Gunatillake, Pathiraja A; Meagher, Laurence

    2015-08-30

    RAFT- mediated polymerization, providing control over polymer length and architecture as well as facilitating post polymerization modification of end groups, has been applied to virtually every facet of biomedical materials research. RAFT polymers have seen particularly extensive use in drug delivery research. Facile generation of functional and telechelic polymers permits straightforward conjugation to many therapeutic compounds while synthesis of amphiphilic block copolymers via RAFT allows for the generation of self-assembled structures capable of carrying therapeutic payloads. With the large and growing body of literature employing RAFT polymers as drug delivery aids and vehicles, concern over the potential toxicity of RAFT derived polymers has been raised. While literature exploring this complication is relatively limited, the emerging consensus may be summed up in three parts: toxicity of polymers generated with dithiobenzoate RAFT agents is observed at high concentrations but not with polymers generated with trithiocarbonate RAFT agents; even for polymers generated with dithiobenzoate RAFT agents, most reported applications call for concentrations well below the toxicity threshold; and RAFT end-groups may be easily removed via any of a variety of techniques that leave the polymer with no intrinsic toxicity attributable to the mechanism of polymerization. The low toxicity of RAFT-derived polymers and the ability to remove end groups via straightforward and scalable processes make RAFT technology a valuable tool for practically any application in which a polymer of defined molecular weight and architecture is desired. Copyright © 2015. Published by Elsevier B.V.

  10. The Lipid Raft Proteome of African Trypanosomes Contains Many Flagellar Proteins.

    Science.gov (United States)

    Sharma, Aabha I; Olson, Cheryl L; Engman, David M

    2017-08-24

    Lipid rafts are liquid-ordered membrane microdomains that form by preferential association of 3-β-hydroxysterols, sphingolipids and raft-associated proteins often having acyl modifications. We isolated lipid rafts of the protozoan parasite Trypanosoma brucei and determined the protein composition of lipid rafts in the cell. This analysis revealed a striking enrichment of flagellar proteins and several putative signaling proteins in the lipid raft proteome. Calpains and intraflagellar transport proteins, in particular, were found to be abundant in the lipid raft proteome. These findings provide additional evidence supporting the notion that the eukaryotic cilium/flagellum is a lipid raft-enriched specialized structure with high concentrations of sterols, sphingolipids and palmitoylated proteins involved in environmental sensing and cell signaling.

  11. Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

    Science.gov (United States)

    Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

    2008-10-01

    Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

  12. Effectiveness of combinations of raft foundation with aprons as a ...

    Indian Academy of Sciences (India)

    B A Vijayasree

    2018-03-10

    Mar 10, 2018 ... bridge projects vary from exposed hard rock to pure sand for a considerable .... arrangements were made to dissipate the energy during inletting of water .... This endorsed the fact that the use of raft foundation in Case 2 reduced the ... at the downstream side, which extended to the wake region of horseshoe ...

  13. Direct surface PEGylation of nanodiamond via RAFT polymerization

    International Nuclear Information System (INIS)

    Shi, Yingge; Liu, Meiying; Wang, Ke; Huang, Hongye; Wan, Qing; Tao, Lei; Fu, Lihua; Zhang, Xiaoyong; Wei, Yen

    2015-01-01

    Graphical abstract: In this paper, we describe an efficient, practical and novel method to modify ND via direct immobilization of chain transfer agent for RAFT polymerization. - Highlights: • Surface PEGylation of ND via RAFT polymerization. • ND with high water dispersibility and excellent biocompatibility. • Controlled living polymerization. - Abstract: Nanodiamond (ND) is a novel class of carbon nanomaterials, which has been extensively investigated for biomedical applications because of its small size, high surface area and excellent biocompatibility. However, the biomedical applications of unmodified ND are still largely restricted because of their poor dispersibility in both aqueous and organic medium. In this work, we reported a novel strategy for the surface modification of ND via reversible addition fragmentation chain transfer (RAFT) polymerization. For preparation of the PEGylated ND (pPEGMA-ND), chain transfer agent (CTA) was immobilized onto ND through reaction between the hydroxyl group of ND and the carboxyl group of CTA, which was used as the initiator for surface-initiated RAFT polymerization. The successful preparation of pPEGMA-ND was characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectra and thermal gravimetric analysis in detail. Results demonstrated that pPEGMA-ND exhibited enhanced water dispersibility and desirable biocompatibility, making it promising for biomedical applications.

  14. Toward a Value for Guided Rafting on Southern Rivers

    Science.gov (United States)

    J. Michael Bowker; Donald B.K. English; Jason A. Donovan

    1996-01-01

    This study examines per trip consumer surplus associated with guided whitewater rafting on two southern rivers. First, household recreation demand functions are estimated based on the individual travel cost model using truncated count data regression methods and alternative price specifications. Findings show mean per trip consumer surplus point estimates between $89...

  15. Research Applications for Teaching (RAFT) Project. Final Report.

    Science.gov (United States)

    Thomson, James R., Jr.; Handley, Herbert M.

    A report is given of the development and progress of the Research Applications for Teaching (RAFT) project, developed at Mississippi State University. Based upon research findings relative to effective teaching and effective schooling, five curriculum modules were prepared and implemented in instruction. In the second year of the project the…

  16. NMR spectroscopy in the optimization and evaluation of RAFT agents

    NARCIS (Netherlands)

    Klumperman, B.; McLeary, J.B.; van den Dungen, E.; Pound, G.

    2007-01-01

    The selection of a suitable mediating agent in Reversible Addition-Fragmentation Chain Transfer (RAFT) mediated polymerization is crucial to the degree of control that can be achieved. An overview of work from the Stellenbosch group is presented in which the use of NMR spectroscopy as a tool for

  17. Radical-induced oxidation of RAFT agents : a kinetic study

    NARCIS (Netherlands)

    Li, Changxi; He, Junpo; Zhou, Yanwu; Gu, Yuankai; Yang, Yuliang

    2011-01-01

    Radical-induced oxidn. of reversible addn.-fragmentation chain transfer (RAFT) agents is studied with respect to the effect of mol. structure on oxidn. rate. The radicals are generated by homolysis of either azobisisobutyronitrile or alkoxyamine and transformed in situ immediately into peroxy

  18. Direct surface PEGylation of nanodiamond via RAFT polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yingge [Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004 (China); Liu, Meiying [Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Wang, Ke [Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084 (China); Huang, Hongye; Wan, Qing [Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Tao, Lei [Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084 (China); Fu, Lihua [School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004 (China); Zhang, Xiaoyong, E-mail: xiaoyongzhang1980@gmail.com [Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Wei, Yen, E-mail: weiyen@tsinghua.edu.cn [Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084 (China)

    2015-12-01

    Graphical abstract: In this paper, we describe an efficient, practical and novel method to modify ND via direct immobilization of chain transfer agent for RAFT polymerization. - Highlights: • Surface PEGylation of ND via RAFT polymerization. • ND with high water dispersibility and excellent biocompatibility. • Controlled living polymerization. - Abstract: Nanodiamond (ND) is a novel class of carbon nanomaterials, which has been extensively investigated for biomedical applications because of its small size, high surface area and excellent biocompatibility. However, the biomedical applications of unmodified ND are still largely restricted because of their poor dispersibility in both aqueous and organic medium. In this work, we reported a novel strategy for the surface modification of ND via reversible addition fragmentation chain transfer (RAFT) polymerization. For preparation of the PEGylated ND (pPEGMA-ND), chain transfer agent (CTA) was immobilized onto ND through reaction between the hydroxyl group of ND and the carboxyl group of CTA, which was used as the initiator for surface-initiated RAFT polymerization. The successful preparation of pPEGMA-ND was characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectra and thermal gravimetric analysis in detail. Results demonstrated that pPEGMA-ND exhibited enhanced water dispersibility and desirable biocompatibility, making it promising for biomedical applications.

  19. Lipid rafts and their roles in T-cell activation

    Czech Academy of Sciences Publication Activity Database

    Hořejší, Václav

    2005-01-01

    Roč. 7, č. 2 (2005), s. 310-316 ISSN 1286-4579 R&D Projects: GA MŠk(CZ) LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : lipid rafts * T- cell * immunoreceptor signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.154, year: 2005

  20. The roles of membrane microdomains (rafts) in T cell activation

    Czech Academy of Sciences Publication Activity Database

    Hořejší, Václav

    2003-01-01

    Roč. 191, - (2003), s. 148-164 ISSN 0105-2896 R&D Projects: GA MŠk LN00A026 Grant - others:Wellcome Trust(GB) J1116W24Z Institutional research plan: CEZ:AV0Z5052915 Keywords : membrane microdomain * raft * T cell Subject RIV: EC - Immunology Impact factor: 7.052, year: 2003

  1. Modifying Lipid Rafts Promotes Regeneration and Functional Recovery

    Directory of Open Access Journals (Sweden)

    Nardos G. Tassew

    2014-08-01

    Full Text Available Ideal strategies to ameliorate CNS damage should promote both neuronal survival and axon regeneration. The receptor Neogenin promotes neuronal apoptosis. Its ligand prevents death, but the resulting repulsive guidance molecule a (RGMa-Neogenin interaction also inhibits axonal growth, countering any prosurvival benefits. Here, we explore strategies to inhibit Neogenin, thus simultaneously enhancing survival and regeneration. We show that bone morphogenetic protein (BMP and RGMa-dependent recruitment of Neogenin into lipid rafts requires an interaction between RGMa and Neogenin subdomains. RGMa or Neogenin peptides that prevent this interaction, BMP inhibition by Noggin, or reduction of membrane cholesterol all block Neogenin raft localization, promote axon outgrowth, and prevent neuronal apoptosis. Blocking Neogenin raft association influences axonal pathfinding, enhances survival in the developing CNS, and promotes survival and regeneration in the injured adult optic nerve and spinal cord. Moreover, lowering cholesterol disrupts rafts and restores locomotor function after spinal cord injury. These data reveal a unified strategy to promote both survival and regeneration in the CNS.

  2. Astronaut Tamara Jernigan deploys life raft during WETF training

    Science.gov (United States)

    1994-01-01

    Astronaut Tamara E. Jernigan, STS-67 payload commander, deploys a life raft during a session of emergency bailout training. The training took place in the 25-feet deep pool at JSC's Weightless Environment Training Facility (WETF). Jernigan was joined by her crew mates for the training session. Several SCUBA-equipped divers who assisted in the training can be seen in this photograph.

  3. Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells.

    Science.gov (United States)

    Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel; Yallampalli, Chandra

    2012-10-01

    Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the G(M1)-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.

  4. Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H

    2003-01-01

    of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol...

  5. Lipid raft association restricts CD44-ezrin interaction and promotion of breast cancer cell migration.

    LENUS (Irish Health Repository)

    Donatello, Simona

    2012-12-01

    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.

  6. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

    Science.gov (United States)

    McGraw, Kathy L; Basiorka, Ashley A; Johnson, Joseph O; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F

    2014-01-01

    Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

  7. Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

    NARCIS (Netherlands)

    Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; van Dam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

    2010-01-01

    We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal

  8. Effect of diesel exhaust generated by a city bus engine on stress responses and innate immunity in primary bronchial epithelial cell cultures.

    Science.gov (United States)

    Zarcone, M C; Duistermaat, E; Alblas, M J; van Schadewijk, A; Ninaber, D K; Clarijs, V; Moerman, M M; Vaessen, D; Hiemstra, P S; Kooter, I M

    2018-04-01

    Harmful effects of diesel emissions can be investigated via exposures of human epithelial cells, but most of previous studies have largely focused on the use of diesel particles or emission sources that are poorly representative of engines used in current traffic. We studied the cellular response of primary bronchial epithelial cells (PBECs) at the air-liquid interface (ALI) to the exposure to whole diesel exhaust (DE) generated by a Euro V bus engine, followed by treatment with UV-inactivated non-typeable Haemophilus influenzae (NTHi) bacteria to mimic microbial exposure. The effect of prolonged exposures was investigated, as well as the difference in the responses of cells from COPD and control donors and the effect of emissions generated during a cold start. HMOX1 and NQO1 expression was transiently induced after DE exposure. DE inhibited the NTHi-induced expression of human beta-defensin-2 (DEFB4A) and of the chaperone HSPA5/BiP. In contrast, expression of the stress-induced PPP1R15A/GADD34 and the chemokine CXCL8 was increased in cells exposed to DE and NTHi. HMOX1 induction was significant in both COPD and controls, while inhibition of DEFB4A expression by DE was significant only in COPD cells. No significant differences were observed when comparing cellular responses to cold engine start and prewarmed engine emissions. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes

    DEFF Research Database (Denmark)

    Calay, Damien; Vind-Kezunovic, Dina; Frankart, Aurelie

    2010-01-01

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...... of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect...... in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid...

  10. Study on design method and vibration reduction characteristic of floating raft with periodic structure

    Science.gov (United States)

    Fang, Yuanyuan; Zuo, Yanyan; Xia, Zhaowang

    2018-03-01

    The noise level is getting higher with the development of high-power marine power plant. Mechanical noise is one of the most obvious noise sources which not only affect equipment reliability, riding comfort and working environment, but also enlarge underwater noise. The periodic truss type device which is commonly applied in fields of aerospace and architectural is introduced to floating raft construction in ship. Four different raft frame structure are designed in the paper. The vibration transmissibility is taken as an evaluation index to measure vibration isolation effect. A design scheme with the best vibration isolation effect is found by numerical method. Plate type and the optimized periodic truss type raft frame structure are processed to experimental verify vibration isolation effect of the structure of the periodic raft. The experimental results demonstrate that the same quality of the periodic truss floating raft has better isolation effect than that of the plate type floating raft.

  11. Lipid rafts generate digital-like signal transduction in cell plasma membranes.

    Science.gov (United States)

    Suzuki, Kenichi G N

    2012-06-01

    Lipid rafts are meso-scale (5-200 nm) cell membrane domains where signaling molecules assemble and function. However, due to their dynamic nature, it has been difficult to unravel the mechanism of signal transduction in lipid rafts. Recent advanced imaging techniques have revealed that signaling molecules are frequently, but transiently, recruited to rafts with the aid of protein-protein, protein-lipid, and/or lipid-lipid interactions. Individual signaling molecules within the raft are activated only for a short period of time. Immobilization of signaling molecules by cytoskeletal actin filaments and scaffold proteins may facilitate more efficient signal transmission from rafts. In this review, current opinions of how the transient nature of molecular interactions in rafts generates digital-like signal transduction in cell membranes, and the benefits this phenomenon provides, are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Effect analysis of geometric parameters of floating raft on isolation performance

    Directory of Open Access Journals (Sweden)

    LI Shangda

    2017-12-01

    Full Text Available [Objectives] This paper focuses on the effects of the geometric parameters of a floating raft on isolation performance.[Methods] Based on the idea that the weight of a floating raft remains constant, a parametric finite element model is established using geometric parameters, and the effects of the geometric parameters when isolation performance is measured by vibration level difference are discussed.[Results] The effects of the geometric parameters of a floating raft on isolation performance are mainly reflected in the middle and high frequency areas. The most important geometric parameters which have an impact on isolation performance are the raft's height, length to width ratio and number of ribs. Adjusting the geometric parameters of the raft is one effective way to avoid the vibration frequency of mechanical equipment.[Conclusions] This paper has some practical value for the engineering design of floating raft isolation systems.

  13. Circular Raft Footings Strengthened by Stone Columns under Static Loads

    OpenAIRE

    R. Ziaie Moayed; B. Mohammadi-Haji

    2016-01-01

    Stone columns have been widely employed to improve the load-settlement characteristics of soft soils. The results of two small scale displacement control loading tests on stone columns were used in order to validate numerical finite element simulations. Additionally, a series of numerical calculations of static loading have been performed on strengthened raft footing to investigate the effects of using stone columns on bearing capacity of footings. The bearing capacity of single and group of ...

  14. The foundation mass concrete construction technology of Hongyun Building B tower raft

    Science.gov (United States)

    Liu, Yu; Yin, Suhua; Wu, Yanli; Zhao, Ying

    2017-08-01

    The foundation of Hongyun building B tower is made of raft board foundation which is 3300mm in the thickness and 2800mm beside side of the core tube. It is researched that the raft foundation mass concrete construction technology is expatiated from temperature and cracks of the raft foundation and the temperature control and monitoring of the concrete base slab construction and concrete curing.

  15. Effect of modeling of super-structure on the behaviour of reactor building raft

    International Nuclear Information System (INIS)

    Mondal, A.; Singh, A.K.; Roy, Raghupati; Verma, U.S.P.; Warudkar, A.S.

    2003-01-01

    The behaviour of the reactor building raft was studied when the stiffness of the super-structural elements is included in the analysis as compared to the results of conventional analysis ignoring the stiffness of the super-structural elements. The effect of the stiffness of the super-structures on the loss of contact of the raft under seismic environment was also investigated. In order to study the effect of horizontal springs on the behaviour of the raft particularly near the stressing gallery under seismic environment, a separate study has been carried out considering a 3D model consisting of solid elements supported on both horizontal and vertical springs. The model was analysed for all the forces applied at the top of the raft and the analysis results were compared with those of shell model. The following conclusions are drawn: (i) Idealisation of the reactor building raft using shell elements is adequate for estimating the design forces/moments on the raft. The design forces/moments obtained from FE model consisting of solid elements closely matches with those obtained from FE model with shell elements. Idealisation of the RB raft using shell elements will also reduce the problem size and the related computational efforts. (ii) The stiffness of the super-structure has significant effect on the behaviour of the raft. Consideration of the stiffness of the super structure reduces the design forces/moments significantly and hence, modelling of the stiffness of the super structure is necessary for economical design. (iii) Modelling of horizontal stiffness of the raft in terms of horizontal springs at the interface of the raft and the rock does not have significant effect on the behaviour of the raft and as such, is not required to be considered in the FE model. However, it is necessary to ensure adequate factor of safety against the overall stability of the raft

  16. Evaluation of RPE65, CRALBP, VEGF, CD68, and tyrosinase gene expression in human retinal pigment epithelial cells cultured on amniotic membrane.

    Science.gov (United States)

    Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Khalooghi, Keynoush; Ahmadieh, Hamid; Kanavi, Mojgan Rezaie; Samiei, Shahram; Pakravesh, Jalil

    2011-06-01

    The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

  17. Lipid alterations in lipid rafts from Alzheimer's disease human brain cortex.

    Science.gov (United States)

    Martín, Virginia; Fabelo, Noemí; Santpere, Gabriel; Puig, Berta; Marín, Raquel; Ferrer, Isidre; Díaz, Mario

    2010-01-01

    Lipid rafts are membrane microdomains intimately associated with cell signaling. These biochemical microstructures are characterized by their high contents of sphingolipids, cholesterol and saturated fatty acids and a reduced content of polyunsaturated fatty acids (PUFA). Here, we have purified lipid rafts of human frontal brain cortex from normal and Alzheimer's disease (AD) and characterized their biochemical lipid composition. The results revealed that lipid rafts from AD brains exhibit aberrant lipid profiles compared to healthy brains. In particular, lipid rafts from AD brains displayed abnormally low levels of n-3 long chain polyunsaturated fatty acids (LCPUFA, mainly 22:6n-3, docosahexaenoic acid) and monoenes (mainly 18:1n-9, oleic acid), as well as reduced unsaturation and peroxidability indexes. Also, multiple relationships between phospholipids and fatty acids were altered in AD lipid rafts. Importantly, no changes were observed in the mole percentage of lipid classes and fatty acids in rafts from normal brains throughout the lifespan (24-85 years). These indications point to the existence of homeostatic mechanisms preserving lipid raft status in normal frontal cortex. The disruption of such mechanisms in AD brains leads to a considerable increase in lipid raft order and viscosity, which may explain the alterations in lipid raft signaling observed in AD.

  18. Formulation and optimisation of raft-forming chewable tablets containing H2 antagonist.

    Science.gov (United States)

    Prajapati, Shailesh T; Mehta, Anant P; Modhia, Ishan P; Patel, Chhagan N

    2012-10-01

    The purpose of this research work was to formulate raft-forming chewable tablets of H2 antagonist (Famotidine) using a raft-forming agent along with an antacid- and gas-generating agent. Tablets were prepared by wet granulation and evaluated for raft strength, acid neutralisation capacity, weight variation, % drug content, thickness, hardness, friability and in vitro drug release. Various raft-forming agents were used in preliminary screening. A 2(3) full-factorial design was used in the present study for optimisation. The amount of sodium alginate, amount of calcium carbonate and amount sodium bicarbonate were selected as independent variables. Raft strength, acid neutralisation capacity and drug release at 30 min were selected as responses. Tablets containing sodium alginate were having maximum raft strength as compared with other raft-forming agents. Acid neutralisation capacity and in vitro drug release of all factorial batches were found to be satisfactory. The F5 batch was optimised based on maximum raft strength and good acid neutralisation capacity. Drug-excipient compatibility study showed no interaction between the drug and excipients. Stability study of the optimised formulation showed that the tablets were stable at accelerated environmental conditions. It was concluded that raft-forming chewable tablets prepared using an optimum amount of sodium alginate, calcium carbonate and sodium bicarbonate could be an efficient dosage form in the treatment of gastro oesophageal reflux disease.

  19. Surface chemistry of lipid raft and amyloid Aβ (1-40) Langmuir monolayer.

    Science.gov (United States)

    Thakur, Garima; Pao, Christine; Micic, Miodrag; Johnson, Sheba; Leblanc, Roger M

    2011-10-15

    Lipid rafts being rich in cholesterol and sphingolipids are considered to provide ordered lipid environment in the neuronal membranes, where it is hypothesized that the cleavage of amyloid precursor protein (APP) to Aβ (1-40) and Aβ (1-42) takes place. It is highly likely that the interaction of lipid raft components like cholesterol, sphingomylein or GM1 leads to nucleation of Aβ and results in aggregation or accumulation of amyloid plaques. One has investigated surface pressure-area isotherms of the lipid raft and Aβ (1-40) Langmuir monolayer. The compression-decompression cycles and the stability of the lipid raft Langmuir monolayer are crucial parameters for the investigation of interaction of Aβ (1-40) with the lipid raft Langmuir monolayer. It was revealed that GM1 provides instability to the lipid raft Langmuir monolayer. Adsorption of Aβ (1-40) onto the lipid raft Langmuir monolayer containing neutral (POPC) or negatively charged phospholipid (DPPG) was examined. The adsorption isotherms revealed that the concentration of cholesterol was important for adsorption of Aβ (1-40) onto the lipid raft Langmuir monolayer containing POPC whereas for the lipid raft Langmuir monolayer containing DPPG:cholesterol or GM1 did not play any role. In situ UV-vis absorption spectroscopy supported the interpretation of results for the adsorption isotherms. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

    Science.gov (United States)

    Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Uhr, Manfred; Wagner, Eva-Maria; Gilling, Kate E; Parsons, Chris G; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer; Rammes, Gerhard

    2013-07-01

    Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

  1. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  2. DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles

    DEFF Research Database (Denmark)

    Danielsen, Pernille Høgh; Loft, Steffen; Møller, Peter

    2008-01-01

    ABSTRACT: BACKGROUND: Exposure to air pollution particles has been acknowledged to be associated with excess generation of oxidative damage to DNA in experimental model systems and humans. The use of standard reference material (SRM), such as SRM1650 and SRM2975, is advantageous because experiments...... collected at a traffic intensive road in Copenhagen, Denmark. RESULTS: All of the particles generated strand breaks and oxidized purines in A549 lung epithelial cells in a dose-dependent manner and there were no overt differences in their potency. The exposures also yielded dose-dependent increase...... of cytotoxicity (as lactate dehydrogenase release) and reduced colony forming ability with slightly stronger cytotoxicity of SRM1650 than of the other particles. In contrast, only the authentic street particles were able to generate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA, which might...

  3. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

    Directory of Open Access Journals (Sweden)

    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  4. Culture.

    Science.gov (United States)

    Smith, Timothy B; Rodríguez, Melanie Domenech; Bernal, Guillermo

    2011-02-01

    This article summarizes the definitions, means, and research of adapting psychotherapy to clients' cultural backgrounds. We begin by reviewing the prevailing definitions of cultural adaptation and providing a clinical example. We present an original meta-analysis of 65 experimental and quasi-experimental studies involving 8,620 participants. The omnibus effect size of d = .46 indicates that treatments specifically adapted for clients of color were moderately more effective with that clientele than traditional treatments. The most effective treatments tended to be those with greater numbers of cultural adaptations. Mental health services targeted to a specific cultural group were several times more effective than those provided to clients from a variety of cultural backgrounds. We recommend a series of research-supported therapeutic practices that account for clients' culture, with culture-specific treatments being more effective than generally culture-sensitive treatments. © 2010 Wiley Periodicals, Inc.

  5. Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

    Directory of Open Access Journals (Sweden)

    Kennedy Colleen

    2011-12-01

    Full Text Available Abstract Background Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

  6. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

    Full Text Available Micropatterning of bacteria using aqueous two phase system (ATPS enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

  7. Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

    Science.gov (United States)

    Wang, Zhen; Schey, Kevin L

    2015-12-01

    Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

  8. High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.B.; Harney, J.; Hennessy, T.P.

    1994-01-01

    When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in open-quotes edgeclose quotes cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed. 33 refs., 2 figs., 3 tabs

  9. Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air-liquid interface method for nasal drug transport study.

    Science.gov (United States)

    Cho, Hyun-Jong; Choi, Min-Koo; Lin, Hongxia; Kim, Jung Sun; Chung, Suk-Jae; Shim, Chang-Koo; Kim, Dae-Duk

    2011-03-01

    P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  10. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukai, Atsushi [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan); Kurisaki, Tomohiro [Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Sato, Satoshi B. [Research Center for Low Temperature and Material Sciences, Kyoto University, Yoshida-honmachi, Kyoto 606-8501 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198 (Japan); Kondoh, Gen [Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Hashimoto, Naohiro, E-mail: nao@nils.go.jp [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan)

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  11. Lipid raft microdomains: key sites for Coxsackievirus A9 infectious cycle

    International Nuclear Information System (INIS)

    Triantafilou, Kathy; Triantafilou, Martha

    2003-01-01

    Lipid rafts have an important property to preferentially concentrate some proteins, while excluding others. Lipid rafts can also act as functional platforms for multiple signalling and trafficking processes. Several reports have shown that lipid rafts play a crucial role in the assembly of several enveloped viruses and possibly their cell entry. In this study we investigated the importance of lipid raft formation in Coxsackievirus A9 (CAV-9) entry and cell infection. Here by using a variety of biochemical and biophysical methods, we report that receptor molecules integrin αvβ3 and GRP78, which are implicated in CAV-9 infection as well as accessory molecules such as MHC class I, are accumulated in increased concentrations in lipid rafts following CAV-9 infection. In addition our studies revealed that raft integrity is essential for this virus since CAV-9 activates the Raf/MAPK signalling pathway within the raft and raft-disrupting drugs such as nystatin and MCD can successfully inhibit CAV-9 infection

  12. Lipid raft organization and function in the small intestinal brush border

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge

    2008-01-01

    proteinases, are protected from untimely release into the gut lumen. Finally, anti-glycosyl antibodies, synthesized by plasma cells locally in the gut, are deposited on the brush border glycolipid rafts, protecting the epithelium from lumenal pathogens that exploit lipid rafts as portals for entry...... to the organism....

  13. Characterization of Z-RAFT star polymerization of butyl acrylate by size-exclusion chromatography

    NARCIS (Netherlands)

    Boschmann, D.; Edam, R.; Schoenmakers, P.J.; Vana, P.

    2009-01-01

    Z-RAFT star polymerization of butyl acrylate using multifunctional trithiocarbonate-type RAFT agents carrying methyl propionate as the leaving group were used to form star polymers having 3, 4, and 6 arms. The polymerizations showed well controlled behavior up to high monomer conversions. By using a

  14. Are lipid rafts involved in ABC transporter-mediated drug resistance of tumor cells?

    NARCIS (Netherlands)

    Kok, Jan Willem; Klappe, Karin; Hummel, Ina; Kroesen, Bart-Jan; Sietsma, Hannie; Meszaros, Peter

    2008-01-01

    Since their discovery, lipid rafts have been implicated in several cellular functions, including protein transport in polarized cells and signal transduction. Also in multidrug resistance lipid rafts may be important with regard to the localization of ATP-binding cassette (ABC) transporters in these

  15. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-01-01

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  16. Separation of actin-dependent and actin-independent lipid rafts

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Kok, Jan Willem

    2013-01-01

    Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting

  17. 33 CFR 100.102 - Great Connecticut River Raft Race, Middletown, CT.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Great Connecticut River Raft Race, Middletown, CT. 100.102 Section 100.102 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND... Raft Race, Middletown, CT. (a) Regulated Area. That section of the Connecticut River between Dart...

  18. Probing the RAFT process using a model reaction between alkoxyamine and dithioester

    NARCIS (Netherlands)

    Zhou, Y.

    2012-01-01

    A small-molecular model reaction was designed to probe the reversible addition–fragmentation chain transfer (RAFT) process. In this reaction, alkoxyamine releases radicals that react in situ with dithioester through the RAFT process, generating new radicals through the fragmentation of the

  19. Research Note 2: Egg raft density and feeding preference of Culex ...

    African Journals Online (AJOL)

    ... useful for estimating the number of eggs in a culicine egg raft, making a decision on larvae food preference and using the easily available table sugar instead of the more expensive sucrose in adult rearing. Keywords:mosquito eggs, mosquito larvae, culicine mosquito, egg raft density. Nigerian Journal of Parasitology Vol.

  20. Economic impacts of guided whitewater rafting: a study of five rivers

    Science.gov (United States)

    Donald B.K. English; J. Michael Bowker

    1996-01-01

    This paper presents estimates of the statewide economic impacts of guided whitewater rafting on five rivers in six states: the Nantahala (North Carolina), Gauley (West Virginia), Kennebec (Maine), Middle Fork of the Salmon (Idaho), and Chattooga (Georgia-South Carolina). Except for the Chattooga and Middle Fork, rafting is dependent on upstream dam releases. Guide fees...

  1. RAFTing with Raptors: Connecting Science, English Language Arts, and the Common Core State Standards

    Science.gov (United States)

    Senn, Gary J.; McMurtrie, Deborah H.; Coleman, Bridget K.

    2013-01-01

    This article explores using the RAFT strategy (Role, Audience, Format, Topic) for writing in science classes. The framework of the RAFT strategy will be explained, and connections with Common Core State Standards (CCSS) for ELA/Literacy will be discussed. Finally, there will be a discussion of a professional learning experience for teachers in…

  2. The raft foundation reinforcement construction technology of Hongyun Building B tower

    Science.gov (United States)

    Liu, Yu; Yin, Suhua; Wu, Yanli; Zhao, Ying

    2017-08-01

    The foundation of Hongyun building B tower is made of raft board foundation which is 3300mm in the thickness include four kinds of reinforcement Φ32, Φ28, Φ12 and 12 steel grade two, in respective. It is researched that the raft foundation mass concrete construction technology is expatiated from temperature and cracks of the raft foundation and the temperature control and monitoring of the concrete base slab construction and concrete curing. According to the characteristics with large volume and thickness of the engineering of raft foundation, the construction of the reinforced force was calculated and the quality control measures were used to the reinforcement binding and connection, so it is success that Hongyun Building B tower raft foundation reinforced construction.

  3. Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Immerdal, Lissi; Thorsen, Evy

    2001-01-01

    Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from...... rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations...... of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p

  4. Aminopeptidase N/CD13 is associated with raft membrane microdomains in monocytes

    DEFF Research Database (Denmark)

    Navarrete Santos, A; Roentsch, J; Danielsen, E M

    2000-01-01

    as in adhesion and cell-cell interactions. Here, we report for the first time that aminopeptidase N/CD13 in monocytes is partially localized in detergent-insoluble membrane microdomains enriched in cholesterol, glycolipids, and glycosylphosphoinositol-anchored proteins, referred to as "rafts." Raft fractions...... of monocytes were characterized by the presence of GM1 ganglioside as raft marker molecule and by the high level of tyrosine-phosphorylated proteins. Furthermore, similar to polarized cells, rafts in monocytic cells lack Na(+), K(+)-ATPase. Cholesterol depletion of monocytes by methyl-beta-cyclodextrin greatly...... reduces raft localization of aminopeptidase N/CD13 without affecting ala-p-nitroanilide cleaving activity of cells....

  5. Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1.

    Science.gov (United States)

    Li, Yuan; Shan, Fei; Chen, Jinglong

    2017-03-21

    The mechanisms of lipid raft regulation by microRNAs in breast cancer are not fully understood. This work focused on the evaluation and identification of miR-3908, which may be a potential biomarker related to the migration of breast cancer cells, and elucidates lipid-raft-regulating cell migration in breast cancer. To confirm the prediction that miR-3908 is matched with AdipoR1, we used 3'-UTR luciferase activity of AdipoR1 to assess this. Then, human breast cancer cell line MCF-7 was cultured in the absence or presence of the mimics or inhibitors of miR-3908, after which the biological functions of MCF-7 cells were analyzed. The protein expression of AdipoR1, AMPK, and SIRT-1 were examined. The interaction between AdipoR1 and Flotillin-1, or its effects on lipid rafts on regulating cell migration of MCF-7, was also investigated. AdipoR1 is a direct target of miR-3908. miR-3908 suppresses the expression of AdipoR1 and its downstream pathway genes, including AMPK, p-AMPK, and SIRT-1. miR-3908 enhances the process of breast cancer cell clonogenicity. miR-3908 exerts its effects on the proliferation and migration of MCF-7 cells, which are mediated by lipid rafts regulating AdipoR1's ability to bind Flotillin-1. miR-3908 is a crucial mediator of the migration process in breast cancer cells. Lipid rafts regulate the interactions between AdipoR1 and Flotillin-1 and then the migration process associated with miR-3908 in MCF-7 cells. Our findings suggest that targeting miR-3908 and the lipid raft, may be a promising strategy for the treatment and prevention of breast cancer.

  6. Activation of different pathways of apoptosis by air pollution particulate matter (PM2.5) in human epithelial lung cells (L132) in culture

    International Nuclear Information System (INIS)

    Dagher, Zeina; Garcon, Guillaume; Billet, Sylvain; Gosset, Pierre; Ledoux, Frederic; Courcot, Dominique; Aboukais, Antoine; Shirali, Pirouz

    2006-01-01

    Epidemiological studies have associated the increase of respiratory and cardiovascular mortality and morbidity with high levels of air pollution particulate matter (PM). However, the underlying mechanisms of actions by which PM induce adverse health effects are still unclear. We have recently undertaken an extensive investigation of the adverse health effects of air pollution PM 2.5 , and shown that in vitro short-term exposure to PM 2.5 induced oxidative stress and inflammation in human lung epithelial cells (L132). Hence, it was convenient to complete the physical and chemical characterization of PM and to investigate whether in vitro short-term exposure to PM could be imply in the activation of apoptosis. Accordingly, we found that 92.15% of PM were equal or smaller than 2.5 μm and their specific surface area was 1 m 2 /g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. polycyclic aromatic hydrocarbons) chemicals were found in PM, suggesting that much of them derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. In other respects, we showed that PM exposure induced apoptosis by activating not only the tumor necrosis factor-alpha (TNF-α)-induced pathway (i.e. TNF-α secretion, caspase-8 and -3 activation), but also the mitochondrial pathway (i.e. 8-hydroxy-2'-desoxyguanosine formation, cytochrome c release from mitochondria, caspase-9 and -3 activation). Moreover, changes in the transcription rates of p53, bcl-2, and bax genes, on the one hand, and DNA fragmentation, on the other hand, were reported in PM-exposed proliferating L132 cells, revealing the occurrence of apoptotic events. Taken together, these findings suggested that in vitro short-term exposure to PM 2.5 induced apoptosis in L132 cells

  7. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

    International Nuclear Information System (INIS)

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.

    2006-01-01

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis

  8. Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

    Science.gov (United States)

    Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

    2015-01-01

    Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

    Science.gov (United States)

    Donatello, Simona; Babina, Irina S.; Hazelwood, Lee D.; Hill, Arnold D.K.; Nabi, Ivan R.; Hopkins, Ann M.

    2012-01-01

    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration. PMID:23031255

  10. Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

    DEFF Research Database (Denmark)

    Braccia, Anita; Villani, Maristella; Immerdal, Lissi

    2003-01-01

    rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction...... and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other...

  11. 7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Pereira Paulo

    2004-06-01

    Full Text Available Abstract Background Connexin43 (Cx43 is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols affect gap junction intercellular communication (GJIC. Results Primary cultures of lens epithelial cells (LEC were incubated with 7-ketocholesterol (7-Keto, 25-hydroxycholesterol (25-OH or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. Conclusions Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication

  12. Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

    Science.gov (United States)

    Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

    2016-10-07

    Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

  13. Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells.

    Science.gov (United States)

    Dessus-Babus, Sophie; Moore, Cheryl G; Whittimore, Judy D; Wyrick, Priscilla B

    2008-04-01

    A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

  14. The Relationship between fenestrations, sieve plates and rafts in liver sinusoidal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Dmitri Svistounov

    Full Text Available Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.

  15. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  16. Human Papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    Science.gov (United States)

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    2012-01-01

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone. PMID:22284893

  17. Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

    Science.gov (United States)

    Marquet-de Rougé, Perrine; Clamagirand, Christine; Facchinetti, Patricia; Rose, Christiane; Sargueil, Françoise; Guihenneuc-Jouyaux, Chantal; Cynober, Luc; Moinard, Christophe; Allinquant, Bernadette

    2013-10-01

    The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

  18. Lipid raft disarrangement as a result of neuropathological progresses: a novel strategy for early diagnosis?

    Science.gov (United States)

    Marin, R; Rojo, J A; Fabelo, N; Fernandez, C E; Diaz, M

    2013-08-15

    Lipid rafts are the preferential site of numerous membrane signaling proteins which are involved in neuronal functioning and survival. These proteins are organized in multiprotein complexes, or signalosomes, in close contact with lipid classes particularly represented in lipid rafts (i.e. cholesterol, sphingolipids and saturated fatty acids), which may contribute to physiological responses leading to neuroprotection. Increasing evidence indicates that alteration of lipid composition in raft structures as a consequence of neuropathologies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), causes a dramatic increase in lipid raft order. These phenomena may correlate with perturbation of signalosome activities, likely contributing to neurodegenerative progression. Interestingly, significant disruption of stable raft microenvironments has been already observed in the first stages of either AD or PD, suggesting that these alterations may represent early events in the neuropathological development. In this regard, the search for biochemical markers, such as specific metabolic products altered in the brain at the first steps of the disease, presently represents an important challenge for early diagnostic strategies. Alterations of these biomarkers may be reflected in either plasma or cerebrospinal fluid, thus representing a potential strategy to predict an accurate diagnosis. We propose that pathologically-linked lipid raft markers may be interesting candidates to be explored at this level, although it has not been studied so far to what extent alteration of different signalosome components may be reflected in peripheral fluids. In this mini-review, we will discuss on relevant aspects of lipid rafts that contribute to the modulation of neuropathological events related to AD and PD. An interesting hypothesis is that anomalies on raft biomarkers measured at peripheral fluids might mirror the lipid raft pathology observed in early stages of AD and PD. Copyright

  19. Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

    Science.gov (United States)

    Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

    2012-04-15

    A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

  20. Glypican-1 mediates both prion protein lipid raft association and disease isoform formation.

    Directory of Open Access Journals (Sweden)

    David R Taylor

    2009-11-01

    Full Text Available In prion diseases, the cellular form of the prion protein, PrP(C, undergoes a conformational conversion to the infectious isoform, PrP(Sc. PrP(C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs. We show that heparin displaces PrP(C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C. We then utilised a transmembrane-anchored form of PrP (PrP-TM, which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C from rafts, promoting its endocytosis. Glypican-1 and PrP(C colocalised on the cell surface and both PrP(C and PrP(Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C and PrP(Sc in lipid rafts.

  1. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    International Nuclear Information System (INIS)

    Jin, Hua; Shen, Shuijie; Chen, Xiaoyan; Zhong, Dafang; Zheng, Jiang

    2012-01-01

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  2. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hua; Shen, Shuijie [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States); Chen, Xiaoyan; Zhong, Dafang [Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203 (China); Zheng, Jiang, E-mail: jiang.zheng@seattlechildrens.org [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States)

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  3. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Directory of Open Access Journals (Sweden)

    Priscila Cardoso Cristovam

    2008-10-01

    Full Text Available OBJETIVO: Avaliar a importância da presença de células 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo obtido de rimas córneo-esclerais. MÉTODOS: Rimas de diferentes doadores tiveram seus estroma posterior e endotélio removidos (n=6. Cada rima foi dividida em três segmentos iguais, que foram colocados em cultura em três diferentes condições: um segmento foi colocado na placa de cultura com o lado epitelial para cima (Grupo A. Os dois segmentos restantes foram tripsinizados e a suspensão de células obtida foi cultivada com (Grupo B ou sem (Grupo C células 3T3 irradiadas. As células foram mantidas em meio de cultura "supplemental hormonal epithelial médium" (SHEM, a migração epitelial e a formação de clones nos grupos A, B e C foram avaliadas pela microscopia de contraste de fase e por coloração pela rodamina B. Os resultados foram comparados estatisticamente. RESULTADOS: O crescimento de células epiteliais foi observado em 4/6 rimas (Grupo A. Todas as suspensões de células epiteliais que foram cultivadas com células 3T3 (Grupo B formaram clones. Nenhuma adesão ou formação de clones verdadeiros (holo ou meroclones foi observada na cultura de células que foi cultivada sem 3T3 (Grupo C (p=0,009. CONCLUSÕES: Suspensão de células epiteliais límbicas obtidas de rimas córneo-esclerais no modelo utilizado precisa ser cultivada com células 3T3 para formar clones e estabelecer colônias epiteliais com perspectivas para uso terapêutico na reconstrução da superfície ocular.PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR. METHODS: Corneo-scleral rims from different donors (n=6 had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the

  4. Selective association of outer surface lipoproteins with the lipid rafts of Borrelia burgdorferi.

    Science.gov (United States)

    Toledo, Alvaro; Crowley, Jameson T; Coleman, James L; LaRocca, Timothy J; Chiantia, Salvatore; London, Erwin; Benach, Jorge L

    2014-03-11

    Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ospA, ospB, and ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism's adaptation to changing environments. IMPORTANCE Lipid rafts are cholesterol-rich clusters within the membranes of cells. Lipid rafts contain proteins that have functions in sensing the cell environment and transmitting signals. Although selective proteins are present in

  5. Engineering stromal-epithelial interactions in vitro for ...

    Science.gov (United States)

    Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo tissue recombination, and in vitro co-cultures. Although these approaches have elucidated signaling mechanisms underlying morphogenetic processes and adult mammalian epithelial tissue function, they are limited by the availability of human tissue, low throughput, and human developmental or physiological relevance. Objectives: Bioengineering strategies to promote EMIs using human epithelial and mesenchymal cells have enabled the development of human in vitro models of adult epidermal and glandular tissues. In this review, we describe recent bioengineered models of human epithelial tissue and organs that can instruct the design of organotypic models of human developmental processes.Methods: We reviewed current bioengineering literature and here describe how bioengineered EMIs have enabled the development of human in vitro epithelial tissue models.Discussion: Engineered models to promote EMIs have recapitulated the architecture, phenotype, and function of adult human epithelial tissue, and similar engineering principles could be used to develop models of developmental morphogenesis. We describe how bioengineering strategies including bioprinting and spheroid culture could be implemented to

  6. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  7. Differential association of the Na+/H+ Exchanger Regulatory Factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3.

    Science.gov (United States)

    Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E; Donowitz, Mark; Yun, C Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

    2013-01-01

    Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Detergent resistant membranes ("lipid rafts") were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3(-) mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. © 2013 S. Karger AG, Basel.

  8. Accelerated Combinatorial High Throughput Star Polymer Synthesis via a Rapid One-Pot Sequential Aqueous RAFT (rosa-RAFT) Polymerization Scheme.

    Science.gov (United States)

    Cosson, Steffen; Danial, Maarten; Saint-Amans, Julien Rosselgong; Cooper-White, Justin J

    2017-04-01

    Advanced polymerization methodologies, such as reversible addition-fragmentation transfer (RAFT), allow unprecedented control over star polymer composition, topology, and functionality. However, using RAFT to produce high throughput (HTP) combinatorial star polymer libraries remains, to date, impracticable due to several technical limitations. Herein, the methodology "rapid one-pot sequential aqueous RAFT" or "rosa-RAFT," in which well-defined homo-, copolymer, and mikto-arm star polymers can be prepared in very low to medium reaction volumes (50 µL to 2 mL) via an "arm-first" approach in air within minutes, is reported. Due to the high conversion of a variety of acrylamide/acrylate monomers achieved during each successive short reaction step (each taking 3 min), the requirement for intermediary purification is avoided, drastically facilitating and accelerating the star synthesis process. The presented methodology enables RAFT to be applied to HTP polymeric bio/nanomaterials discovery pipelines, in which hundreds of complex polymeric formulations can be rapidly produced, screened, and scaled up for assessment in a wide range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Atom-scale molecular interactions in lipid raft mixtures

    DEFF Research Database (Denmark)

    Niemelä, Perttu S; Hyvönen, Marja T; Vattulainen, Ilpo

    2009-01-01

    We review the relationship between molecular interactions and the properties of lipid environments. A specific focus is given on bilayers which contain sphingomyelin (SM) and sterols due to their essential role for the formation of lipid rafts. The discussion is based on recent atom-scale molecular...... dynamics simulations, complemented by extensive comparison to experimental data. The discussion is divided into four sections. The first part investigates the properties of one-component SM bilayers and compares them to bilayers with phosphatidylcholine (PC), the focus being on a detailed analysis...... examples of this issue. The third part concentrates on the specificity of intermolecular interactions in three-component mixtures of SM, PC and cholesterol (CHOL) under conditions where the concentrations of SM and CHOL are dilute with respect to that of PC. The results show how SM and CHOL favor one...

  10. Fault and joint geometry at Raft River Geothermal Area, Idaho

    Science.gov (United States)

    Guth, L. R.; Bruhn, R. L.; Beck, S. L.

    1981-07-01

    Raft River geothermal reservoir is formed by fractures in sedimentary strata of the Miocene and Pliocene salt lake formation. The fracturing is most intense at the base of the salt lake formation, along a decollement that dips eastward at less than 50 on top of metamorphosed precambrian and lower paleozoic rocks. Core taken from less than 200 m above the decollement contains two sets of normal faults. The major set of faults dips between 500 and 700. These faults occur as conjugate pairs that are bisected by vertical extension fractures. The second set of faults dips 100 to 200 and may parallel part of the basal decollement or reflect the presence of listric normal faults in the upper plate. Surface joints form two suborthogonal sets that dip vertically. East-northeast-striking joints are most frequent on the limbs of the Jim Sage anticline, a large fold that is associated with the geothermal field.

  11. Prion protein accumulation in lipid rafts of mouse aging brain.

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    Federica Agostini

    Full Text Available The cellular form of the prion protein (PrP(C is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C. In old mice, this change favors PrP(C accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C translocation into detergent-resistant membranes (DRMs, we looked at PrP(C compartmentalization in hippocampi from acid sphingomyelinase (ASM knockout (KO mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.

  12. Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

    Science.gov (United States)

    Davari, Maliheh; Soheili, Zahra-Soheila; Samiei, Shahram; Sharifi, Zohreh; Pirmardan, Ehsan Ranaei

    2017-01-29

    miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. A methodology for distinguishing divergent cell fates within a common progenitor population: adenoma- and neuroendocrine-like cells are confounders of rat ileal epithelial cell (IEC-18 culture

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    Paxton Jessica B

    2005-01-01

    Full Text Available Abstract Background IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation / transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage. Results IEC-18 cells were cultured for 72 hours in serum free media (SFM, with and without various insulin-like growth factor agonists to differentially boost the basal rate of proliferation. A strategy was employed to identify constitutive genes as markers of divergent fates through gene array analysis by cross-referencing fold-change trends for individual genes against crypt cell abundance in each treatment. We then confirmed the cell-specific phenotype by immunolocalization of proteins corresponding to those genes. The majority of IEC-18 cells in SFM alone had a loss in expression of the adenomatous polyposis coli (APC gene at the mRNA and protein levels, consistent with adenoma-like transformation. In addition, a small subset of cells expressed the serotonin receptor 2A gene and had neuroendocrine-like morphology. Conclusions IEC-18 cells commonly undergo a change in cell fate prior to reaching confluence. The most common fate switch that we were able to detect correlates with a down regulation of the APC gene and transformation into an adenoma-like phenotype.

  14. cultural

    Directory of Open Access Journals (Sweden)

    Irene Kreutz

    2006-01-01

    Full Text Available Es un estudio cualitativo que adoptó como referencial teorico-motodológico la antropología y la etnografía. Presenta las experiencias vivenciadas por mujeres de una comunidad en el proceso salud-enfermedad, con el objetivo de comprender los determinantes sócio-culturales e históricos de las prácticas de prevención y tratamiento adoptados por el grupo cultural por medio de la entrevista semi-estructurada. Los temas que emergieron fueron: la relación entre la alimentación y lo proceso salud-enfermedad, las relaciones con el sistema de salud oficial y el proceso salud-enfermedad y lo sobrenatural. Los dados revelaron que los moradores de la comunidad investigada tienen un modo particular de explicar sus procedimientos terapéuticos. Consideramos que es papel de los profesionales de la salud en sus prácticas, la adopción de abordajes o enfoques que consideren al individuo en su dimensión sócio-cultural e histórica, considerando la enorme diversidad cultural en nuestro país.

  15. Mechanism and kinetics of dithiobenzoate-mediated RAFT polymerization. I. The current situation

    NARCIS (Netherlands)

    Barner-Kowollik, C.; Buback, M.; Charleux, B.; Coote, M.L.; Drache, M.; Fukuda, T.; Goto, A.; Klumperman, B.; Lowe, A.B.; McLeary, J.B.; Moad, G.; Monteiro, M.J.; Sanderson, R.D.; Tonge, M.P.; Vana, P.

    2006-01-01

    Investigations into the kinetics and mechanism of dithiobenzoate-mediated Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerizations, which exhibit nonideal kinetic behavior, such as induction periods and rate retardation, are comprehensively reviewed. The appreciable uncertainty in the

  16. Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

    Science.gov (United States)

    Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

    2010-12-01

    Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

  17. FEM analysis of foundation raft for 500 MWe pressurized heavy water reactor building

    International Nuclear Information System (INIS)

    Kulkarni, N.N.; Goray, J.S.; Joshi, M.H.; Paramasivam, V.

    1989-01-01

    Foundation raft supports the containment structure and internals for 500 MWe PHW reactor building. It also serves as bottom envelop of the containment structure. In view of this, the design of foundation raft assumes great importance. The foundation raft is subjected to various load, most significant of them are dead load of structure, equipment loads transferred through a system of floors, walls and structural steel columns, pressure load during accident conditions, seismic loads, earth pressure, uplift due to buoyancy loads, foundation reaction etc. In order to achieve optimum design, the detailed structural analysis is required to be performed methodically and in most realistic manner. Finite element methods which have come in vogue with the developments in digital computers can be successfully applied in this area. The paper describes the above methods in detail for the analysis of foundation raft for the various load combinations required to be considered for safe and optimum design

  18. Intermediate Radical Termination Theory in Elucidation of RAFT Kinetics and Comparison to Experimental Data

    Directory of Open Access Journals (Sweden)

    M. Baqeri-Jagharq

    2008-12-01

    Full Text Available In current work a comprehensive mechanism based on intermediate radical termination theory is assumed for RAFT polymerization of styrene over cumyl dithiobenzoate as RAFT agent. Rate constants for addition (ka and fragmentation reactions (kf are set to 6×106 and 5×104 respectively, which lead to an equilibrium constant value of K = ka/kf = 1.2 x 102. Moment equations method was used to model this mechanism and the results were compared to experimental data to verify modeling. The effects of changing RAFT agent concentration on conversion, molecular weight and polydispersity index of the final product were investigated through the modeling. According to the results, the likelihood of living polymerization increases with raising RAFT agent concentration which leads to linearity of conversion and molecular weight curves and therefore lowering the polydispersity index and narrowing the molecular weight distribution.

  19. Phase diagrams of lipid mixtures relevant to the study of membrane rafts

    DEFF Research Database (Denmark)

    Goni, Felix; Alonso, Alicia; Bagatolli, Luis

    2008-01-01

    The present paper reviews the phase properties of phosphatidylcholine-sphingomyelin-cholesterol mixtures, that are often used as models for membrane "raft" microdomains. The available data based on X-ray, microscopic and spectroscopic observations, surface pressure and calorimetric measurements, ...

  20. Final Technical Resource Confirmation Testing at the Raft River Geothermal Project, Cassia County, Idaho

    Energy Technology Data Exchange (ETDEWEB)

    Glaspey, Douglas J.

    2008-01-30

    Incorporates the results of flow tests for geothermal production and injection wells in the Raft River geothermal field in southern Idaho. Interference testing was also accomplished across the wellfield.

  1. Experimental and Numerical Investigation of Towing Resistance of the Innovative Pneumatic Life Raft

    Directory of Open Access Journals (Sweden)

    Burciu Zbigniew

    2017-06-01

    Full Text Available The paper presents the experimental and numerical investigations of a novel design of a pneumatic life raft in calm water conditions. Two main issues were considered: the life raft performance under tow and its resistance in calm water conditions. The experiment and CFD simulations were performed by using the full scale prototype to eliminate the scale effect. The towing tank tests confirmed the results of numerical computations. The compatibility of the results of numerical and experimental tests was high mainly because the new life raft does not deform under the hydrodynamic and aerodynamic loads. The characteristics similar to rigid body behaviour result mainly from a new construction and materials used for manufacturing the life raft.

  2. Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

    Science.gov (United States)

    Danielson, Alex P; Bailey-Van Kuren, Dylan; Lucius, Melissa E; Makaroff, Katherine; Williams, Cameron; Page, Richard C; Berberich, Jason A; Konkolewicz, Dominik

    2016-02-01

    Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Application of a movable active vibration control system on a floating raft

    Science.gov (United States)

    Wang, Zhen; Mak, Cheuk Ming

    2018-02-01

    This paper presents a theoretical study of an inertial actuator connected to an accelerometer by a local feedback loop for active vibration control on a floating raft. On the criterion of the minimum power transmission from the vibratory machines to the flexible foundation in the floating raft, the best mounting positions for the inertial actuator on the intermediate mass of the floating raft are investigated. Simulation results indicate that the best mounting positions for the inertial actuator vary with frequency. To control time-varying excitations of vibratory machines on a floating raft effectively, an automatic control system based on real-time measurement of a cost function and automatically searching the best mounting position of the inertial actuator is proposed. To the best of our knowledge, it is the first time that an automatic control system is proposed to move an actuator automatically for controlling a time-varying excitation.

  4. Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

    Directory of Open Access Journals (Sweden)

    Ai Ushiyama

    Full Text Available Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.

  5. Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

    Science.gov (United States)

    Hu, Xiaolong; Zhu, Min; Liang, Zi; Kumar, Dhiraj; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2017-04-01

    The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

  6. Surfactant-Free RAFT Emulsion Polymerization of Styrene Using Thermoresponsive macroRAFT Agents: Towards Smart Well-Defined Block Copolymers with High Molecular Weights

    Directory of Open Access Journals (Sweden)

    Steffen Eggers

    2017-12-01

    Full Text Available The combination of reversible addition–fragmentation chain transfer (RAFT and emulsion polymerization has recently attracted much attention as a synthetic tool for high-molecular-weight block copolymers and their micellar nano-objects. Up to recently, though, the use of thermoresponsive polymers as both macroRAFT agents and latex stabilizers was impossible in aqueous media due to their hydrophobicity at the usually high polymerization temperatures. In this work, we present a straightforward surfactant-free RAFT emulsion polymerization to obtain thermoresponsive styrenic block copolymers with molecular weights of around 100 kDa and their well-defined latexes. The stability of the aqueous latexes is achieved by adding 20 vol % of the cosolvent 1,4-dioxane (DOX, increasing the phase transition temperature (PTT of the used thermoresponsive poly(N-acryloylpyrrolidine (PAPy macroRAFT agents above the polymerization temperature. Furthermore, this cosolvent approach is combined with the use of poly(N,N-dimethylacrylamide-block-poly(N-acryloylpiperidine-co-N-acryloylpyrrolidine (PDMA-b-P(APi-co-APy as the macroRAFT agent owning a short stabilizing PDMA end block and a widely adjustable PTT of the P(APi-co-APy block in between 4 and 47 °C. The temperature-induced collapse of the latter under emulsion polymerization conditions leads to the formation of RAFT nanoreactors, which allows for a very fast chain growth of the polystyrene (PS block. In dynamic light scattering (DLS, as well as cryo-transmission electron microscopy (cryoTEM, moreover, all created latexes indeed reveal a high (temperature stability and a reversible collapse of the thermoresponsive coronal block upon heating. Hence, this paper pioneers a versatile way towards amphiphilic thermoresponsive high-molecular-weight block copolymers and their nano-objects with tailored corona switchability.

  7. Analysis of raft foundations for spent fuel pool in nuclear facilities

    International Nuclear Information System (INIS)

    Subramanian, K.V.; Kashikar, A.V.; Nath, C.; Shintre, C.C.

    2005-01-01

    Foundation rafts are analysed as a plate on elastic foundation with the representation of the foundation media using the Winkler idealisation i.e. series of linear uncoupled springs. The elastic constant of the Winkler springs is derived using the sub-grade modulus. However, the Winkler approach has limitations due to incompatibility of the deflections at raft-soil interface. The deflection of the raft at the point of contact and the deformation of the foundation media at this point of contact are incompatible in this approach. This particularly influences flexible rafts and further if the foundation media is soil. This paper discusses the analysis of raft, in general, and the analysis of the foundation raft for a Spent Fuel pool facility using 'variable k approach' where deformations at a node and influencing nodes are computed using Boussinesq's theory. The limitations stated above are overcome in this approach. Some studies on the sensitivity of parameters were carried out in the form of variation of moduli of elasticity of concrete and deformation modulus of soil. Analysis is also performed with conventional method using 'Winkler' soil springs. It is concluded that the Winkler model does not correctly predict the behaviour of the mat both qualitatively and quantitatively and could lead to underestimation of soil pressures leading to unconservative design. The approach involving soil structure interaction like the one presented here is hence recommended for important structures like those involved in Nuclear facilities. (authors)

  8. The shedding activity of ADAM17 is sequestered in lipid rafts

    International Nuclear Information System (INIS)

    Tellier, Edwige; Canault, Matthias; Rebsomen, Laure; Bonardo, Bernadette; Juhan-Vague, Irene; Nalbone, Gilles; Peiretti, Franck

    2006-01-01

    The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts

  9. Constitutive modeling of creep behavior in single crystal superalloys: Effects of rafting at high temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ya-Nan, E-mail: fanyn12@mails.tsinghua.edu.cn; Shi, Hui-Ji, E-mail: shihj@mail.tsinghua.edu.cn; Qiu, Wen-Hui

    2015-09-17

    Rafting and creep modeling of single crystal superalloys at high temperatures are important for the safety assessment and life prediction in practice. In this research, a new model has been developed to describe the rafting evolution and incorporated into the Cailletaud single crystal plasticity model to simulate the creep behavior. The driving force of rafting is assumed to be the relaxation of the strain energy, and it is calculated with the local stress state, a superposition of the external and misfit stress tensors. In addition, the isotropic coarsening is introduced by the cube root dependence of the microstructure periodicity on creep time based on Ostwal ripening. Then the influence of rafting on creep deformation is taken into account as the Orowan stress in the single crystal plasticity model. The capability of the proposed model is validated with creep experiments of CMSX-4 at 950 °C and 1050 °C. It is able to predict the rafting direction at complex loading conditions and evaluate the channel width during rafting. For [001] tensile creep tests, good agreement has been shown between the model predictions and experimental results at different temperatures and stress levels. The creep acceleration can be captured with this model and is attributed to the microstructure degradation caused by the precipitate coarsening.

  10. Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

    Science.gov (United States)

    Lu, Stella M; Fairn, Gregory D

    2018-04-01

    The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

  11. Cholesterol, sphingolipids, and glycolipids: What do we know about their role in raft-like membranes?

    DEFF Research Database (Denmark)

    Rog, T.; Vattulainen, I.

    2014-01-01

    Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units with pote......Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units...... with potential specific functions. Although the understanding of the structure of rafts in living cells is quite limited, the possible functions of rafts are widely discussed in the literature, highlighting their importance in cellular functions. In this review, we discuss the understanding of rafts that has...... emerged based on recent atomistic and coarse-grained molecular dynamics simulation studies on the key lipid raft components, which include cholesterol, sphingolipids, glycolipids, and the proteins interacting with these classes of lipids. The simulation results are compared to experiments when possible...

  12. Direct access to dithiobenzoate RAFT agent fragmentation rate coefficients by ESR spin-trapping.

    Science.gov (United States)

    Ranieri, Kayte; Delaittre, Guillaume; Barner-Kowollik, Christopher; Junkers, Thomas

    2014-12-01

    The β-scission rate coefficient of tert-butyl radicals fragmenting off the intermediate resulting from their addition to tert-butyl dithiobenzoate-a reversible addition-fragmentation chain transfer (RAFT) agent-is estimated via the recently introduced electron spin resonance (ESR)-trapping methodology as a function of temperature. The newly introduced ESR-trapping methodology is critically evaluated and found to be reliable. At 20 °C, a fragmentation rate coefficient of close to 0.042 s(-1) is observed, whereas the activation parameters for the fragmentation reaction-determined for the first time-read EA = 82 ± 13.3 kJ mol(-1) and A = (1.4 ± 0.25) × 10(13) s(-1) . The ESR spin-trapping methodology thus efficiently probes the stability of the RAFT adduct radical under conditions relevant for the pre-equilibrium of the RAFT process. It particularly indicates that stable RAFT adduct radicals are indeed formed in early stages of the RAFT poly-merization, at least when dithiobenzoates are employed as controlling agents as stipulated by the so-called slow fragmentation theory. By design of the methodology, the obtained fragmentation rate coefficients represent an upper limit. The ESR spin-trapping methodology is thus seen as a suitable tool for evaluating the fragmentation rate coefficients of a wide range of RAFT adduct radicals. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Requirement of transmembrane domain for CD154 association to lipid rafts and subsequent biological events.

    Directory of Open Access Journals (Sweden)

    Nadir Benslimane

    Full Text Available Interaction of CD40 with CD154 leads to recruitment of both molecules into lipid rafts, resulting in bi-directional cell activation. The precise mechanism by which CD154 is translocated into lipid rafts and its impact on CD154 signaling remain largely unknown. Our aim is to identify the domain of CD154 facilitating its association to lipid rafts and the impact of such association on signaling events and cytokine production. Thus, we generated Jurkat cell lines expressing truncated CD154 lacking the cytoplasmic domain or chimeric CD154 in which the transmembrane domain was replaced by that of transferrin receptor I, known to be excluded from lipid rafts. Our results show that cell stimulation with soluble CD40 leads to the association of CD154 wild-type and CD154-truncated, but not CD154-chimera, with lipid rafts. This is correlated with failure of CD154-chimera to activate Akt and p38 MAP kinases, known effectors of CD154 signaling. We also found that CD154-chimera lost the ability to promote IL-2 production upon T cell stimulation with anti-CD3/CD28 and soluble CD40. These results demonstrate the implication of the transmembrane domain of CD154 in lipid raft association, and that this association is necessary for CD154-mediated Akt and p38 activation with consequent enhancement of IL-2 production.

  14. Silk Film Topography Directs Collective Epithelial Cell Migration

    Science.gov (United States)

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  15. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  16. Genotype-induced changes in biophysical properties of frontal cortex lipid raft from APP/PS1 transgenic mice

    Directory of Open Access Journals (Sweden)

    Mario L Diaz

    2012-11-01

    Full Text Available Alterations in the lipid composition of lipid rafts have been demonstrated both in human brain and transgenic mouse models, and it has been postulated that aberrant lipid composition in lipid rafts is partly responsible for neuronal degeneration. In order to assess the impact of lipid changes on lipid raft functional properties, we have aimed at determining relevant physicochemical modifications in lipid rafts purified from frontal cortex of wild type (WT and APP/PS1 double transgenic mice. By means of steady-state fluorescence anisotropy analyses using two lipid soluble fluorescent probes, TMA-DPH (1-[(4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene and DPH (1,6-diphenyl-1,3,5-hexatriene, we demonstrate that cortical lipid rafts from WT and APP/PS1 animals exhibit different biophysical behaviours, depending on genotype but also on age. Thus, aged APP/PS1 animals exhibited slightly more liquid-ordered lipid rafts than WT counterparts. Membrane microviscosity napp analyses demonstrate that WT lipid rafts are more fluid than APP/PS1 animals of similar age, both at the aqueous interface and hydrophobic core of the membrane. napp in APP/PS1 animals was higher for DPH than for TMA-DPH under similar experimental conditions, indicating that the internal core of the membrane is more viscous than the raft membrane at the aqueous interface. The most dramatic changes in biophysical properties of lipid rafts were observed when membrane cholesterol was depleted with methyl-beta-cyclodextrin. Overall, our results indicate that APP/PS1 genotype strongly affects physicochemical properties of lipid raft. Such alterations appear not to be homogeneous across the raft membrane axis, but rather are more prominent at the membrane plane. These changes correlate with aberrant proportions of sphingomyelin, cholesterol and saturated fatty acids, as well as polyunsaturated fatty acids, measured in lipid rafts from frontal cortex in this familial model of

  17. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  18. ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

    Science.gov (United States)

    Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

    2012-01-01

    The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

  19. Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gαs.

    Science.gov (United States)

    Erb, Samuel J; Schappi, Jeffrey M; Rasenick, Mark M

    2016-09-16

    Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify Gαs localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of Gαs to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    Science.gov (United States)

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Association between tetrodotoxin resistant channels and lipid rafts regulates sensory neuron excitability.

    Directory of Open Access Journals (Sweden)

    Alessandro Pristerà

    Full Text Available Voltage-gated sodium channels (VGSCs play a key role in the initiation and propagation of action potentials in neurons. Na(V1.8 is a tetrodotoxin (TTX resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. Na(V1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for Na(V1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated Na(V1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that Na(V1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that Na(V1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-β-cyclodextrin (MβCD and 7-ketocholesterol (7KC led to the dissociation between rafts and Na(V1.8. By calcium imaging we demonstrated that the lack of association between rafts and Na(V1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of Na(V1.8 in nociceptors and highlight the importance of the association between Na(V1.8 and lipid rafts in the control of nociceptor excitability.

  2. Ethanol Enhances TGF-β Activity by Recruiting TGF-β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

    Science.gov (United States)

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Johnson, Frank E; Huang, Jung San

    2016-04-01

    Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼ 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼ 2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues. © 2015 Wiley Periodicals, Inc.

  3. Development of a new anti-cancer agent for targeted radionuclide therapy: β- radiolabeled RAFT-RGD

    International Nuclear Information System (INIS)

    Petitprin, A.

    2013-01-01

    β-emitters radiolabeled RAFT-RGD as new agents for internal targeted radiotherapy. The αvβ3 integrin is known to play an important role in tumor-induced angiogenesis, tumor proliferation, survival and metastasis. Because of its overexpression on neo-endothelial cells such as those present in growing tumors, as well as on tumor cells of various origins, αvβ3 integrin is an attractive molecular target for diagnosis and therapy of the rapidly growing and metastatic tumors. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin αvβ3 in vitro and in vivo. RAFT-RGD has been used for tumor imaging and drug targeting. This study is the first to evaluate the therapeutic potential of the β-emitters radiolabeled tetrameric RGD peptide RAFT-RGD in a Nude mouse model of αvβ3 -expressing tumors. An injection of 37 MBq of 90 Y-RAFT-RGD or 177 Lu-RAFT-RGD in mice with αvβ3 -positive tumors caused a significant growth delay as compared with mice treated with 37 MBq of 90 Y-RAFT-RAD or 177 Lu-RAFT-RAD or untreated mice. In comparison, an injection of 30 MBq of 90 Y-RAFT-RGD had no efficacy for the treatment of αvβ3 -negative tumors. 90 Y-RAFT-RGD and 177 Lu-RAFT-RGD are potent αvβ3 -expressing tumor targeting agents for internal targeted radiotherapy. (author)

  4. Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease.

    Science.gov (United States)

    Hattersley, Kathryn J; Hein, Leanne K; Fuller, Maria

    2013-12-06

    Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells.

    Science.gov (United States)

    Wu, Li; Huang, Jie; Yu, Xiaoxue; Zhou, Xiaoqing; Gan, Chaoye; Li, Ming; Chen, Yong

    2014-02-01

    The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.

  6. The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

    Science.gov (United States)

    Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

    2013-09-24

    The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial

  7. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

    Science.gov (United States)

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  8. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  9. Juvenile-onset loss of lipid-raft domains in attractin-deficient mice

    International Nuclear Information System (INIS)

    Azouz, Abdallah; Gunn, Teresa M.; Duke-Cohan, Jonathan S.

    2007-01-01

    Mutations at the attractin (Atrn) locus in mice result in altered pigmentation on an agouti background, higher basal metabolic rate and juvenile-onset hypomyelination leading to neurodegeneration, while studies on human immune cells indicate a chemotaxis regulatory function. The underlying biochemical defect remains elusive. In this report we identify a role for attractin in plasma membrane maintenance. In attractin's absence there is a decline in plasma membrane glycolipid-enriched rafts from normal levels at 8 weeks to a complete absence by 24 weeks. The structural integrity of lipid rafts depends upon cholesterol and sphingomyelin, and can be identified by partitioning within of ganglioside GM 1 . Despite a significant fall in cellular cholesterol with maturity, and a lesser fall in both membrane and total cellular GM 1 , these parameters lag behind raft loss, and are normal when hypomyelination/neurodegeneration has already begun thus supporting consequence rather than cause. These findings can be recapitulated in Atrn-deficient cell lines propagated in vitro. Further, signal transduction through complex membrane receptor assemblies is not grossly disturbed despite the complete absence of lipid rafts. We find these results compatible with a role for attractin in plasma membrane maintenance and consistent with the proposal that the juvenile-onset hypomyelination and neurodegeneration represent a defect in attractin-mediated raft-dependent myelin biogenesis

  10. Intercalation and structural aspects of macroRAFT agents into MgAl layered double hydroxides

    Directory of Open Access Journals (Sweden)

    Dessislava Kostadinova

    2016-12-01

    Full Text Available Increasing attention has been devoted to the design of layered double hydroxide (LDH-based hybrid materials. In this work, we demonstrate the intercalation by anion exchange process of poly(acrylic acid (PAA and three different hydrophilic random copolymers of acrylic acid (AA and n-butyl acrylate (BA with molar masses ranging from 2000 to 4200 g mol−1 synthesized by reversible addition-fragmentation chain transfer (RAFT polymerization, into LDH containing magnesium(II and aluminium(III intralayer cations and nitrates as counterions (MgAl-NO3 LDH. At basic pH, the copolymer chains (macroRAFT agents carry negative charges which allowed the establishment of electrostatic interactions with the LDH interlayer and their intercalation. The resulting hybrid macroRAFT/LDH materials displayed an expanded interlamellar domain compared to pristine MgAl-NO3 LDH from 1.36 nm to 2.33 nm. Depending on the nature of the units involved into the macroRAFT copolymer (only AA or AA and BA, the intercalation led to monolayer or bilayer arrangements within the interlayer space. The macroRAFT intercalation and the molecular structure of the hybrid phases were further characterized by Fourier transform infrared (FTIR and solid-state 13C, 1H and 27Al nuclear magnetic resonance (NMR spectroscopies to get a better description of the local structure.

  11. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

    Science.gov (United States)

    Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

    2012-01-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

  12. Clomipramine counteracts lipid raft disturbance due to short-term muscle disuse.

    Science.gov (United States)

    Bryndina, Irina G; Shalagina, Maria N; Sekunov, Alexey V; Zefirov, Andrei L; Petrov, Alexey M

    2018-01-18

    Disuse-induced skeletal muscle dysfunction is a serious consequence of long-term spaceflight, numerous diseases and conditions for which treatment possibilities are still strictly limited. We have previously shown that acute hindlimb suspension (HS)-mediated disuse disrupts membrane lipid rafts in the unloaded muscle. Here, we investigated whether pretreatment of rats with the inhibitor of acid sphingomyelinase, clomipramine (1.25mg/g/day, intramuscularly, for 5days before HS), is able to hinder the loss in lipid raft integrity in response to 12h of HS. Clomipramine pretreatment significantly counteracted the decrease in labeling of the plasma membranes with lipid raft markers (fluorescent cholera toxin B subunit and bodipy-GM1-ganglioside) specifically in the junctional regions of the suspended soleus muscle. This was associated with: a) enhancing raft disrupting potential of exogenous sphingomyelinase in the junctional membranes; b) prevention of both ceramide accumulation and cholesterol loss; c) prevention of decline in nicotinic acetylcholine receptor labeling in the unloaded muscle. Our data suggest that sphingomyelinase-mediated raft disturbance serves as one of the earlier events in HS effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle.

    Science.gov (United States)

    Chakraborty, Atanu; Jana, Nikhil R

    2015-09-17

    Nanoparticle interacts with live cells depending on their surface chemistry, enters into cell via endocytosis, and is commonly trafficked to an endosome/lysozome that restricts subcellular targeting options. Here we show that nanoparticle surface chemistry can be tuned to alter their cell uptake mechanism and subcellular trafficking. Quantum dot based nanoprobes of 20-30 nm hydrodynamic diameters have been synthesized with tunable surface charge (between +15 mV to -25 mV) and lipophilicity to influence their cellular uptake processes and subcellular trafficking. It is observed that cationic nanoprobe electrostatically interacts with cell membrane and enters into cell via clathrin-mediated endocytosis. At lower surface charge (between +10 mV to -10 mV), the electrostatic interaction with cell membrane becomes weaker, and additional lipid raft endocytosis is initiated. If a lipophilic functional group is introduced on a weakly anionic nanoparticle surface, the uptake mechanism shifts to predominant lipid raft-mediated endocytosis. In particular, the zwitterionic-lipophilic nanoprobe has the unique advantage as it weakly interacts with anionic cell membrane, migrates toward lipid rafts for interaction through lipophilic functional group, and induces lipid raft-mediated endocytosis. While predominate or partial clathrin-mediated entry traffics most of the nanoprobes to lysozome, predominate lipid raft-mediated entry traffics them to perinuclear region, particularly to the Golgi apparatus. This finding would guide in designing appropriate nanoprobe for subcellular targeting and delivery.

  14. Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

    Institute of Scientific and Technical Information of China (English)

    Takashi Nakamura; Yuta Chiba; Masahiro Naruse; Kan Saito; Hidemitsu Harada; Satoshi Fukumoto

    2016-01-01

    Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

  15. Lipid Raft Size and Lipid Mobility in Non-raft Domains Increase during Aging and Are Exacerbated in APP/PS1 Mice Model of Alzheimer's Disease. Predictions from an Agent-Based Mathematical Model

    Science.gov (United States)

    Santos, Guido; Díaz, Mario; Torres, Néstor V.

    2016-01-01

    A connection between lipid rafts and Alzheimer's disease has been studied during the last decades. Mathematical modeling approaches have recently been used to correlate the effects of lipid composition changes in the physicochemical properties of raft-like membranes. Here we propose an agent based model to assess the effect of lipid changes in lipid rafts on the evolution and progression of Alzheimer's disease using lipid profile data obtained in an established model of familial Alzheimer's disease. We have observed that lipid raft size and lipid mobility in non-raft domains are two main factors that increase during age and are accelerated in the transgenic Alzheimer's disease mouse model. The consequences of these changes are discussed in the context of neurotoxic amyloid β production. Our agent based model predicts that increasing sterols (mainly cholesterol) and long-chain polyunsaturated fatty acids (LCPUFA) (mainly DHA, docosahexaenoic acid) proportions in the membrane composition might delay the onset and progression of the disease. PMID:27014089

  16. Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Pedersen, Esben D K; Immerdal, Lissi

    2005-01-01

    a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin...

  17. Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

    Science.gov (United States)

    Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

    2015-02-01

    Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

  18. Sulfonated macro-RAFT agents for the surfactant-free synthesis of cerium oxide-based hybrid latexes

    NARCIS (Netherlands)

    Garnier, J.; Warnant, J.; Lacroix-Desmazes, P.; Dufils, P.E.; Vinas, J.; Herk, van A.M.

    2013-01-01

    Three types of amphiphatic macro-RAFT agents were employed as compatibilizers to promote the polymerization reaction at the surface of nanoceria for the synthesis of CeO2-based hybrid latexes. Macro-RAFT copolymers and terpolymers were first synthesized employing various combinations of butyl

  19. Detergent-Based Isolation of Yeast Membrane Rafts: An Inquiry-Based Laboratory Series for the Undergraduate Cell Biology or Biochemistry Lab

    Science.gov (United States)

    Willhite, D. Grant; Wright, Stephen E.

    2009-01-01

    Lipid rafts have been implicated in numerous cellular processes including cell signaling, endocytosis, and even viral infection. Isolation of these lipid rafts often involves detergent treatment of the membrane to dissolve nonraft components followed by separation of raft regions in a density gradient. We present here an inquiry-based lab series…

  20. SAN-b-P4VP block copolymer synthesis by chain extension from RAFT-functional poly(4-vinylpyridine) in solution and in emulsion

    NARCIS (Netherlands)

    Bozovic, J.S.; Tello Manon, H.M; Meuldijk, J.; Koning, C.E.; Klumperman, B.

    2007-01-01

    Reversible addition fragmentation chain transfer (RAFT)-mediated polymerization was successfully applied for the synthesis of poly(4-vinylpyridine) (P4VP) polymers of predetermined molar mass and of low polydispersity index. These RAFT end-functionalized polymers were then used as macro-RAFT agents

  1. Activation of c-Src and Fyn kinases by protein tyrosine phosphatase RPTPalpha is substrate-specific and compatible with lipid raft localization

    DEFF Research Database (Denmark)

    Vacaresse, Nathalie; Møller, Bente; Danielsen, Erik Michael

    2008-01-01

    and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPa is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPa. Forced concentration of RPTPa into lipid rafts is compatible...

  2. Progressive friction mobilization and enhanced Janssen's screening in confined granular rafts

    Science.gov (United States)

    Saavedra V., Oscar; Elettro, Hervé; Melo, Francisco

    2018-04-01

    Confined two-dimensional assemblies of floating particles, known as granular rafts, are prone to develop a highly nonlinear response under compression. Here we investigate the transition to the friction-dominated jammed state and map the gradual development of the internal stress profile with flexible pressure sensors distributed along the raft surface. Surprisingly, we observe that the surface stress screening builds up much more slowly than previously thought and that the typical screening distance later dramatically decreases. We explain this behavior in terms of progressive friction mobilization, where the full amplitude of the frictional forces is only reached after a macroscopic local displacement. At further stages of compression, rafts of large length-to-width aspect ratio experience much stronger screenings than the full mobilization limit described by the Janssen's model. We solve this paradox using a simple mathematical analysis and show that such enhanced screening can be attributed to a localized compaction front, essentially shielding the far field from compressive stresses.

  3. Tsunami-driven rafting: Transoceanic species dispersal and implications for marine biogeography.

    Science.gov (United States)

    Carlton, James T; Chapman, John W; Geller, Jonathan B; Miller, Jessica A; Carlton, Deborah A; McCuller, Megan I; Treneman, Nancy C; Steves, Brian P; Ruiz, Gregory M

    2017-09-29

    The 2011 East Japan earthquake generated a massive tsunami that launched an extraordinary transoceanic biological rafting event with no known historical precedent. We document 289 living Japanese coastal marine species from 16 phyla transported over 6 years on objects that traveled thousands of kilometers across the Pacific Ocean to the shores of North America and Hawai'i. Most of this dispersal occurred on nonbiodegradable objects, resulting in the longest documented transoceanic survival and dispersal of coastal species by rafting. Expanding shoreline infrastructure has increased global sources of plastic materials available for biotic colonization and also interacts with climate change-induced storms of increasing severity to eject debris into the oceans. In turn, increased ocean rafting may intensify species invasions. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  4. Elucidation of the Key Role of [Ru(bpy)3 ](2+) in Photocatalyzed RAFT Polymerization.

    Science.gov (United States)

    Christmann, Julien; Ibrahim, Ahmad; Charlot, Vincent; Croutxé-Barghorn, Céline; Ley, Christian; Allonas, Xavier

    2016-08-04

    Photocatalysis reactions using [Ru(II) (bpy)3 ](2+) were studied on the example of visible-light-sensitized reversible addition-fragmentation chain transfer (RAFT) polymerization. Although both photoinduced electron- and energy-transfer mechanisms are able to describe this interaction, no definitive experimental proof has been presented so far. This paper investigates the actual mechanism governing this reaction. A set of RAFT agents was selected, their redox potentials measured by cyclic voltammetry, and relaxed triplet energies calculated by quantum mechanics. Gibbs free-energy values were calculated for both electron- and energy-transfer mechanisms. Quenching rate constants were determined by laser flash photolysis. The results undoubtedly evidence the involvement of a photoinduced energy-transfer reaction. Controlled photopolymerization experiments are discussed in the light of the primary photochemical process and photodissociation ability of RAFT agent triplet states. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Uptake of raft components into amyloid β-peptide aggregates and membrane damage.

    Science.gov (United States)

    Sasahara, Kenji; Morigaki, Kenichi; Mori, Yasuko

    2015-07-15

    Amyloid aggregation and deposition of amyloid β-peptide (Aβ) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aβ with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aβ deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aβ in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aβ, uptake of the raft components into Aβ aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aβ deposits induces macroscopic heterogeneity of the membrane structure. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi.

    Science.gov (United States)

    Malinsky, Jan; Opekarová, Miroslava; Grossmann, Guido; Tanner, Widmar

    2013-01-01

    The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.

  7. Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

    OpenAIRE

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

  8. A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

    Directory of Open Access Journals (Sweden)

    Melanie L. Hutton

    2017-06-01

    Full Text Available The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248 with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01. HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05, and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05. Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

  9. Membrane rafts: a potential gateway for bacterial entry into host cells.

    Science.gov (United States)

    Hartlova, Anetta; Cerveny, Lukas; Hubalek, Martin; Krocova, Zuzana; Stulik, Jiri

    2010-04-01

    Pathogenic bacteria have developed various mechanisms to evade host immune defense systems. Invasion of pathogenic bacteria requires interaction of the pathogen with host receptors, followed by activation of signal transduction pathways and rearrangement of the cytoskeleton to facilitate bacterial entry. Numerous bacteria exploit specialized plasma membrane microdomains, commonly called membrane rafts, which are rich in cholesterol, sphingolipids and a special set of signaling molecules which allow entry to host cells and establishment of a protected niche within the host. This review focuses on the current understanding of the raft hypothesis and the means by which pathogenic bacteria subvert membrane microdomains to promote infection.

  10. Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

    Directory of Open Access Journals (Sweden)

    Joshua F. Ceñido

    2017-07-01

    Full Text Available Lipid microdomains (‘lipid rafts’ are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR, but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13 and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. 1H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing

  11. The concrete technology of post pouring zone of raft foundation of Hongyun Building B tower

    Science.gov (United States)

    Yin, Suhua; Yu, Liu; Wu, Yanli; Zhao, Ying

    2017-08-01

    The foundation of Hongyun building B tower is made of raft board foundation which is 3300mm in the thickness concreted pouring amount of large and the late poured band in the pouring settlement formed. The temperature of the pouring settlement was controlled in order to prevent the crack of the construction of the late poured band. The steel of post pouring band was designed and monitorred. The quality of post pouring band quality is guaranteed in the raft concrete foundation of Hongyun Building B tower.

  12. Synthesis of click-reactive HPMA copolymers using RAFT polymerization for drug delivery applications

    DEFF Research Database (Denmark)

    Ebbesen, Morten F; Schaffert, D.H.; Crowley, Michael L

    2013-01-01

    This study describes a versatile strategy combining reversible addition fragmentation transfer (RAFT) polymerization and click chemistry to synthesize well-defined, reactive copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) for drug delivery applications. A novel azide containing monomer N-(3......-azidopropyl)methacrylamide (AzMA) was synthesized and copolymerized with HPMA using RAFT polymerization to provide p(HPMA-co-AzMA) copolymers with high control of molecular weight (∼10–54 kDa) and polydispersity (≤1.06). The utility of the side-chain azide functionality by Cu(I)-catalyzed azide...

  13. Gravity-induced encapsulation of liquids by destabilization of granular rafts

    Science.gov (United States)

    Abkarian, Manouk; Protière, Suzie; Aristoff, Jeffrey M.; Stone, Howard A.

    2013-05-01

    Droplets and bubbles coated by a protective armour of particles find numerous applications in encapsulation, stabilization of emulsions and foams, and flotation techniques. Here we study the role of a body force, such as in flotation, as a means of continuous encapsulation by particles. We use dense particles, which self-assemble into rafts, at oil-water interfaces. We show that these rafts can be spontaneously or controllably destabilized into armoured oil-in-water droplets, which highlights a possible role for common granular materials in environmental remediation. We further present a method for continuous production and discuss the generalization of our approach towards colloidal scales.

  14. On ripples and rafts: Curvature induced nanoscale structures in lipid membranes

    International Nuclear Information System (INIS)

    Schmid, Friederike; Dolezel, Stefan; Meinhardt, Sebastian; Lenz, Olaf

    2014-01-01

    We develop an elastic theory that predicts the spontaneous formation of nanoscale structures in lipid bilayers which locally phase separate between two phases with different spontaneous monolayer curvature. The theory rationalizes in a unified manner the observation of a variety of nanoscale structures in lipid membranes: Rippled states in one-component membranes, lipid rafts in multicomponent membranes. Furthermore, we report on recent observations of rippled states and rafts in simulations of a simple coarse-grained model for lipid bilayers, which are compatible with experimental observations and with our elastic model

  15. Graft Copolymerization of Styrene from Poly(vinyl alcohol via RAFT Process

    Directory of Open Access Journals (Sweden)

    Gholam Ali Koohmareh

    2011-01-01

    Full Text Available Polystyrene, PS, was grafted from poly(vinyl alcohol, PVA, backbone by reversible addition-fragmentation chain transfer (RAFT polymerization. The hydroxyl groups of the PVA were converted into aromatic dithioester RAFT agent and polymerization began in the presence of this agent. The structure of compounds was confirmed by FT-IR and 1HNMR spectroscopy. The graft copolymer was characterized by thermogravimetric analysis (TGA, X-ray diffraction (XRD, and scanning electron microscopy (SEM. Grafted polystyrene chains were cleaved from the PVA backbone by acidic hydrolysis of the PVA-g-PS, and its polydispersity index, PDI, was determined by gel permeation chromatography (GPC showing narrow molecular weight distribution.

  16. Telemedicina en Bolivia: proyecto RAFT-Altiplano, experiencias, perspectivas y recomendaciones

    OpenAIRE

    Vargas, Alejandro; Ugalde, Miguel; Vargas, Reynaldo; Narvaez, Ramiro; Geissbuhler, Antoine

    2014-01-01

    OBJETIVO: El objetivo del proyecto RAFT-Altiplano (RAFT, Red Africana Francófona de Telemedicina) es evaluar la viabilidad, potencialidad y riesgos de la implementación y el desarrollo de una red de telemedicina en el contexto de un país en desarrollo, como es el caso del Altiplano de Bolivia, para mejorar el acceso a la atención médica y la formación continua en el área rural. En este estudio se describe el desarrollo de dicho proyecto entre 2011 y 2013. MÉTODOS: Se donaron equipos de teleme...

  17. Multifocal Epithelial Hyperplasia.

    Science.gov (United States)

    Agnew, Caitlin; Alexander, Sherene; Prabhu, Neeta

    2017-01-15

    Multifocal epithelial hyperplasia is a rare disease associated with human papilloma virus types 13 and 32. Diagnosis is based on clinical and histopathological findings, and most lesions are asymptomatic and regress spontaneously with time. The purpose of this paper is to describe a five-year-old girl who presented with multiple intraoral lesions on the buccal mucosa and tongue, which regressed spontaneously in 15 months.

  18. Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

    Science.gov (United States)

    Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

    2009-04-01

    Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

  19. Solo and keratin filaments regulate epithelial tubule morphology.

    Science.gov (United States)

    Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

    2018-04-28

    Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

  20. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    Directory of Open Access Journals (Sweden)

    Chi-Chin Sun

    Full Text Available Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  1. HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

    Science.gov (United States)

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina; Lien, Kathy; Tugizov, Sharof M.

    2018-01-01

    Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30–40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission. PMID:29277006

  2. Adsorption study of a macro-RAFT agent onto SiO2-coated Gd2O3:Eu3+ nanorods: Requirements and limitations

    Science.gov (United States)

    Zou, Hua; Melro, Liliana; de Camargo Chaparro, Thaissa; de Souza Filho, Isnaldi Rodrigues; Ananias, Duarte; Bourgeat-Lami, Elodie; dos Santos, Amilton Martins; Barros-Timmons, Ana

    2017-02-01

    The use of a macromolecular RAFT (macro-RAFT) agent to encapsulate anisotropic nano-objects via emulsion polymerization is an emerging route to prepare polymer/inorganic colloidal nanocomposites. However, a number of requirements have to be fulfilled. This work aims at highlighting the effects of the preparative procedure and dispersion method on the amount of macro-RAFT agent adsorbed onto SiO2-coated Gd2O3:Eu3+ nanorods. The adsorption of macro-RAFT agent was studied using the depletion method with UV-vis spectrophotometry. Measurements were performed at a fixed concentration of nanorods and varying concentrations of the macro-RAFT agent in aqueous dispersion at room temperature. The adsorption isotherms showed that for the same initial macro-RAFT agent concentration, the highest adsorption capacity of the macro-RAFT agent on nanorods was usually achieved for non-calcined thin SiO2-coated nanorods under mild bath sonication.

  3. Effect of tamoxifen in RAFT miniemulsion polymerization during the synthesis of polymer nanoparticles

    Directory of Open Access Journals (Sweden)

    Tailane Sant'Anna Moreira

    2014-01-01

    Full Text Available Tamoxifen (TXF is currently the only hormonal agent used for treatment of breast cancer. Although very effective, TXF presents low solubility in water, which affects its absorption and bioavailability. A common strategy to overcome this barrier is the formulation of a drug delivery system (DDS in order to increase the drug stability and improve the treatment effectiveness. Reversible addition-fragmentation chain transfer (RAFT polymerization is the most versatile method of controlled/living radical polymerization (CLRP, allowing for synthesis of well-defined polymers and being adapted to a wide range of polymerization systems. Miniemulsion polymerization is a dispersed system that is commonly used to prepare nanoparticles (NP with 50 to 500 nm of diameter. The aim of this work was to evaluate the effect of the in situ incorporation of TXF during miniemulsion conventional and RAFT polymerizations, using methyl methacrylate (MMA as monomer. Although the in situ addition of TXF promoted a slight reduction of the reaction rate, it did not affect the final particle size distribution of the latex or the molecular weight control exerted by the RAFT agent. The obtained results suggest that in situ incorporation of TXF during the synthesis of polymer NP via RAFT polymerization allows for production of a polymer DDS for different uses, such as the breast cancer treatment.

  4. In Plant and Animal Cells, Detergent-Resistant Membranes Do Not Define Functional Membrane Rafts

    Czech Academy of Sciences Publication Activity Database

    Tanner, W.; Malínský, Jan; Opekarová, Miroslava

    2011-01-01

    Roč. 23, č. 4 (2011), s. 1191-1193 ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50200510 Keywords : plasma-membrane * lipod rafts * proteins Subject RIV: EA - Cell Biology Impact factor: 8.987, year: 2011

  5. A new type of membrane raft-like microdomains and their possible involvement in TCR signaling

    Czech Academy of Sciences Publication Activity Database

    Otáhal, Pavel; Angelisová, Pavla; Hrdinka, Matouš; Brdička, Tomáš; Novák, Petr; Drbal, Karel; Hořejší, Václav

    2010-01-01

    Roč. 184, č. 7 (2010), s. 3689-3696 ISSN 0022-1767 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50200510 Keywords : membrane rafts * LAT * T-receptor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.745, year: 2010

  6. Designing materials for advanced microelectronic patterning applications using controlled polymerization RAFT technology

    Science.gov (United States)

    Sheehan, Michael T.; Farnham, William B.; Chambers, Charles R.; Tran, Hoang V.; Okazaki, Hiroshi; Brun, Yefim; Romberger, Matthew L.; Sounik, James R.

    2011-04-01

    Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization technology enables the production of polymers possessing low polydispersity (PD) in high yield for many applications. RAFT technology also enables control over polymer architecture. With synthetic control over these polymer characteristics, a variety of polymers can be designed and manufactured for use in advanced electronic applications. By matching the specific RAFT reagent and monomer combinations, we can accommodate monomer reactivity and optimize acrylate or methacrylate polymerizations (193 and 193i photoresist polymers) or optimize styrenic monomer systems (248 nm photoresist polymers) to yield polymers with PD as low as 1.05. For 193i lithography, we have used RAFT technology to produce block copolymers comprising of a random "resist" block with composition and size based on conventional dry photoresist materials and a "low surface energy" block The relative block lengths and compositions may be varied to tune solution migration behavior, surface energy, contact angles, and solubility in developer. Directed self assembly is proving to be an interesting and innovative method to make 2- and even 3-dimensional periodic, uniform patterns. Two keys to acceptable performance of directed self assembly from block copolymers are the uniformity and the purity of the materials will be discussed.

  7. Fabrication of cell outer membrane mimetic polymer brush on polysulfone surface via RAFT technique

    International Nuclear Information System (INIS)

    Ma Qian; Zhang Hui; Zhao Jiang; Gong Yongkuan

    2012-01-01

    Highlights: ► Cell membrane mimetic antifouling polymer brush was grown on polysulfone surface. ► Graft density and polymerization degree were calculated from XPS results. ► Water contact angle measurements showed an extremely hydrophilic surface. ► Platelet adhesion and protein adsorption results suggested excellent antifouling ability. - Abstract: Cell membrane mimetic antifouling polymer brush was grown on polysulfone (PSF) membrane by surface-induced reversible addition–fragmentation chain transfer (RAFT) polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC). The RAFT agent immobilized PSF substrate was prepared by successive chloromethylation, amination with ethylenediamine (EDA) and amidation of the amine group of grafted EDA with the carboxylic group of 4-cyanopentanoic acid dithiobenzoate (CPAD). The surface RAFT polymerization of MPC was initiated in aqueous solution by 4,4′-azobis-4-cyanopentanoic acid (ACPA). The formation of PMPC brush coating is evidenced by X-ray photoelectron spectroscopy and water contact angle measurements. The degree of polymerization of PMPC and the polymer grafting density were calculated from the high resolution XPS spectra. The platelet adhesion and protein adsorption results showed that the PMPC-grafted PSF surface has excellent antifouling ability to resist platelet adhesion completely and suppress protein adsorption significantly. This biomimetic and bio-friendly surface RAFT polymerization strategy could be promising for a variety of biomedical applications.

  8. Clinical and laboratory studies of the antacid and raft-forming properties of Rennie alginate suspension.

    Science.gov (United States)

    Tytgat, G N; Simoneau, G

    2006-03-15

    Acid pockets at the gastro-oesophageal junction escape buffering from meals in the stomach. Combining high-dose antacid with alginate may therefore be of benefit in gastro-oesophageal reflux disease. To characterize the antacid and raft-forming properties of Rennie alginate suspension (containing high-dose antacid and alginate; Bayer Consumer Care, Bladel, the Netherlands). The in vitro acid-neutralizing capacity of Rennie algniate was compared with Gaviscon (Reckitt Benckiser, Slough, UK) by pH-recorded HCl titration. Alginate raft weight formed in vitro at different pH was used to evaluate the pH dependency of raft formation with each product. A double-blind, placebo-controlled, randomized crossover study also compared the antacid activity of Rennie alginate vs. placebo in vivo using continuous intragastric pH monitoring in 12 healthy fasting volunteers. Compared with Gaviscon, Rennie alginate had a higher acid-neutralizing capacity, greater maximum pH and longer duration of antacid activity in vitro. However, the two products produced comparable alginate rafts at each pH evaluated. In vivo, Rennie alginate provided rapid, effective and long-lasting acid neutralization, with an onset of action of <5 min, and duration of action of almost 90 min. The dual mode of action of Rennie alginate offers an effective treatment option for mild symptomatic gastro-oesophageal reflux disease particularly considering recent findings regarding 'acid pockets'.

  9. Clinical and laboratory studies of the antacid and raft-forming properties of Rennie alginate suspension

    NARCIS (Netherlands)

    Tytgat, G. N.; Simoneau, G.

    2006-01-01

    BACKGROUND: Acid pockets at the gastro-oesophageal junction escape buffering from meals in the stomach. Combining high-dose antacid with alginate may therefore be of benefit in gastro-oesophageal reflux disease. AIM: To characterize the antacid and raft-forming properties of Rennie alginate

  10. Influence of lipid rafts on CD1d presentation by dendritic cells

    DEFF Research Database (Denmark)

    Peng, Wei; Martaresche, Cecile; Escande-Beillard, Nathalie

    2011-01-01

    corresponding to lipid rafts and we describe that alpha-GalCer enhanced CD1d amount in the low density detergent insoluble fraction. We conclude that the membrane environment of CD1d can influence antigen presentation mainly when the endocytic pathway is required. Flow cytometry analysis can provide additional...

  11. Internal Technical Report, Safety Analysis Report 5 MW(e) Raft River Research and Development Plant

    Energy Technology Data Exchange (ETDEWEB)

    Brown, E.S.; Homer, G.B.; Shaber, C.R.; Thurow, T.L.

    1981-11-17

    The Raft River Geothermal Site is located in Southern Idaho's Raft River Valley, southwest of Malta, Idaho, in Cassia County. EG and G idaho, Inc., is the DOE's prime contractor for development of the Raft River geothermal field. Contract work has been progressing for several years towards creating a fully integrated utilization of geothermal water. Developmental progress has resulted in the drilling of seven major DOE wells. Four are producing geothermal water from reservoir temperatures measured to approximately 149 C (approximately 300 F). Closed-in well head pressures range from 69 to 102 kPa (100 to 175 psi). Two wells are scheduled for geothermal cold 60 C (140 F) water reinjection. The prime development effort is for a power plant designed to generate electricity using the heat from the geothermal hot water. The plant is designated as the ''5 MW(e) Raft River Research and Development Plant'' project. General site management assigned to EG and G has resulted in planning and development of many parts of the 5 MW program. Support and development activities have included: (1) engineering design, procurement, and construction support; (2) fluid supply and injection facilities, their study, and control; (3) development and installation of transfer piping systems for geothermal water collection and disposal by injection; and (4) heat exchanger fouling tests.

  12. Internal Technical Report, Safety Analysis Report 5 MW(e) Raft River Pilot Plant

    Energy Technology Data Exchange (ETDEWEB)

    Brown, E.S.; Homer, G.B.; Spencer, S.G.; Shaber, C.R.

    1980-05-30

    The Raft River Geothermal Site is located in Southern Idaho's Raft River Valley, southwest of Malta, Idaho, in Cassia County. EG and G idaho, Inc., is the DOE's prime contractor for development of the Raft River geothermal field. Contract work has been progressing for several years towards creating a fully integrated utilization of geothermal water. Developmental progress has resulted in the drilling of seven major DOE wells. Four are producing geothermal water from reservoir temperatures measured to approximately 149 C (approximately 300 F). Closed-in well head pressures range from 69 to 102 kPa (100 to 175 psi). Two wells are scheduled for geothermal cold 60 C (140 F) water reinjection. The prime development effort is for a power plant designed to generate electricity using the heat from the geothermal hot water. The plant is designated as the ''5 MW(e) Raft River Research and Development Plant'' project. General site management assigned to EG and G has resulted in planning and development of many parts of the 5 MW program. Support and development activities have included: (1) engineering design, procurement, and construction support; (2) fluid supply and injection facilities, their study, and control; (3) development and installation of transfer piping systems for geothermal water collection and disposal by injection; and (4) heat exchanger fouling tests.

  13. Multiblock Copolymers of Styrene and Butyl Acrylate via Polytrithiocarbonate-Mediated RAFT Polymerization

    Directory of Open Access Journals (Sweden)

    Bastian Ebeling

    2011-03-01

    Full Text Available When linear polytrithiocarbonates as Reversible Addition-Fragmentation chain Transfer (RAFT agents are employed in a radical polymerization, the resulting macromolecules consist of several homogeneous polymer blocks, interconnected by the functional groups of the respective RAFT agent. Via a second polymerization with another monomer, multiblock copolymers—polymers with alternating segments of both monomers—can be prepared. This strategy was examined mechanistically in detail based on subsequent RAFT polymerizations of styrene and butyl acrylate. Size-exclusion chromatography (SEC of these polymers showed that the examined method yields low-disperse products. In some cases, resolved peaks for molecules with different numbers of blocks (polymer chains separated by the trithiocarbonate groups could be observed. Cleavage of the polymers at the trithiocarbonate groups and SEC analysis of the products showed that the blocks in the middle of the polymers are longer than those at the ends and that the number of blocks corresponds to the number of functional groups in the initial RAFT agent. Furthermore, the produced multiblock copolymers were analyzed via differential scanning calorimetry (DSC. This work underlines that the examined methodology is very well suited for the synthesis of well-defined multiblock copolymers.

  14. Ethanol alters cellular activation and CD14 partitioning in lipid rafts

    International Nuclear Information System (INIS)

    Dai Qun; Zhang Jun; Pruett, Stephen B.

    2005-01-01

    Alcohol consumption interferes with innate immunity. In vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor 4 (TLR4) and inhibits TLR4 signaling. Actually, EtOH exhibits a generalized suppressive effect on signaling and cytokine responses induced by through most TLRs. However, the underlying mechanism remains unknown. RAW264.7 cells were treated with LPS or co-treated with EtOH or with lipid raft-disrupting drugs. TNF-α production, IRAK-1 activation, and CD14 partition were evaluated. EtOH or nystatin, a lipid raft-disrupting drug, suppressed LPS-induced production of TNF-α. The suppressive effect of EtOH on LPS-induced TNF-α production was additive with that of methyl-β-cyclodextrin (MCD), another lipid raft-disrupting drug. EtOH interfered with IRAK-1 activation, an early TLR4 intracellular signaling event. Cell fractionation analyses show that acute EtOH altered LPS-related partition of CD14, a critical component of the LPS receptor complex. These results suggest a novel mechanism of EtOH action that involves interference with lipid raft clustering induced by LPS. This membrane action of EtOH might be one of the mechanisms by which EtOH acts as a generalized suppressor for TLR signaling

  15. RAFT Miniemulsion Polymerization of MMA with Cumyl Dithiobenzoate as Chain Transfer Agent

    Institute of Scientific and Technical Information of China (English)

    Tian Ying GUO; Dong Lin TANG; Jing Wei ZHU; Mou Dao SONG; Bang Hua ZHANG

    2006-01-01

    Reversible addition-fragmentation transfer (RAFT) miniemulsion polymerizations for PMMA with cumyl dithiobenzoate (CDB) as a chain transfer agent (CTA) has been carried out.Higher temperature made the polymerization much faster and the PDI remained below 1.20, when the temperature was upon 70 ℃.

  16. Incidence, predictors, and procedural results of upgrade to resynchronization therapy: the RAFT upgrade substudy.

    Science.gov (United States)

    Essebag, Vidal; Joza, Jacqueline; Birnie, David H; Sapp, John L; Sterns, Laurence D; Philippon, Francois; Yee, Raymond; Crystal, Eugene; Kus, Teresa; Rinne, Claus; Healey, Jeffrey S; Sami, Magdi; Thibault, Bernard; Exner, Derek V; Coutu, Benoit; Simpson, Chris S; Wulffhart, Zaev; Yetisir, Elizabeth; Wells, George; Tang, Anthony S L

    2015-02-01

    The resynchronization-defibrillation for ambulatory heart failure trial (RAFT) study demonstrated that adding cardiac resynchronization therapy (CRT) in selected patients requiring de novo implantable cardiac defibrillators (ICD) reduced mortality as compared with ICD therapy alone, despite an increase in procedure-related adverse events. Data are lacking regarding the management of patients with ICD therapy who develop an indication for CRT upgrade. Participating RAFT centers provided data regarding de novo CRT-D (CRT with ICD) implant, upgrade to CRT-D during RAFT (study upgrade), and upgrade within 6 months after presentation of study results (substudy). Substudy centers enrolled 1346 (74.9%) patients in RAFT, including 644 de novo, 80 study upgrade, and 60 substudy CRT attempts. The success rate (initial plus repeat attempts) was 95.2% for de novo versus 96.3% for study upgrade and 90.0% for substudy CRT attempts (P=0.402). Acute complications occurred among 26.2% of de novo versus 18.8% of study upgrade and 3.4% of substudy CRT implantation attempts (PRAFT study and other trials. © 2014 American Heart Association, Inc.

  17. Controlled growth of protein resistant PHEMA brushes via S-RAFT polymerization

    Czech Academy of Sciences Publication Activity Database

    Zamfir, M.; Rodriguez-Emmenegger, Cesar; Bauer, S.; Barner, L.; Rosenhahn, A.; Barner-Kowollik, C.

    2013-01-01

    Roč. 1, č. 44 (2013), s. 6027-6034 ISSN 2050-750X R&D Projects: GA ČR GAP205/12/1702; GA ČR GAP106/12/1451 Institutional support: RVO:61389013 Keywords : surface RAFT * polymer brushes * antifouling Subject RIV: CD - Macromolecular Chemistry

  18. Hydroperoxide Traces in Common Cyclic Ethers as Initiators for Controlled RAFT Polymerizations.

    Science.gov (United States)

    Eggers, Steffen; Abetz, Volker

    2018-04-01

    Herein, a reversible addition-fragmentation chain transfer (RAFT) polymerization is introduced for reactive monomers like N-acryloylpyrrolidine or N,N-dimethylacrylamide working without a conventional radical initiator. As a very straightforward proof of principle, the method takes advantage of the usually inconvenient radical-generating hydroperoxide contaminations in cyclic ethers like tetrahydrofuran or 1,4-dioxane, which are very common solvents in polymer sciences. The polymerizations are surprisingly well controlled and the polymers can be extended with a second block, indicating their high livingness. "Solvent-initiated" RAFT polymerizations hence prove to be a feasible access to tailored materials with minimal experimental effort and standard laboratory equipment, only requiring the following ingredients: hydroperoxide-contaminated solvent, monomer, and RAFT agent. In other respects, however, the potential coinitiating ability of the used solvent is to be considered when investigating the kinetics of RAFT polymerizations or aiming for the synthesis of high-livingness polymers, e.g., multiblock copolymers. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Preparation for Instruction. A Module of Instruction in Teacher Education. Prepared for Project RAFT.

    Science.gov (United States)

    Handley, Herbert M., Ed.

    This module, developed by the Research Applications for Teaching (RAFT) project, was written to assist students to write lesson plans that are effective and interactive. Students are given directions for the preparation of behavioral objectives and for the selection of appropriate instructional methodologies to meet the widely varying needs of…

  20. MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

    Science.gov (United States)

    Biernatowska, Agnieszka; Augoff, Katarzyna; Podkalicka, Joanna; Tabaczar, Sabina; Gajdzik-Nowak, Weronika; Czogalla, Aleksander; Sikorski, Aleksander F

    2017-11-01

    Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  1. Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

    DEFF Research Database (Denmark)

    Hansen, Gert H; Dalskov, Stine-Mathilde; Rasmussen, Christina Rehné

    2005-01-01

    The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internaliz......The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its...... accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating...... that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border...

  2. Sensitivity of whitewater rafting consumers surplus to pecuniary travel cost specifications

    Science.gov (United States)

    Donald B.K. English; J. Michael Bowker

    1996-01-01

    Considerable research has examined how different ways of accounting for onsite and travel time affect surplus estimates from travel cost models. However, little has been done regarding different definitions of out-of-pocket costs. Estimates of per trip consumer surplus are developed for a zonal travel cost model for outfitted rafting on the Chattooga River. Nine price...

  3. Polymer@gold Nanoparticles Prepared via RAFT Polymerization for Opto-Biodetection

    Directory of Open Access Journals (Sweden)

    Sónia O. Pereira

    2018-02-01

    Full Text Available Colloidal gold nanoparticles (Au NPs have been used in several biological applications, which include the exploitation of size- and shape-dependent Localized Surface Plasmon Resonance (LSPR in biosensing devices. In order to obtain functional and stable Au NPs in a physiological medium, surface modification and functionalization are crucial steps in these endeavors. Reversible addition-fragmentation chain transfer (RAFT polymerization meets this need offering the possibility of control over the composition and architecture of polymeric shells coating Au NPs. Furthermore, playing with a careful choice of monomers, RAFT polymerization allows the possibility to design a polymer shell with the desired functional groups aiming at Au based nanocomposites suitable for biorecognition and biotargeting. This review provides important aspects concerning the synthesis and optical properties of Au NPs as well as concepts of RAFT polymerization. Understanding these concepts is crucial to appreciate the chemical strategies available towards RAFT-polymer coated Au core-shell nanostructures, which are here reviewed. Finally, examples of applications in opto-biodetection devices are provided and the potential of responsive “smart” nanomaterials based on such structures can be applied to other biological applications.

  4. Astronaut Curtis L. Brown, Jr., pilot, works with his life raft during emergency bailout training

    Science.gov (United States)

    1996-01-01

    STS-77 TRAINING VIEW --- Astronaut Curtis L. Brown, Jr., pilot, works with his life raft during emergency bailout training for crew members in the Johnson Space Centers (JSC) Weightless Environment Training Facility (WET-F). Brown will join five other astronauts for nine days aboard the Space Shuttle Endeavour next month.

  5. The Role of Lipid Rafts in the Early Stage of Enterovirus 71 Infection

    Directory of Open Access Journals (Sweden)

    Yong-Zhe Zhu

    2015-02-01

    Full Text Available Background/Aims: Although it has been widely accepted that Enterovirus 71 (EV71 enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. Methods: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. Results: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. Conclusion: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.

  6. Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

    International Nuclear Information System (INIS)

    Kosicek, Marko; Malnar, Martina; Goate, Alison; Hecimovic, Silva

    2010-01-01

    It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1 -/- cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.

  7. Peripheral epithelial odontogenic tumor

    International Nuclear Information System (INIS)

    Carzoglio, J.; Tancredi, N.; Capurro, S.; Ravecca, T.; Scarrone, P.

    2006-01-01

    A new case of peripheral epithelial odontogenic tumor (Pindborg tumor) is reported. It is localized in the superior right gingival region, a less frequent site, and has the histopathological features previously reported. Immunochemical studies were performed, revealing a differential positive stain to cytokeratins in tumor cells deeply seated in the tumor mass, probably related to tumoral cell heterogeneity.Interestingly, in this particular case S-100 protein positive reactivity was also detected in arborescent cells intermingled with tumoral cells, resembling Langerhans cells. Even though referred in the literature in central Pindborg tumors, no references were found about their presence in peripheral tumors, like the one that is presented here

  8. Roles of Raft-Anchored Adaptor Cbp/PAG1 in Spatial Regulation of c-Src Kinase

    Science.gov (United States)

    Oneyama, Chitose; Suzuki, Takashi; Okada, Masato

    2014-01-01

    The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed. PMID:24675741

  9. Transcriptional networks in epithelial-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Christo Venkov

    Full Text Available Epithelial-mesenchymal transition (EMT changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis.Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells.Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts.