WorldWideScience

Sample records for organotypic epithelial raft

  1. 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

  2. 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

  3. 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.

  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. 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. 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.

  7. 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.

  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. 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

  10. 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

  11. 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.

  12. Human papillomavirus type 45 propagation, infection, and neutralization

    International Nuclear Information System (INIS)

    McLaughlin-Drubin, Margaret E.; Wilson, Susan; Mullikin, Brian; Suzich, JoAnn; Meyers, Craig

    2003-01-01

    The organotypic (raft) culture system has allowed the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs), including virion morphogenesis. We introduced linearized HPV45 genomic DNA into primary keratinocytes, where it recircularized and maintained episomally at a range of 10-50 copies of HPV genomic DNA. Following epithelial stratification and differentiation in organotypic culture, virion morphogenesis occurred. HPV45 virions were purified from raft cultures and were able to infect keratinocytes in vitro. By testing a panel of HPV VLP antisera, we were able to demonstrate that the infection was neutralized not only with human HPV45 VLP-specific antiserum, but also with human HPV18 VLP-specific antiserum, demonstrating serological cross-reactivity between HPV18 and HPV45

  13. Chemotherapy-induced oral mucositis and associated infections in a novel organotypic model.

    Science.gov (United States)

    Sobue, T; Bertolini, M; Thompson, A; Peterson, D E; Diaz, P I; Dongari-Bagtzoglou, A

    2018-06-01

    Oral mucositis is a common side effect of cancer chemotherapy, with significant adverse impact on the delivery of anti-neoplastic treatment. There is a lack of consensus regarding the role of oral commensal microorganisms in the initiation or progression of mucositis because relevant experimental models are non-existent. The goal of this study was to develop an in vitro mucosal injury model that mimics chemotherapy-induced mucositis, where the effect of oral commensals can be studied. A novel organotypic model of chemotherapy-induced mucositis was developed based on a human oral epithelial cell line and a fibroblast-embedded collagen matrix. Treatment of organotypic constructs with 5-fluorouracil (5-FU) reproduced major histopathologic characteristics of oral mucositis, such as DNA synthesis inhibition, apoptosis and cytoplasmic vacuolation, without compromising the three-dimensional structure of the multilayer organotypic mucosa. Although structural integrity of the model was preserved, 5-FU treatment resulted in a widening of epithelial intercellular spaces, characterized by E-cadherin dissolution from adherens junctions. In a neutrophil transmigration assay we discovered that this treatment facilitated transport of neutrophils through epithelial layers. Moreover, 5-FU treatment stimulated key proinflammatory cytokines that are associated with the pathogenesis of oral mucositis. 5-FU treatment of mucosal constructs did not significantly affect fungal or bacterial biofilm growth under the conditions tested in this study; however, it exacerbated the inflammatory response to certain bacterial and fungal commensals. These findings suggest that commensals may play a role in the pathogenesis of oral mucositis by amplifying the proinflammatory signals to mucosa that is injured by cytotoxic chemotherapy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. 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...

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Three-dimensional lithographically-defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Inman, Jamie L.; Bissell, Mina J.

    2008-02-13

    Here we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following one to three days. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.

  1. 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.

  2. 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

  3. In-vitro replication of Chelonid herpesvirus 5 in organotypic skin cultures from Hawaiian green turtles (Chelonia mydas)

    Science.gov (United States)

    Work, Thierry M.; Dagenais, Julie; Weatherby, Tina; Ackermann, Mathias; Balazs, George H.

    2017-01-01

    Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with Chelonid herpesvirus 5 (ChHV5) that has historically been refractory to growth in tissue culture. Here, we show for the first time de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative for active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included 1) either in-vitro culturing of ChHV5-positive tumor biopsies (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and 2) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies revealing intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegumentation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign for active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures where most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as model to culture other viruses that are resistant to replication in conventional cell culture.

  4. 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

  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. 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.

  7. Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis.

    Science.gov (United States)

    Li, Jingting; Sen, George L

    2015-12-14

    Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

  8. 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

  9. Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition

    OpenAIRE

    Liu, Jing; Saponjian, Yero; Mahoney, Mark M.; Staley, Kevin J.; Berdichevsky, Yevgeny

    2017-01-01

    Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic ...

  10. 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.

  11. 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.

  12. Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model.

    Science.gov (United States)

    Khoh-Reiter, Su; Jessen, Bart A

    2009-07-28

    Benzalkonium chloride (BAC) is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D) corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC) and olopatadine (0.01% BAC) was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T) cell cultures, expression levels (mRNA and protein) of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 microL drops twice daily in 1 eye for 1 year) in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC) and untreated eyes. The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC contained in ophthalmic solutions are not likely to cause

  13. Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model

    Directory of Open Access Journals (Sweden)

    Jessen Bart A

    2009-07-01

    Full Text Available Abstract Background Benzalkonium chloride (BAC is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. Methods The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC and olopatadine (0.01% BAC was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T cell cultures, expression levels (mRNA and protein of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 μL drops twice daily in 1 eye for 1 year in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. Results In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC and untreated eyes. Conclusion The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC

  14. 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.

  15. 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.

  16. Prevention of Bronchial Hyperplasia by EGFR Pathway Inhibitors in an Organotypic Culture Model

    Science.gov (United States)

    Lee, Jangsoon; Ryu, Seung-Hee; Kang, Shin Myung; Chung, Wen-Cheng; Gold, Kathryn Ann; Kim, Edward S.; Hittelman, Walter N.; Hong, Waun Ki; Koo, Ja Seok

    2011-01-01

    Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. The current study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured using the 3-dimensional organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands EGF, transforming growth factor-alpha, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MEK inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we demonstrated that bronchial hyperplasia can be modeled in vitro using primary NHTBE cells maintained in a 3-dimensional (3-D) organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role. PMID:21505178

  17. 3D Organotypic Culture Model to Study Components of ERK Signaling.

    Science.gov (United States)

    Chioni, Athina-Myrto; Bajwa, Rabia Tayba; Grose, Richard

    2017-01-01

    Organotypic models are 3D in vitro representations of an in vivo environment. Their complexity can range from an epidermal replica to the establishment of a cancer microenvironment. These models have been used for many years, in an attempt to mimic the structure and function of cells and tissues found inside the body. Methods for developing 3D organotypic models differ according to the tissue of interest and the experimental design. For example, cultures may be grown submerged in culture medium and or at an air-liquid interface. Our group is focusing on an air-liquid interface 3D organotypic model. These cultures are grown on a nylon membrane-covered metal grid with the cells embedded in a Collagen-Matrigel gel. This allows cells to grow in an air-liquid interface to enable diffusion and nourishment from the medium below. Subsequently, the organotypic cultures can be used for immunohistochemical staining of various components of ERK signaling, which is a key player in mediating communication between cells and their microenvironment.

  18. Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

    Science.gov (United States)

    Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

    2013-01-01

    Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

  19. Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

    Directory of Open Access Journals (Sweden)

    Jing Liu

    Full Text Available Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium, which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF. Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH levels (biomarkers of ictal activity and cell death, respectively in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

  20. Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

    Science.gov (United States)

    Liu, Jing; Saponjian, Yero; Mahoney, Mark M; Staley, Kevin J; Berdichevsky, Yevgeny

    2017-01-01

    Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

  1. 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.

  2. 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

  3. 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

  4. 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)

  5. Segmentation of Image Data from Complex Organotypic 3D Models of Cancer Tissues with Markov Random Fields.

    Science.gov (United States)

    Robinson, Sean; Guyon, Laurent; Nevalainen, Jaakko; Toriseva, Mervi; Åkerfelt, Malin; Nees, Matthias

    2015-01-01

    Organotypic, three dimensional (3D) cell culture models of epithelial tumour types such as prostate cancer recapitulate key aspects of the architecture and histology of solid cancers. Morphometric analysis of multicellular 3D organoids is particularly important when additional components such as the extracellular matrix and tumour microenvironment are included in the model. The complexity of such models has so far limited their successful implementation. There is a great need for automatic, accurate and robust image segmentation tools to facilitate the analysis of such biologically relevant 3D cell culture models. We present a segmentation method based on Markov random fields (MRFs) and illustrate our method using 3D stack image data from an organotypic 3D model of prostate cancer cells co-cultured with cancer-associated fibroblasts (CAFs). The 3D segmentation output suggests that these cell types are in physical contact with each other within the model, which has important implications for tumour biology. Segmentation performance is quantified using ground truth labels and we show how each step of our method increases segmentation accuracy. We provide the ground truth labels along with the image data and code. Using independent image data we show that our segmentation method is also more generally applicable to other types of cellular microscopy and not only limited to fluorescence microscopy.

  6. Segmentation of Image Data from Complex Organotypic 3D Models of Cancer Tissues with Markov Random Fields.

    Directory of Open Access Journals (Sweden)

    Sean Robinson

    Full Text Available Organotypic, three dimensional (3D cell culture models of epithelial tumour types such as prostate cancer recapitulate key aspects of the architecture and histology of solid cancers. Morphometric analysis of multicellular 3D organoids is particularly important when additional components such as the extracellular matrix and tumour microenvironment are included in the model. The complexity of such models has so far limited their successful implementation. There is a great need for automatic, accurate and robust image segmentation tools to facilitate the analysis of such biologically relevant 3D cell culture models. We present a segmentation method based on Markov random fields (MRFs and illustrate our method using 3D stack image data from an organotypic 3D model of prostate cancer cells co-cultured with cancer-associated fibroblasts (CAFs. The 3D segmentation output suggests that these cell types are in physical contact with each other within the model, which has important implications for tumour biology. Segmentation performance is quantified using ground truth labels and we show how each step of our method increases segmentation accuracy. We provide the ground truth labels along with the image data and code. Using independent image data we show that our segmentation method is also more generally applicable to other types of cellular microscopy and not only limited to fluorescence microscopy.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Blood-group-related carbohydrates are expressed in organotypic cultures of human skin and oral mucosa

    DEFF Research Database (Denmark)

    Grøn, B; Andersson, A; Dabelsteen, Erik

    1999-01-01

    cultures. The organotypic skin and oral mucosa cultures showed a histological differentiation pattern analogous to that of normal skin and buccal mucosa, and a tissue-specific expression of carbohydrate structures and cytokeratins. However, both types of organotypic cultures also expressed markers which...... are normally seen during wound healing, including Lewis y, cytokeratin 16, and cytokeratin 19. We conclude that the organotypic cultures of oral mucosa and skin are suitable models for future studies of the function of cell-surface carbohydrates, although the expression of wound healing markers has to be taken...... the function of cell-surface carbohydrates, we established organotypic cultures of skin and buccal mucosa. In these cultures, keratinocytes are grown at the air-liquid interface on a supporting matrix consisting of homologous fibroblasts embedded in a collagen type I gel. We examined the expression of blood...

  12. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Adamchik, Y; Frantseva, M V; Weisspapir, M; Carlen, P L; Perez Velazquez, J L

    2000-04-01

    Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30

  13. OCT as a convenient tool to assess the quality and application of organotypic retinal samples

    Science.gov (United States)

    Gater, Rachel; Khoshnaw, Nicholas; Nguyen, Dan; El Haj, Alicia J.; Yang, Ying

    2016-03-01

    Eye diseases such as macular degeneration and glaucoma have profound consequences on the quality of human life. Without treatment, these diseases can lead to loss of sight. To develop better treatments for retinal diseases, including cell therapies and drug intervention, establishment of an efficient and reproducible 3D native retinal tissue system, enabled over a prolonged culture duration, will be valuable. The retina is a complex tissue, consisting of ten layers with a different density and cellular composition to each. Uniquely, as a light transmitting tissue, retinal refraction of light differs among the layers, forming a good basis to use optical coherence tomography (OCT) in assessing the layered structure of the retina and its change during the culture and treatments. In this study, we develop a new methodology to generate retinal organotypic tissues and compare two substrates: filter paper and collagen hydrogel, to culture the organotypic tissue. Freshly slaughtered pig eyes have been obtained for use in this study. The layered morphology of intact organotypic retinal tissue cultured on two different substrates has been examined by spectral domain OCT. The viability of the tissues has been examined by live/dead fluorescence dye kit to cross validate the OCT images. For the first time, it is demonstrated that the use of a collagen hydrogel supports the viability of retinal organotypic tissue, capable of prolonged culture up to 2 weeks. OCT is a convenient tool for appraising the quality and application of organotypic retinal samples and is important in the development of current organotypic models.

  14. 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

  15. The effects of insulin and hyperglycemia on surfactant phospholipid synthesis in organotypic cultures of type II pneumocytes.

    Science.gov (United States)

    Engle, M J; Langan, S M; Sanders, R L

    1983-08-29

    Organotypic cultures of fetal type II epithelial cells were incubated in media containing insulin at concentrations ranging from 10 to 400 microunits/ml. Exposure to insulin resulted in increased glucose uptake from the media and in the rate of glucose conversion to CO2. Furthermore, both glucose uptake and CO2 production were dependent on the glucose concentration in the media. Surfactant and residual phosphatidylcholine fractions were isolated from the organotypic cultures by sucrose density centrifugation. The presence of low doses of insulin (10-25 microunits/ml) caused a significant increase in the incorporation of glucose into both surfactant and residual phosphatidylcholine. Insulin at levels of 100 microunits/ml or higher resulted in a significant decrease in glucose incorporation into both phosphatidylcholine fractions. Increasing the media glucose concentration from 5.6 to 20 mM caused a 2- to 2.5-fold increase in glucose utilization for surfactant and residual phospholipid synthesis, but did not produce any significant changes in choline incorporation into either surfactant or residual phosphatidylcholine. The addition of 400 microunits/ml of insulin to media containing 20 mM glucose, however, resulted in a 20% decrease in choline incorporation into surfactant phosphatidylcholine but had no effect on choline incorporation into residual phosphatidylcholine. These results suggest that insulin is an important hormone regulating fetal lung maturation and that hyperinsulinemia may be responsible for the delayed lung development in infants of diabetic mothers.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. Cryopreservation of organotypic multicellular spheroids from human gliomas

    NARCIS (Netherlands)

    Kaaijk, P.; van den Berg, F.; van Amstel, P.; Troost, D.

    1996-01-01

    Fresh human glioma tissue can be cultured on agarose to form organotypic multicellular spheroids (OMS). The major advantage of OMS is the preservation of the cellular heterogeneity and the tumour architecture, which is lost in conventional monolayer cultures. The present study was undertaken to

  6. 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...

  7. 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

  8. In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas).

    Science.gov (United States)

    Work, Thierry M; Dagenais, Julie; Weatherby, Tina M; Balazs, George H; Ackermann, Mathias

    2017-09-01

    Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with chelonid herpesvirus 5 (ChHV5), which has historically been refractory to growth in tissue culture. Here we show, for the first time, de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative of active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included (i) either in vitro cultures of ChHV5-positive tumor biopsy specimens (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and (ii) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies that revealed intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegument formation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign of active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures, in which most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as a model for culture of other viruses that are resistant to replication in conventional cell culture. IMPORTANCE A major challenge in virology is the study of viruses that cannot be grown in the laboratory. One example is chelonid herpesvirus 5 (ChHV5), which is associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of

  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. Microfluidic culture chamber for the long-term perfusion and precise chemical stimulation of organotypic brain tissue slices

    DEFF Research Database (Denmark)

    Caicedo, H. H.; Vignes, M.; Brugg, B.

    2010-01-01

    We have developed a microfluidic perfusion-based culture system to study long-term in-vitro responses of organo-typic brain slices exposed to localized neurochemical stimulation. Using this microperfusion chamber we show that hip-pocampal organotypic brain slices cultures grown on nitrocellulose ...

  11. 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.

  12. 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.

  13. 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.

  14. 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....

  15. 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.

  16. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    Energy Technology Data Exchange (ETDEWEB)

    De Abrew, K. Nadira [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Thomas-Virnig, Christina L.; Rasmussen, Cathy A. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Bolterstein, Elyse A. [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Schlosser, Sandy J. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Allen-Hoffmann, B. Lynn, E-mail: blallenh@wisc.edu [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States)

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  17. 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.

  18. 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.

  19. 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.

  20. Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures.

    Science.gov (United States)

    Forsberg, David; Thonabulsombat, Charoensri; Jäderstad, Johan; Jäderstad, Linda Maria; Olivius, Petri; Herlenius, Eric

    2017-08-14

    Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. 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.

  2. 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...

  3. In vitro formation of the Merkel cell-neurite complex in embryonic mouse whiskers using organotypic co-cultures.

    Science.gov (United States)

    Ishida, Kentaro; Saito, Tetsuichiro; Mitsui, Toshiyuki

    2018-06-01

    A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method. © 2018 Japanese Society of Developmental Biologists.

  4. 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...

  5. Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.

    Directory of Open Access Journals (Sweden)

    Agneta Mewes

    Full Text Available BACKGROUND: Organotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer's disease (AD. AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs, surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1 and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH- and the MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO(2 concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue. CONCLUSION/SIGNIFICANCE: Until now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study

  6. 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

  7. A novel organotypic 3D sweat gland model with physiological functionality.

    Directory of Open Access Journals (Sweden)

    Patricia Klaka

    Full Text Available Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5, muscarinic acetylcholine receptor M3 (CHRM3, Na+-K+-Cl- cotransporter 1 (NKCC1, calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A, and aquaporin-5 (AQP5 are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential

  8. Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

    Science.gov (United States)

    Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

    2017-03-15

    Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

  9. 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.

  10. 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.

  11. 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.

  12. 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....

  13. 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...

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

    Science.gov (United States)

    Drevets, Peter; Chucair-Elliott, Ana; Shrestha, Priyadarsini; Jinkins, Jeremy; Karamichos, Dimitrios; Carr, Daniel J J

    2015-10-01

    To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

  10. 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.

  11. 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.

  12. 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....

  13. 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

  14. 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

  15. Regulation of Prostate Development and Benign Prostatic Hyperplasia by Autocrine Cholinergic Signaling via Maintaining the Epithelial Progenitor Cells in Proliferating Status.

    Science.gov (United States)

    Wang, Naitao; Dong, Bai-Jun; Quan, Yizhou; Chen, Qianqian; Chu, Mingliang; Xu, Jin; Xue, Wei; Huang, Yi-Ran; Yang, Ru; Gao, Wei-Qiang

    2016-05-10

    Regulation of prostate epithelial progenitor cells is important in prostate development and prostate diseases. Our previous study demonstrated a function of autocrine cholinergic signaling (ACS) in promoting prostate cancer growth and castration resistance. However, whether or not such ACS also plays a role in prostate development is unknown. Here, we report that ACS promoted the proliferation and inhibited the differentiation of prostate epithelial progenitor cells in organotypic cultures. These results were confirmed by ex vivo lineage tracing assays and in vivo renal capsule recombination assays. Moreover, we found that M3 cholinergic receptor (CHRM3) was upregulated in a large subset of benign prostatic hyperplasia (BPH) tissues compared with normal tissues. Activation of CHRM3 also promoted the proliferation of BPH cells. Together, our findings identify a role of ACS in maintaining prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Regulation of Prostate Development and Benign Prostatic Hyperplasia by Autocrine Cholinergic Signaling via Maintaining the Epithelial Progenitor Cells in Proliferating Status

    Directory of Open Access Journals (Sweden)

    Naitao Wang

    2016-05-01

    Full Text Available Regulation of prostate epithelial progenitor cells is important in prostate development and prostate diseases. Our previous study demonstrated a function of autocrine cholinergic signaling (ACS in promoting prostate cancer growth and castration resistance. However, whether or not such ACS also plays a role in prostate development is unknown. Here, we report that ACS promoted the proliferation and inhibited the differentiation of prostate epithelial progenitor cells in organotypic cultures. These results were confirmed by ex vivo lineage tracing assays and in vivo renal capsule recombination assays. Moreover, we found that M3 cholinergic receptor (CHRM3 was upregulated in a large subset of benign prostatic hyperplasia (BPH tissues compared with normal tissues. Activation of CHRM3 also promoted the proliferation of BPH cells. Together, our findings identify a role of ACS in maintaining prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes

    DEFF Research Database (Denmark)

    Jahnsen, Henrik; Kristensen, Bjarne Winther; Thiébaud, P

    1999-01-01

    Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish...

  8. 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.

  9. Organotypic Cultures as a Model to Study Adult Neurogenesis in CNS Disorders

    Directory of Open Access Journals (Sweden)

    Fabio Cavaliere

    2016-01-01

    Full Text Available Neural regeneration resides in certain specific regions of adult CNS. Adult neurogenesis occurs throughout life, especially from the subgranular zone of hippocampus and the subventricular zone, and can be modulated in physiological and pathological conditions. Numerous techniques and animal models have been developed to demonstrate and observe neural regeneration but, in order to study the molecular and cellular mechanisms and to characterize multiple types of cell populations involved in the activation of neurogenesis and gliogenesis, investigators have to turn to in vitro models. Organotypic cultures best recapitulate the 3D organization of the CNS and can be explored taking advantage of many techniques. Here, we review the use of organotypic cultures as a reliable and well defined method to study the mechanisms of neurogenesis under normal and pathological conditions. As an example, we will focus on the possibilities these cultures offer to study the pathophysiology of diseases like Alzheimer disease, Parkinson’s disease, and cerebral ischemia.

  10. Prolonged Minocycline Treatment Impairs Motor Neuronal Survival and Glial Function in Organotypic Rat Spinal Cord Cultures

    Science.gov (United States)

    Pinkernelle, Josephine; Fansa, Hisham; Ebmeyer, Uwe; Keilhoff, Gerburg

    2013-01-01

    Background Minocycline, a second-generation tetracycline antibiotic, exhibits anti-inflammatory and neuroprotective effects in various experimental models of neurological diseases, such as stroke, Alzheimer’s disease, amyotrophic lateral sclerosis and spinal cord injury. However, conflicting results have prompted a debate regarding the beneficial effects of minocycline. Methods In this study, we analyzed minocycline treatment in organotypic spinal cord cultures of neonatal rats as a model of motor neuron survival and regeneration after injury. Minocycline was administered in 2 different concentrations (10 and 100 µM) at various time points in culture and fixed after 1 week. Results Prolonged minocycline administration decreased the survival of motor neurons in the organotypic cultures. This effect was strongly enhanced with higher concentrations of minocycline. High concentrations of minocycline reduced the number of DAPI-positive cell nuclei in organotypic cultures and simultaneously inhibited microglial activation. Astrocytes, which covered the surface of the control organotypic cultures, revealed a peripheral distribution after early minocycline treatment. Thus, we further analyzed the effects of 100 µM minocycline on the viability and migration ability of dispersed primary glial cell cultures. We found that minocycline reduced cell viability, delayed wound closure in a scratch migration assay and increased connexin 43 protein levels in these cultures. Conclusions The administration of high doses of minocycline was deleterious for motor neuron survival. In addition, it inhibited microglial activation and impaired glial viability and migration. These data suggest that especially high doses of minocycline might have undesired affects in treatment of spinal cord injury. Further experiments are required to determine the conditions for the safe clinical administration of minocycline in spinal cord injured patients. PMID:23967343

  11. 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

  12. 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)

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  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. 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.

  5. 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.

  6. 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.

  7. Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system.

    Science.gov (United States)

    McCaughey-Chapman, Amy; Connor, Bronwen

    2017-02-01

    Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures. Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss. We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson's disease. Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks. Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson's disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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.

  9. 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

  10. 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.

  11. Electrophysiology of Hypothalamic Magnocellular Neurons In vitro: A Rhythmic Drive in Organotypic Cultures and Acute Slices.

    Science.gov (United States)

    Israel, Jean-Marc; Oliet, Stéphane H; Ciofi, Philippe

    2016-01-01

    Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.

  12. Electrophysiology of hypothalamic magnocellular neurons in vitro: a rhythmic drive in organotypic cultures and acute slices

    Directory of Open Access Journals (Sweden)

    Jean-Marc eIsrael

    2016-03-01

    Full Text Available Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.

  13. 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.

  14. 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.

  15. 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.

  16. 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)

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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...

  5. 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

  6. 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...

  7. 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....

  8. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  9. 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.

  10. 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

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. 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.

  17. 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.

  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. 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. Daunorubicin and doxorubicin but not BCNU have deleterious effects on organotypic multicellular spheroids of gliomas

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; de Boer, O. J.; van Amstel, P.; Bakker, P. J.; Leenstra, S.; Bosch, D. A.

    1996-01-01

    In the present study organotypic multicellular spheroids (OMS) were used to study the effects of chemotherapeutic agents on malignant gliomas. Compared with the frequently used cell line models, OMS have several advantages with respect to the preservation of the cellular heterogeneity and the

  1. 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

  2. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells.

    Science.gov (United States)

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

  3. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

    Science.gov (United States)

    Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

    2012-01-01

    Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

  4. 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.

  5. 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...

  6. Rare human papillomavirus 16 E6 variants reveal significant oncogenic potential

    Directory of Open Access Journals (Sweden)

    Tommasino Massimo

    2011-06-01

    Full Text Available Abstract The aim of this study was to determine whether low prevalence human papillomavirus (HPV 16 E6 variants differ from high prevalence types in their functional abilities. We evaluated functions relevant to carcinogenesis for the rarely-detected European variants R8Q, R10G and R48W as compared to the commonly detected L83V. Human immortalized keratinocytes (NIKS stably transduced with the E6 variants were used in most functional assays. Low and high prevalence E6 variants displayed similar abilities in abrogation of growth arrest and inhibition of p53 elevation induced by actinomycin D. Differences were detected in the abilities to dysregulate stratification and differentiation of NIKS in organotypic raft cultures, modulate detachment induced apoptosis (anoikis and hyperactivate Wnt signaling. No distinctive phenotype could be assigned to include all rare variants. Like L83V, raft cultures derived from variants R10G and R48W similarly induced hyperplasia and aberrantly expressed keratin 5 in the suprabasal compartment with significantly lower expression of keratin 10. Unlike L83V, both variants, and particularly R48W, induced increased levels of anoikis upon suspension in semisolid medium. R8Q induced a unique phenotype characterized by thin organotypic raft cultures, low expression of keratin 10, and high expression of keratins 5 and 14 throughout all raft layers. Interestingly, in a reporter based assay R8Q exhibited a higher ability to augment TCF/β-catenin transcription. The data suggests that differences in E6 variant prevalence in cervical carcinoma may not be related to the carcinogenic potential of the E6 protein.

  7. 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.

  8. 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...

  9. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B

    2000-01-01

    Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation...

  10. 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

  11. 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

  12. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    NSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology......We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h......NSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation....

  13. 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.

  14. 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.

  15. Development of striatal patch/matrix organization in organotypic co-cultures of perinatal striatum, cortex and substantia nigra.

    Science.gov (United States)

    Snyder-Keller, A; Costantini, L C; Graber, D J

    2001-01-01

    Organotypic cultures of fetal or early postnatal striatum were used to assess striatal patch formation and maintenance in the presence or absence of dopaminergic and glutamatergic influences. Vibratome-cut slices of the striatum prepared from embryonic day 19 to postnatal day 4 rat pups were maintained in static culture on clear membrane inserts in Dulbecco's modified Eagle's medium/F12 (1:1) with 20% horse serum. Some were co-cultured with embryonic day 12-16 ventral mesencephalon and/or embryonic day 19 to postnatal day 4 cortex, which produced a dense dopaminergic innervation and a modest cortical innervation. Donors of striatal and cortical tissue were previously injected with bromo-deoxyuridine (BrdU) on embryonic days 13 and 14 in order to label striatal neurons destined to populate the patch compartment of the striatum. Patches of BrdU-immunoreactive cells were maintained in organotypic cultures of late prenatal (embryonic days 20-22) or early postnatal striatum in the absence of nigral dopaminergic or cortical glutamatergic influences. In slices taken from embryonic day 19 fetuses prior to the time of in vivo patch formation, patches were observed to form after 10 days in vitro, in 39% of nigral-striatal co-cultures compared to 6% of striatal slices cultured alone or in the presence of cortex only. Patches of dopaminergic fibers, revealed by tyrosine hydroxylase immunoreactivity, were observed in the majority of nigral-striatal co-cultures. Immunostaining for the AMPA-type glutamate receptor GluR1 revealed a dense patch distribution in nearly all cultures, which developed in embryonic day 19 cultures after at least six days in vitro. These findings indicate that striatal patch/matrix organization is maintained in organotypic culture, and can be induced to form in vitro in striatal slices removed from fetuses prior to the time of in vivo patch formation. Furthermore, dopaminergic innervation from co-cultured pieces of ventral mesencephalon enhances patch

  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. 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.

  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. Multiple single-unit long-term tracking on organotypic hippocampal slices using high-density microelectrode arrays

    Directory of Open Access Journals (Sweden)

    Wei Gong

    2016-11-01

    Full Text Available A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed ‘footprints’ of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times.

  20. 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.

  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. 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

  3. 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...

  4. 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.

  5. Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

    Science.gov (United States)

    Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

    2013-03-05

    One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. Cytolytic effects of autologous lymphokine-activated killer cells on organotypic multicellular spheroids of gliomas in vitro

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; Dast, P. K.; van den Berg, F.; Leenstra, S.; Bosch, D. A.

    1995-01-01

    Knowledge about lymphokine-activated killer (LAK) cell infiltration and LAK cell cytotoxicity is essential to improve the effectiveness of LAK cell therapy against gliomas. In the present study, organotypic multicellular spheroids (OMS) of glioma tissue were used as a culture model to study the

  9. 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...

  10. Implantation of glioblastoma spheroids into organotypic brain slice cultures as a model for investigating effects of irradiation

    DEFF Research Database (Denmark)

    Petterson, Stine Asferg; Jakobsen, Ida Pind; Jensen, Stine Skov

    2016-01-01

    , models for studying the effects of radiotherapy in combination with novel strategies are lacking but important since radiotherapy is the most successful non-surgical treatment of brain tumors. The aim of this study was to establish a glioblastoma spheroid-organotypic rat brain slice culture model...... comprising both tumors, tumor-brain interface and brain tissue to provide a proof of concept that this model is useful for studying effects of radiotherapy. Organotypic brain slice cultures cultured for 1-2 days or 11-16 days corresponding to immature brain and mature brain respectively were irradiated...... with doses between 10 and 50 Gy. There was a high uptake of the cell death marker propidium iodide in the immature cultures. In addition, MAP2 expression decreased whereas GFAP expression increased in these cultures suggesting neuronal death and astrogliosis. We therefore proceeded with the mature cultures...

  11. 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.

  12. 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

  13. 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

  14. 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...

  15. 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

  16. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

    DEFF Research Database (Denmark)

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte

    2003-01-01

    microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident...... lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized...... brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion...

  17. 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.

  18. Nitration of soluble proteins in organotypic culture models of Parkinson's disease

    DEFF Research Database (Denmark)

    Larsen, Trine R; Söderling, Ann-Sofi; Caidahl, Kenneth

    2008-01-01

    Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas...... chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic...

  19. Preparation of well-defined erythromycin imprinted non-woven fabrics via radiation-induced RAFT-mediated grafting

    Science.gov (United States)

    Söylemez, Meshude Akbulut; Barsbay, Murat; Güven, Olgun

    2018-01-01

    Radiation-induced RAFT polymerization technique was applied to synthesize well-defined molecularly imprinted polymers (MIPs) of erythromycin (ERY). Methacrylic acid (MAA) was grafted onto porous polyethylene (PE)/polypropylene (PP) nonwoven fabrics, under γ-irradiation by employing 2-pheny-2-propyl benzodithioate as the RAFT agent and ethylene glycol dimethacrylate (EGDMA) as the crosslinker. MAA/erythromycin ratios of 2/1, 4/1, 6/1 were tested to optimize the synthesis of MIPs. The highest binding capacity was encountered at a MAA/ERY ratio of 4/1. Non-imprinted polymers (NIPs) were also synthesized in the absence of ERY. The MIPs synthesized by RAFT method presented a better binding capacity compared to those prepared by conventional method where no RAFT agent was employed.

  20. Pilocarpine-induced seizure-like activity with increased BNDF and neuropeptide Y expression in organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Poulsen, Frantz Rom; Jahnsen, Henrik; Blaabjerg, Morten

    2002-01-01

    Organotypic hippocampal slice cultures were treated with the muscarinic agonist pilocarpine to study induced seizure-like activity and changes in neurotrophin and neuropeptide expression. For establishment of a seizure-inducing protocol, 2-week-old cultures derived from 6-8-day-old rats were...

  1. 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...

  2. 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.

  3. Evidence for multiphase water-escape during rafting of shelly marine sediments at Clava, Inverness-shire, NE Scotland

    Science.gov (United States)

    Phillips, Emrys; Merritt, Jon

    2008-05-01

    The Pleistocene shelly glaciomarine sediments exposed at Clava, near Inverness, northeast Scotland, occur in a series of thrust-bound rafts accreted at on the up-ice side of a bedrock high (150 m above OD) on the SE side of the valley of the River Nairn. These sediments originally formed part of a coarsening upwards deltaic or subaqueous fan sequence deposited in the marine fjord of the Loch Ness basin, located some 50 km to the SW. The geometry of these allochthonous rafts, coupled with the associated thrusting and large-scale folding of these bodies, are typical of many glacially transported rafts described in literature. However, at Clava, macro- and microscopic evidence indicates that these ice-rafted sediments were not frozen, with liquefaction, hydrofracturing and water-escape occurring repeatedly during their transport and accretion. The presence of large-scale detachments within the sequence has led to the development of a purely glacitectonic model for rafting at Clava. The detachments acted as a focus for fluid flow which lubricated these décollement surfaces, aiding in the subglacial transport of the rafts.

  4. Dopamine release in organotypic cultures of foetal mouse mesencephalon: effects of depolarizing agents, pargyline, nomifensine, tetrodotoxin and calcium

    DEFF Research Database (Denmark)

    Larsen, Trine R; Rossen, Sine; Gramsbergen, Jan B

    2008-01-01

    Organotypic mesencephalic cultures provide an attractive in vitro alternative to study development of the nigrostriatal system and pathophysiological mechanisms related to Parkinson's disease. However, dopamine (DA) release mechanisms have been poorly characterized in such cultures. We report her...

  5. Characterization of organotypic ventral mesencephalic cultures from embryonic mice and protection against MPP toxicity by GDNF

    DEFF Research Database (Denmark)

    Jakobsen, B; Gramsbergen, J B; Møller Dall, A

    2005-01-01

    We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP(+)) and ...

  6. 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.

  7. Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

    Science.gov (United States)

    Yates, Laura L.; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M. Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N.; Niswander, Lee A.; Greenfield, Andy; Dean, Charlotte H.

    2013-01-01

    During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen

  8. 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.

  9. Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

    Science.gov (United States)

    Kinoshita, Masanao; Suzuki, Kenichi G.N.; Takada, Misa; Ano, Hikaru; Abe, Mitsuhiro; Makino, Asami; Kobayashi, Toshihide; Hirosawa, Koichiro M.; Fujiwara, Takahiro K.; Murata, Michio

    2017-01-01

    Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM. PMID:28330937

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. αVβ3 Integrin-Targeted Radionuclide Therapy with 64Cu-cyclam-RAFT-c(-RGDfK-)4.

    Science.gov (United States)

    Jin, Zhao-Hui; Furukawa, Takako; Degardin, Mélissa; Sugyo, Aya; Tsuji, Atsushi B; Yamasaki, Tomoteru; Kawamura, Kazunori; Fujibayashi, Yasuhisa; Zhang, Ming-Rong; Boturyn, Didier; Dumy, Pascal; Saga, Tsuneo

    2016-09-01

    The transmembrane cell adhesion receptor αVβ3 integrin (αVβ3) has been identified as an important molecular target for cancer imaging and therapy. We have developed a tetrameric cyclic RGD (Arg-Gly-Asp) peptide-based radiotracer (64)Cu-cyclam-RAFT-c(-RGDfK-)4, which successfully captured αVβ3-positive tumors and angiogenesis by PET. Here, we subsequently evaluated its therapeutic potential and side effects using an established αVβ3-positive tumor mouse model. Mice with subcutaneous U87MG glioblastoma xenografts received single administrations of 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 (37 MBq/nmol), peptide control, or vehicle solution and underwent tumor growth evaluation. Side effects were assessed in tumor-bearing and tumor-free mice in terms of body weight, routine hematology, and hepatorenal functions. Biodistribution of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 with ascending peptide doses (0.25-10 nmol) and with the therapeutic dose of 2 nmol were determined at 3 hours and at various time points (2 minutes-24 hours) postinjection, respectively, based on which radiation-absorbed doses were estimated. The results revealed that (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently slowed down the tumor growth. The mean tumor doses were 1.28 and 1.81 Gy from 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, respectively. Peptide dose study showed that the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently decreased at doses ≥1 nmol, indicating a saturation of αVβ3 with the administered therapeutic doses (1 and 2 nmol). Combined analysis of the data from tumor-bearing and tumor-free mice revealed no significant toxicity caused by 37-74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 Our study demonstrates the therapeutic efficacy and safety of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 for αVβ3-targeted radionuclide therapy. (64)Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising theranostic drug for cancer imaging and therapy. Mol Cancer Ther; 15(9); 2076-85. ©2016 AACR

  16. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages

    Directory of Open Access Journals (Sweden)

    Dagmar A. Kuhn

    2014-09-01

    Full Text Available Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1 and a human alveolar epithelial type II cell line (A549. In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis. Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. A guide to the synthesis of block copolymers using reversible-addition fragmentation chain transfer (RAFT) polymerization.

    Science.gov (United States)

    Keddie, Daniel J

    2014-01-21

    The discovery of reversible-deactivation radical polymerization (RDRP) has provided an avenue for the synthesis of a vast array of polymers with a rich variety of functionality and architecture. The preparation of block copolymers has received significant focus in this burgeoning research field, due to their diverse properties and potential in a wide range of research environments. This tutorial review will address the important concepts behind the design and synthesis of block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT polymerization is arguably the most versatile of the RDRP methods due to its compatibility with a wide range of functional monomers and reaction media along with its relative ease of use. With an ever increasing array of researchers that possess a variety of backgrounds now turning to RDRP, and RAFT in particular, to prepare their required polymeric materials, it is pertinent to discuss the important points which enable the preparation of high purity functional block copolymers with targeted molar mass and narrow molar mass distribution using RAFT polymerization. The key principles of appropriate RAFT agent selection, the order of monomer addition in block synthesis and potential issues with maintaining high end-group fidelity are addressed. Additionally, techniques which allow block copolymers to be accessed using a combination of RAFT polymerization and complementary techniques are touched upon.

  2. 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.

  3. Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

    International Nuclear Information System (INIS)

    McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie; Rixon, Helen W. McL.; Sugrue, Richard J.

    2004-01-01

    The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells

  4. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Valeria de Turris

    Full Text Available Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  5. 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…

  6. 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

  7. Effect of glycyrrhetinic acid on lipid raft model at the air/water interface.

    Science.gov (United States)

    Sakamoto, Seiichi; Uto, Takuhiro; Shoyama, Yukihiro

    2015-02-01

    To investigate an interfacial behavior of the aglycon of glycyrrhizin (GC), glycyrrhetinic acid (GA), with a lipid raft model consisting of equimolar ternary mixtures of N-palmitoyl sphingomyelin (PSM), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL), Langmuir monolayer techniques were systematically conducted. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms showed that the adsorbed GA at the air/water interface was desorbed into the bulk upon compression of the lipid monolayer. In situ morphological analysis by Brewster angle microscopy and fluorescence microscopy revealed that the raft domains became smaller as the concentrations of GA in the subphase (CGA) increased, suggesting that GA promotes the formation of fluid networks related to various cellular processes via lipid rafts. In addition, ex situ morphological analysis by atomic force microscopy revealed that GA interacts with lipid raft by lying down at the surface. Interestingly, the distinctive striped regions were formed at CGA=5.0 μM. This phenomenon was observed to be induced by the interaction of CHOL with adsorbed GA and is involved in the membrane-disrupting activity of saponin and its aglycon. A quantitative comparison of GA with GC (Sakamoto et al., 2013) revealed that GA interacts more strongly with the raft model than GC in the monolayer state. Various biological activities of GA are known to be stronger than those of GC. This fact allows us to hypothesize that differences in the interactions of GA/GC with the model monolayer correlate to their degree of exertion for numerous activities. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. 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

  9. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

    Science.gov (United States)

    Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

    2013-11-07

    Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

  10. 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.

  11. 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.

  12. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

    Science.gov (United States)

    Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

    2015-07-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and

  13. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

    Science.gov (United States)

    Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

    2015-01-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

  14. Transcytosis of immunoglobulin A in the mouse enterocyte occurs through glycolipid raft- and rab17-containing compartments

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Immerdal, Lissi

    1999-01-01

    BACKGROUND & AIMS: Glycolipid "rafts" have been shown to play a role in apical membrane trafficking in the enterocyte. The present study characterized the membrane compartments of the enterocyte involved in transepithelial transport of small intestinal immunoglobulin A (IgA). Methods: Immunogold...... electron microscopy and radioactive labeling of mouse small intestinal explants were performed. RESULTS: IgA and the polymeric immunoglobulin receptor/secretory component were present in a raft compartment. Raft association occurred posttranslationally within 30 minutes, preceding secretion...... and were also frequently seen associated with the same vesicular profiles of glycolipid rafts. Colocalization of IgA and rab17, a small guanosine triphosphatase involved in transcytosis, was seen mainly along the basolateral plasma membrane and over basolateral endosomes and vesicles, but also...

  15. Differential antiepileptic effects of the organic calcium antagonists verapamil and flunarizine in neurons of organotypic neocortical explants from newborn rats

    NARCIS (Netherlands)

    Bingmann, D; Speckmann, E J; Baker, R E; Ruijter, J; de Jong, B. M.

    1988-01-01

    Effects of the organic calcium antagonists verapamil and flunarizine on pentylenetetrazol induced paroxysmal depolarizations were tested in organotypic neocortical explants taken from neonatal rats. In these in vitro experiments the papaverin derivative verapamil depressed, and finally abolished,

  16. 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.

  17. Targeted radionuclide therapy with RAFT-RGD radiolabelled with (90)Y or (177)Lu in a mouse model of αvβ3-expressing tumours.

    Science.gov (United States)

    Bozon-Petitprin, A; Bacot, S; Gauchez, A S; Ahmadi, M; Bourre, J C; Marti-Batlle, D; Perret, P; Broisat, A; Riou, L M; Claron, M; Boturyn, D; Fagret, D; Ghezzi, Catherine; Vuillez, J P

    2015-02-01

    The αvβ3 integrin plays an important role in tumour-induced angiogenesis, tumour proliferation, survival and metastasis. The tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets the αvβ3 integrin in vitro and in vivo. The aim of this study was to evaluate the therapeutic potential of RAFT-RGD radiolabelled with β(-) emitters in a nude mouse model of αvβ3 integrin-expressing tumours. Biodistribution and SPECT/CT imaging studies were performed after injection of (90)Y-RAFT-RGD or (177)Lu-RAFT-RGD in nude mice subcutaneously xenografted with αvβ3 integrin-expressing U-87 MG cells. Experimental targeted radionuclide therapy with (90)Y-RAFT-RGD or (177)Lu-RAFT-RGD and (90)Y-RAFT-RAD or (177)Lu-RAFT-RAD (nonspecific controls) was evaluated by intravenous injection of the radionuclides into mice bearing αvβ3 integrin-expressing U-87 MG tumours of different sizes (small or large) or bearing TS/A-pc tumours that do not express αvβ3. Tumour volume doubling time was used to evaluate the efficacy of each treatment. Injection of 37 MBq of (90)Y-RAFT-RGD into mice with large αvβ3-positive tumours or 37 MBq of (177)Lu-RAFT-RGD into mice with small αvβ3-positive tumours caused significant growth delays compared to mice treated with 37 MBq of (90)Y-RAFT-RAD or 37 MBq of (177)Lu-RAFT-RAD or untreated mice. In contrast, injection of 30 MBq of (90)Y-RAFT-RGD had no effect on the growth of αvβ3-negative tumours. (90)Y-RAFT-RGD and (177)Lu-RAFT-RGD are potent agents targeting αvβ3-expressing tumours for internal targeted radiotherapy.

  18. Biomimetic PEGylation of carbon nanotubes through surface-initiated RAFT polymerization.

    Science.gov (United States)

    Shi, Yingge; Zeng, Guanjian; Xu, Dazhuang; Liu, Meiying; Wang, Ke; Li, Zhen; Fu, Lihua; Zhang, Qingsong; Zhang, Xiaoyong; Wei, Yen

    2017-11-01

    Carbon nanotubes (CNTs) are a type of one-dimensional carbon nanomaterials that possess excellent physicochemical properties and have been potentially utilized for a variety of applications. Surface modification of CNTs with polymers is a general route to expand and improve the performance of CNTs and has attracted great research interest over the past few decades. Although many methods have been developed previously, most of these methods still showed some disadvantages, such as low efficiency, complex experimental procedure and harsh reaction conditions etc. In this work, we reported a practical and novel way to fabricate CNTs based polymer composites via the combination of mussel inspired chemistry and reversible addition fragmentation chain transfer (RAFT) polymerization. First, the amino group was introduced onto the surface of CNTs via self-polymerization of dopamine. Then, chain transfer agent can be immobilized on the amino groups functionalized CNTs to obtain CNT-PDA-CTA, which can be utilized for surface-initiated RAFT polymerization. A water soluble and biocompatible monomer poly(ethylene glycol) monomethyl ether methacrylate (PEGMA) was adopted to fabricate pPEGMA functionalized CNTs through RAFT polymerization. The successful preparation of CNTs based polymer composites (CNT-pPEGMA) was confirmed by transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray photoelectron spectroscopy in details. The CNT-pPEGMA showed good dispersibility and desirable biocompatibility, making them highly potential for biomedical applications. More importantly, a large number of CNTs based polymer composites could also be fabricated through the same strategy when different monomers were used due to the good monomer adaptability of RAFT polymerization. Therefore, this strategy should be a general method for preparation of various multifunctional CNTs based polymer composites. Copyright © 2017 Elsevier B.V. All rights

  19. Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

    Science.gov (United States)

    Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

    2018-01-01

    Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

  20. 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.

  1. 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

  2. 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.

  3. Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; Sminia, P.; Hulshof, M. C.; van der Kracht, A. H.; Leenstra, S.; Bosch, D. A.

    1997-01-01

    The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the

  4. 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.

  5. 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…

  6. Revealing the Functions of Tenascin-C in 3-D Breast Cancer Models Using Cell Biological and In Silico Approaches

    National Research Council Canada - National Science Library

    Taraseviciute, Agne; Jones, Peter L

    2007-01-01

    ...) organotypic cultures of human mammary epithelial cells by focusing on cell-cell junctions, adherens junctions in particular, as well as activation of receptor tyrosine kinases, namely EGFR and c-met...

  7. Construction of Hierarchical Polymer Brushes on Upconversion Nanoparticles via NIR-Light-Initiated RAFT Polymerization.

    Science.gov (United States)

    Xie, Zhongxi; Deng, Xiaoran; Liu, Bei; Huang, Shanshan; Ma, Pingan; Hou, Zhiyao; Cheng, Ziyong; Lin, Jun; Luan, Shifang

    2017-09-13

    Photoinduced reversible addition-fragmentation chain transfer (RAFT) polymerization generally adopts high-energy ultraviolet (UV) or blue light. In combination with photoredox catalyst, the excitation light wavelength was extended to the visible and even near-infrared (NIR) region for photoinduced electron transfer RAFT polymerization. In this report, we introduce for the first time a surface NIR-light-initiated RAFT polymerization on upconversion nanoparticles (UCNPs) without adding any photocatalyst and construct a functional inorganic core/polymer shell nanohybrid for application in cancer theranostics. The multilayer core-shell UCNPs (NaYF 4 :Yb/Tm@NaYbF 4 :Gd@NaNdF 4 :Yb@NaYF 4 ), with surface anchorings of chain transfer agents, can serve as efficient NIR-to-UV light transducers for initiating the RAFT polymerization. A hierarchical double block copolymer brush, consisting of poly(acrylic acid) (PAA) and poly(oligo(ethylene oxide)methacrylate-co-2-(2-methoxy-ethoxy)ethyl methacrylate) (PEG for short), was grafted from the surface in sequence. The targeting arginine-glycine-aspartic (RGD) peptide was modified at the end of the copolymer through the trithiolcarbonate end group. After loading of doxorubicin, the UCNPs@PAA-b-PEG-RGD exhibited an enhanced U87MG cancer cell uptake efficiency and cytotoxicity. Besides, the unique upconversion luminescence of the nanohybrids was used for the autofluoresence-free cell imaging and labeling. Therefore, our strategy verified that UCNPs could efficiently activate RAFT polymerization by NIR photoirradiation and construct the complex nanohybrids, exhibiting prospective biomedical applications due to the low phototoxicity and deep penetration of NIR light.

  8. Sulfonated macro-RAFT agents for the surfactant-free synthesis of cerium oxide-based hybrid latexes.

    Science.gov (United States)

    Garnier, Jérôme; Warnant, Jérôme; Lacroix-Desmazes, Patrick; Dufils, Pierre-Emmanuel; Vinas, Jérôme; van Herk, Alex

    2013-10-01

    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 acrylate as a hydrophobic monomer and acrylic acid (AA) and/or 2-acrylamido-2-methylpropane sulfonic acid (AMPS) as hydrophilic monomers. After characterizing the adsorption of these macro-RAFT agents at the cerium oxide surface by UV-visible spectrometry, emulsion copolymerization reactions of styrene and methyl acrylate were then carried out in the presence of the surface-modified nanoceria. Dynamic Light Scattering and cryo-Transmission Electron Microscopy were employed to confirm the hybrid structure of the final CeO2/polymer latexes, and proved that the presence of acrylic acid units in amphiphatic macro-RAFT agents enabled an efficient formation of hybrid structures, while the presence of AMPS units, when combined with AA units, resulted in a better distribution of cerium oxide nanoclusters between latex particles. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

    International Nuclear Information System (INIS)

    Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

    2005-01-01

    We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

  10. pH responsive alginate polymeric rafts for controlled drug release by using box behnken response surface design.

    Science.gov (United States)

    Abbas, Ghulam; Hanif, Muhammad; Khan, Mahtab Ahmad

    2017-01-01

    Aim of the present work was to develop alginate raft forming tablets for controlled release pantoprazole sodium sesquihydrate (PSS). Box behnken design was used to optimize 15 formulations with three independent and three dependent variables. Physical tests of all formulations were within pharmacopoeial limits. Raft was characterized by their strength, thickness, resilience, acid neutralizing capacity, floating lag time and total floating time. Raft strength, thickness and resilience of optimized formulation AR9 were 7.43 ± 0.019 g, 5.8 ± 0.245 cm and greater than 480 min, respectively. Buffering and neutralizing capacity were 11.2 ± 1.01 and 6.5 ± 0.56 meq, respectively. Dissolution studies were performed by using simulated gastric fluid pH 1.2 and cumulative percentage release of optimized formulation AR9 was found 98%. First order release kinetics were followed and non-fickian diffusion was observed as value of n was greater than 0.45 in korsmeyer-peppas model. PSS, polymers, tablets and rafts were further characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). FTIR spectra of PSS, polymers and raft of optimized formulation AR9 showed peaks at 3223.09, 1688.17, 1586.67, 1302.64 and 1027.74 cm -1 due to -OH stretching, ester carbonyl group (C=O) stretching, existence of water and carboxylic group in raft, C-N stretching and -OH bending vibration showed no interaction between them. XRD showed diffraction lines indicates crystalline nature of PSS. DSC thermogram showed endothermic peaks at 250 °C for PSS. The developed raft was suitable for controlled release delivery of PSS.

  11. 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.

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells

    DEFF Research Database (Denmark)

    Dalskov, Stine-Mathilde; Immerdal, Lissi; Niels-Christiansen, Lise-Lotte W

    2005-01-01

    The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98......, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density......, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor...

  17. Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions.

    Science.gov (United States)

    Rose, Suzanne L; Fulton, James M; Brown, Christopher M; Natale, Frank; Van Mooy, Benjamin A S; Bidle, Kay D

    2014-04-01

    Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. 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.

  19. Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity.

    Science.gov (United States)

    Szilagyi, John T; Vetrano, Anna M; Laskin, Jeffrey D; Aleksunes, Lauren M

    2017-07-01

    The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h). BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

    Science.gov (United States)

    Lehrberg, Jeffrey; Gardiner, David M

    2015-01-01

    We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

  1. Regulation of Axolotl (Ambystoma mexicanum Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

    Directory of Open Access Journals (Sweden)

    Jeffrey Lehrberg

    Full Text Available We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

  2. 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

  3. 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…

  4. Lipid raft localization of TLR2 and its co-receptors is independent of membrane lipid composition

    Directory of Open Access Journals (Sweden)

    Christine Hellwing

    2018-01-01

    Full Text Available Background Toll like receptors (TLRs are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. Methods In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA or arachidonic acid (AA and analyzed for receptor expression and microdomain localization in context of TLR stimulation. Results and Conclusions Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

  5. 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.

  6. 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

  7. Ultra-Fast RAFT-HDA Click Conjugation: An Efficient Route to High Molecular Weight Block Copolymers.

    Science.gov (United States)

    Inglis, Andrew J; Stenzel, Martina H; Barner-Kowollik, Christopher

    2009-11-02

    The use of the reversible addition fragmentation chain transfer-hetero Diels-Alder (RAFT-HDA) click reaction for the modular construction of block copolymers is extended to the generation of high molecular weight materials. Cyclopentadienyl end-functionalized polystyrene (PS-Cp) prepared via both atom transfer radical polymerization (ATRP) and the RAFT process are conjugated to poly(isobornyl acrylate) (PiBoA) (also prepared via RAFT polymerization) to achieve well-defined block copolymers with molecular weights ranging from 34 000 to over 100 000 g · mol(-1) and with small polydispersities (PDI HDA click chemistry can provide access to high molecular weight block copolymers in a simple and straight-forward fashion. Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Targeted radionuclide therapy with RAFT-RGD radiolabelled with {sup 90}Y or {sup 177}Lu in a mouse model of αvβ3-expressing tumours

    Energy Technology Data Exchange (ETDEWEB)

    Bozon-Petitprin, A.; Bacot, S.; Ahmadi, M.; Marti-Batlle, D.; Perret, P.; Broisat, A.; Riou, L.M. [INSERM, U1039, Grenoble (France); Universite de Grenoble, UMR-S1039, Grenoble (France); Gauchez, A.S.; Bourre, J.C.; Fagret, D.; Vuillez, J.P. [INSERM, U1039, Grenoble (France); Universite de Grenoble, UMR-S1039, Grenoble (France); CHRU Grenoble, Hopital Michallon, Service de Medecine Nucleaire, Grenoble (France); Claron, M.; Boturyn, D. [CNRS, UMR 5250, Departement de Chimie Moleculaire, Grenoble (France); Ghezzi, Catherine [INSERM, U1039, Grenoble (France); Universite de Grenoble, UMR-S1039, Grenoble (France); INSERM U1039, Radiopharmaceutiques biocliniques, Batiment Jean Roget, Domaine de la Merci, Faculte de Medecine, La Tronche (France)

    2014-08-28

    The αvβ3 integrin plays an important role in tumour-induced angiogenesis, tumour proliferation, survival and metastasis. The tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK]){sub 4} (RAFT-RGD), specifically targets the αvβ3 integrin in vitro and in vivo. The aim of this study was to evaluate the therapeutic potential of RAFT-RGD radiolabelled with β{sup -} emitters in a nude mouse model of αvβ3 integrin-expressing tumours. Biodistribution and SPECT/CT imaging studies were performed after injection of {sup 90}Y-RAFT-RGD or {sup 177}Lu-RAFT-RGD in nude mice subcutaneously xenografted with αvβ3 integrin-expressing U-87 MG cells. Experimental targeted radionuclide therapy with {sup 90}Y-RAFT-RGD or {sup 177}Lu-RAFT-RGD and {sup 90}Y-RAFT-RAD or {sup 177}Lu-RAFT-RAD (nonspecific controls) was evaluated by intravenous injection of the radionuclides into mice bearing αvβ3 integrin-expressing U-87 MG tumours of different sizes (small or large) or bearing TS/A-pc tumours that do not express αvβ3. Tumour volume doubling time was used to evaluate the efficacy of each treatment. Injection of 37 MBq of {sup 90}Y-RAFT-RGD into mice with large αvβ3-positive tumours or 37 MBq of {sup 177}Lu-RAFT-RGD into mice with small αvβ3-positive tumours caused significant growth delays compared to mice treated with 37 MBq of {sup 90}Y-RAFT-RAD or 37 MBq of {sup 177}Lu-RAFT-RAD or untreated mice. In contrast, injection of 30 MBq of {sup 90}Y-RAFT-RGD had no effect on the growth of αvβ3-negative tumours. {sup 90}Y-RAFT-RGD and {sup 177}Lu-RAFT-RGD are potent agents targeting αvβ3-expressing tumours for internal targeted radiotherapy. (orig.)

  9. [Application of rafting K-wire technique for tibial plateau fractures].

    Science.gov (United States)

    Zhang, Xing-zhou; Yu, Wei-zhong; Li, Yun-feng; Liu, Yan-hui

    2015-12-01

    To summarize application of rafting K-wires technique for tibial plateau fractures. From January 2013 to January 2015,45 patients with tibial plateau fractures were treated by locking plate with rafting K-wires, including 33 males and 12 females with an average of 44.2 years old ranging from 22 to 56 years old. According to Schatzker classification, 6 cases were type II, 8 were type Ill, 4 were type IV, 4 were type V, and 5 were type VI. Allogeneic bone graft were performed for bone defects. All patients were fixed with two to five K-wires. Part of weight loading were encouraged at 3 months after operation,and full weight-loading were done at 5 months after operation. Postoperative complications were observed,and Rasmussen clinical and radiological assessment were used to evaluate clinical results. All Patients were followed up from 10 to 23 months with average of 14 months. According to Rasmussen clinical and radiological assessment, clinical scores 23.58 ± 6.33, radiological scores were 14.00 ± 6.33; and excellent and good rates were 82.2% and 77.8% respectively. Four patients occurred severe osteoporosis and collapse of articular surface; 5 patients occurred traumatic arthritis. Rafting K-wires technique with anatomized armor plate could effective fix and support platform collapse and joint bone fragments, increase support surface area and reduce postoperative reduction loss rate.

  10. Development and evaluation of gastroretentive raft forming systems incorporating curcumin-Eudragit® EPO solid dispersions for gastric ulcer treatment.

    Science.gov (United States)

    Kerdsakundee, Nattha; Mahattanadul, Sirima; Wiwattanapatapee, Ruedeekorn

    2015-08-01

    Novel raft forming systems incorporating curcumin-Eudragit® EPO solid dispersions were developed to prolong the gastric residence time and provide for a controlled release therapy of curcumin to treat gastric ulcers. The solid dispersions of curcumin with Eudragit® EPO were prepared by the solvent evaporation method at various ratios to improve the solubility and the dissolution of curcumin. The optimum weight ratio of 1:5 for curcumin to Eudragit® EPO was used to incorporate into the raft forming systems. The raft forming formulations were composed of curcumin-Eudragit® EPO solid dispersions, sodium alginate as a gelling polymer and calcium carbonate for generating divalent Ca(2+) ions and carbon dioxide to form a floating raft. All formulations formed a gelled raft in 1min and sustained buoyancy on the 0.1N hydrochloric acid (pH 1.2) surface with a 60-85% release of curcumin within 8h. The curative effect on the acetic acid-induced chronic gastric ulcer in rats was determined. The curcumin raft forming formulations at 40mg/kg once daily showed a superior curative effect on the gastric ulcer in terms of the ulcer index and healing index than the standard antisecretory agent: lansoprazole (1mg/kg, twice daily) and a curcumin suspension (40mg/kg, twice daily). These studies demonstrated that the new raft forming systems containing curcumin solid dispersions are promising carriers for a stomach-specific delivery of poorly soluble lipophilic compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. 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

  12. 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...

  13. Gag induces the coalescence of clustered lipid rafts and tetraspanin-enriched microdomains at HIV-1 assembly sites on the plasma membrane.

    Science.gov (United States)

    Hogue, Ian B; Grover, Jonathan R; Soheilian, Ferri; Nagashima, Kunio; Ono, Akira

    2011-10-01

    The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominantly at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.

  14. The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

    Directory of Open Access Journals (Sweden)

    Shaoying Lu

    2008-07-01

    Full Text Available Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP experiments, we have developed a finite element (FE method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2/sec than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2/sec and outside: 0.18+/-0.02 microm(2/sec. The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

  15. Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

    International Nuclear Information System (INIS)

    Brown, Gaie; Jeffree, Chris E.; McDonald, Terence; McL Rixon, Helen W.; Aitken, James D.; Sugrue, Richard J.

    2004-01-01

    The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of the virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles

  16. 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.

  17. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    Energy Technology Data Exchange (ETDEWEB)

    Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  18. 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.

  19. 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...

  20. 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...

  1. The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

    Directory of Open Access Journals (Sweden)

    Emma L Smith

    Full Text Available Transforming growth factor-beta3 (TGF-β3 and 1α,25-dihydroxyvitamin D3 (1α,25 (OH 2D3 are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11 were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH 2D3 (25 nM or TGF-β3 (5 ng/mL & 15 ng/mL. Analyses of the femurs were undertaken using micro-computed tomography (μCT, histology and immunohistochemistry. 1α,25 (OH2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

  2. Glacitectonic rafting and associated deformation of mid-Pleistocene glacigenic sediments, near Central Graben, central North Sea; results of a 2D High-Resolution Geophysical Survey

    Science.gov (United States)

    Vaughan-Hirsch, David

    2013-04-01

    Glacitectonic rafts are defined as dislocated slabs of bedrock or unconsolidated sediments, transported from their original position by glacial action. These relatively thin, slab-like bodies feature transport distances ranging from tens of meters to hundreds of kilometers. They occur as either single rafts, or multiple stacked bodies associated with a variety of ice-pushed landforms. Internally, rafts frequently appear undeformed although at a larger scale, they may be folded or cut by shear zones and brittle faults. However, the processes leading to the detachment, transport and subsequent emplacement of the rafts remain uncertain. This work describes the results of a geophysical 2D seismic survey of thrust-bound glacitectonic rafts and associated deformation structures, occurring within mid-Pleistocene glacigenic sediments of the Central Graben, central North Sea. The total shortened length of the rafted section is 2.4km, comprising a series of nine discrete rafts which individually range from 235m to 1018m in length. The principle basal detachment occurs at the erosive contact between Aberdeen Ground Formation and overlying Ling Bank Formation. The ice-proximal (northern) limit of rafting is defined by the presence of a large-scale palaeo-channel oriented perpendicular to the direction of rafting, composed of sediments of the Ling Bank Formation and the Forth Formation. The observed deformation structures infer a mean tectonic direction of 178°, indicating that they are associated with an active glacial advance from the north. The resulting deformation creates a minimum lateral shortening throughout the observed sequence of 35%, typifying a strongly compressional regieme associated with rafting. Throughout the surveyed area, structurally younger rafts are found to be emplaced towards the south, compared to the structurally older rafts which are emplaced towards the south-east. This distinction is suggested to be caused by early rafts creating an obstacle to

  3. Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane ▿

    Science.gov (United States)

    Hogue, Ian B.; Grover, Jonathan R.; Soheilian, Ferri; Nagashima, Kunio; Ono, Akira

    2011-01-01

    The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly. PMID:21813604

  4. Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

    Science.gov (United States)

    Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

    2018-01-01

    It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

  5. 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.

  6. Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

    International Nuclear Information System (INIS)

    Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2009-01-01

    Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P 2 and PI(4,5)P 2 -synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P 2 . PIP5-kinase Iα bound PI(4,5)P 2 , and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P 2 . Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P 2 co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P 2 were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P 2

  7. Modelling glioblastoma tumour-host cell interactions using adult brain organotypic slice co-culture

    Directory of Open Access Journals (Sweden)

    Maria Angeles Marques-Torrejon

    2018-02-01

    Full Text Available Glioblastoma multiforme (GBM is an aggressive incurable brain cancer. The cells that fuel the growth of tumours resemble neural stem cells found in the developing and adult mammalian forebrain. These are referred to as glioma stem cells (GSCs. Similar to neural stem cells, GSCs exhibit a variety of phenotypic states: dormant, quiescent, proliferative and differentiating. How environmental cues within the brain influence these distinct states is not well understood. Laboratory models of GBM can be generated using either genetically engineered mouse models, or via intracranial transplantation of cultured tumour initiating cells (mouse or human. Unfortunately, these approaches are expensive, time-consuming, low-throughput and ill-suited for monitoring live cell behaviours. Here, we explored whole adult brain coronal organotypic slices as an alternative model. Mouse adult brain slices remain viable in a serum-free basal medium for several weeks. GSCs can be easily microinjected into specific anatomical sites ex vivo, and we demonstrate distinct responses of engrafted GSCs to diverse microenvironments in the brain tissue. Within the subependymal zone – one of the adult neural stem cell niches – injected tumour cells could effectively engraft and respond to endothelial niche signals. Tumour-transplanted slices were treated with the antimitotic drug temozolomide as proof of principle of the utility in modelling responses to existing treatments. Engraftment of mouse or human GSCs onto whole brain coronal organotypic brain slices therefore provides a simplified, yet flexible, experimental model. This will help to increase the precision and throughput of modelling GSC-host brain interactions and complements ongoing in vivo studies. This article has an associated First Person interview with the first author of the paper.

  8. Comparison of Polymer Networks Synthesized by Conventional Free Radical and RAFT Copolymerization Processes in Supercritical Carbon Dioxide

    Directory of Open Access Journals (Sweden)

    Patricia Pérez-Salinas

    2017-05-01

    Full Text Available There is a debate in the literature on whether or not polymer networks synthesized by reversible deactivation radical polymerization (RDRP processes, such as reversible addition-fragmentation radical transfer (RAFT copolymerization of vinyl/divinyl monomers, are less heterogeneous than those synthesized by conventional free radical copolymerization (FRP. In this contribution, the syntheses by FRP and RAFT of hydrogels based on 2-hydroxyethylene methacrylate (HEMA and ethylene glycol dimethacrylate (EGDMA in supercritical carbon dioxide (scCO2, using Krytox 157 FSL as the dispersing agent, and the properties of the materials produced, are compared. The materials were characterized by differential scanning calorimetry (DSC, swelling index (SI, infrared spectroscopy (FTIR and scanning electron microscopy (SEM. Studies on ciprofloxacin loading and release rate from hydrogels were also carried out. The combined results show that the hydrogels synthesized by FRP and RAFT are significantly different, with apparently less heterogeneity present in the materials synthesized by RAFT copolymerization. A ratio of experimental (Mcexp to theoretical (Mctheo molecular weight between crosslinks was established as a quantitative tool to assess the degree of heterogeneity of a polymer network.

  9. Tailor-made polyfluoroacrylate and its block copolymer by RAFT polymerization in miniemulsion; improved hydrophobicity in the core-shell block copolymer.

    Science.gov (United States)

    Chakrabarty, Arindam; Singha, Nikhil K

    2013-10-15

    Controlled/living radical polymerization (CRP) of a fluoroacrylate was successfully carried out in miniemulsion by Reversible Addition Fragmentation chain Transfer (RAFT) process. In this case, 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) was polymerized using 2-cyanopropyl dodecyl trithiocarbonate (CPDTC) as RAFT agent, Triton X-405 and sodium dodecyl sulfonate (SDS) as surfactant, and potassium persulphate (KPS) or 2,2'-azobis isobutyronitrile (AIBN) as initiator. Being compatible with hydrophobic fluoroacrylate, this RAFT agent offered very high conversion and good control over the molecular weight of the polymer. The miniemulsion was stable without any costabilizer. The long chain dodecyl group (-C12H25) (Z-group in the RAFT agent) had beneficial effect in stabilizing the miniemulsion. When 2-cyano 2-propyl benzodithioate (CPBD) (Z=-C6H5) was used as RAFT agent, the conversion was less and particle size distribution was very broad. Block copolymerization with butyl acrylate (BA) using PHFBA as macro-RAFT agent showed core-shell morphology with the aggregation of PHFBA segment in the shell. GPC as well as DSC analysis confirmed the formation of block copolymer. The core-shell morphology was confirmed by TEM analysis. The block copolymers (PHFBA-b-PBA) showed significantly higher water contact angle (WCA) showing much better hydrophobicity compared to PHFBA alone. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. "Lipid raft aging" in the human frontal cortex during nonpathological aging: gender influences and potential implications in Alzheimer's disease.

    Science.gov (United States)

    Díaz, Mario; Fabelo, Noemí; Ferrer, Isidre; Marín, Raquel

    2018-07-01

    Lipid rafts are highly dynamic membrane domains featured by distinctive biochemical composition and physicochemical properties compared with the surrounding plasma membrane. These microstructures are associated not only with cellular signaling and communication in normal nerve cells but also with pathological processing of amyloid precursor protein in Alzheimer's disease. Using lipid rafts isolated from human frontal cortex in nondemented subjects aging 24 to 85 years, we demonstrate here that lipid structure of lipid rafts undergo significant alterations of specific lipid classes and phospholipid-bound fatty acids as brain cortex correlating with aging. Main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially docosahexaenoic acid and arachidonic acid), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters). Besides, relevant relationships between main fatty acids and/or lipid classes were altered in an age-related manner. This "lipid raft aging" exhibits clear gender differences and appear to be more pronounced in women than in men, especially in older (postmenopausal) women. The outcomes led us to conclude that human cortical lipid rafts are modified by aging in a gender-dependent fashion. Given the central role of bilayer lipid matrix in lipid rafts functionality and neuronal signaling, we hypothesize that these findings might underlie the higher prevalence of cognitive decline evolving toward Alzheimer's disease in postmenopausal women. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

    Science.gov (United States)

    Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

    2005-04-01

    During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

  12. 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...

  13. 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

  14. A protocol for preparation and transfection of rat entorhinal cortex organotypic cultures for electrophysiological whole-cell recordings

    Directory of Open Access Journals (Sweden)

    Nicholas I. Cilz

    2017-01-01

    Full Text Available Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of “bullets” carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.

  15. FIELD INVESTIGATIONS OF PILED-RAFT FOUNDATIONS WITH SHORT-LENGTH CONIC PILES IN BUILDING AREAS OF MINSK

    Directory of Open Access Journals (Sweden)

    V. A. Sernov

    2015-01-01

    Full Text Available In recent time piled foundations are extensively applied due to an increase of storeys in buildings constructed in Minsk and load increment on the soil. Preference is given to this approach even in the case when relatively firm soil occurs in the top part of the foundation bed. In this case maximum usage of the foundation bed bearing capacity and reduction of foundation cost are considered as top-priority tasks for designers. One of the ways to increase the bearing capacity of piled foundations is the necessity to take into account resistance of foundation bed soil located under raft bottom. The raft as well as a shallow foundation is capable to transfer a significant part of building load into the soil. Such approach makes it possible to reduce a number of piles in the foundation or shorten their length. Then it results in shortening of the construction period and significant reduction in zero cycle. However up to the present moment reliable calculation methods that permit to take into account soil resistance in the raft base. An analysis of previous investigations on the matter executed by various researchers and a number of field investigations have been carried out with the purpose to develop the proposed methods.The paper presents results of field investigations on foundations consisting of short stamped tapered piles which are joined together with the help of the raft fragment. Strength and deformation characteristics of the bases are increasing while making such foundations in the fill-up soil. In this case the filled-up ground layer becomes a bearing layer both for piles and rafts as well. Improvement of high-plastic clay-bearing soil properties is ensured by ramming dry concrete mix under pile foot. The paper describes an experience on application of the piled-raft foundation in complicated engineering and geological conditions while constructing the Orthodox Church in Minsk.

  16. Development of an Autonomous, Dual Chamber Bioreactor for the Growth of 3-Dimensional Epithelial-Stromal Tissues in Microgravity

    Science.gov (United States)

    Patel, Zarana S.; Wettergreen, Matthew A.; Huff, Janice L.

    2014-01-01

    We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3-D organotypic epithelial-stromal cultures that require an air-liquid interface. These complex 3-D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air-liquid interface, these 3-D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue-like human epithelial-stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air-liquid, liquid-liquid, and liquid-air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add-on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.

  17. FE-simulation of the initial stages of rafting in nickel-base superalloys

    International Nuclear Information System (INIS)

    Bioermann, H.; Feng Hua; Mughrabi, H.

    2000-01-01

    In the present work, the model of Socrate and Parks which takes into account the plastic deformation and which applies the method of finite elements (FE) using an energy-perturbation approach will be extended by the introduction of a further contribution to the driving force for rafting, cf. This additional driving force is based on the local variation of the hydrostatic stresses arising from the anisotropic distribution of dislocations after deformation, in combination with the different lattice parameters of the two phases γ and γ'. It is an alternative formulation of the driving force introduced recently. With the present model, the initial stages of rafting and the build-up of internal stresses and strains are determined. (orig.)

  18. 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'.

  19. 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.

  20. Improved Livingness and Control over Branching in RAFT Polymerization of Acrylates: Could Microflow Synthesis Make the Difference?

    Science.gov (United States)

    Derboven, Pieter; Van Steenberge, Paul H M; Vandenbergh, Joke; Reyniers, Marie-Francoise; Junkers, Thomas; D'hooge, Dagmar R; Marin, Guy B

    2015-12-01

    The superior capabilities of structured microreactors over batch reactors are demonstrated for reversible addition-fragmentation chain transfer (RAFT) solution polymerization of n-butyl acrylate with the aid of simulations, explicitly accounting for the chain length distribution of all macrospecies types. Since perfect isothermicity can be established in a microreactor, less side products due to backbiting and β-scission are formed compared to the batch operation in which ineffective heat removal leads to an undesirable temperature spike. For a given RAFT chain transfer agent (CTA), additional microstructural control results under microflow conditions by optimizing the reaction temperature, lowering the dilution degree, or decreasing the initial molar ratio of monomer to RAFT CTA. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

    Science.gov (United States)

    Garofalo, Tina; Ferri, Alberto; Sorice, Maurizio; Azmoon, Pardis; Grasso, Maria; Mattei, Vincenzo; Capozzi, Antonella; Manganelli, Valeria; Misasi, Roberta

    2018-04-01

    Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair

    DEFF Research Database (Denmark)

    Noraberg, Jens; Poulsen, Frantz Rom; Blaabjerg, Morten

    2005-01-01

    Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia......), Alzheimer's disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD......) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimer's disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section...

  3. Chiral twist drives raft formation and organization in membranes composed of rod-like particles

    Science.gov (United States)

    Lubensky, Tom C.

    2017-01-01

    Lipid rafts are hypothesized to facilitate protein interaction, tension regulation, and trafficking in biological membranes, but the mechanisms responsible for their formation and maintenance are not clear. Insights into many other condensed matter phenomena have come from colloidal systems, whose micron-scale particles mimic basic properties of atoms and molecules but permit dynamic visualization with single-particle resolution. Recently, experiments showed that bidisperse mixtures of filamentous viruses can self-assemble into colloidal monolayers with thermodynamically stable rafts exhibiting chiral structure and repulsive interactions. We quantitatively explain these observations by modeling the membrane particles as chiral liquid crystals. Chiral twist promotes the formation of finite-sized rafts and mediates a repulsion that distributes them evenly throughout the membrane. Although this system is composed of filamentous viruses whose aggregation is entropically driven by dextran depletants instead of phospholipids and cholesterol with prominent electrostatic interactions, colloidal and biological membranes share many of the same physical symmetries. Chiral twist can contribute to the behavior of both systems and may account for certain stereospecific effects observed in molecular membranes. PMID:27999184

  4. The effect of low dose ionizing radiation on homeostasis and functional integrity in an organotypic human skin model

    Energy Technology Data Exchange (ETDEWEB)

    Neubeck, Claere von [German Cancer Consortium DKTK partner site Dresden, OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden (Germany); German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Geniza, Matthew J. [Molecular and Cellular Biology Program, Oregon State University, Corvallis OR 97331 (United States); Kauer, Paula M.; Robinson, R. Joe; Chrisler, William B. [Health Impacts and Exposure Science, Pacific Northwest National Laboratory, Richland WA 99352 (United States); Sowa, Marianne B., E-mail: marianne.sowa@pnnl.gov [Health Impacts and Exposure Science, Pacific Northwest National Laboratory, Richland WA 99352 (United States)

    2015-05-15

    Highlights: • Low doses of high LET radiation influence skin homeostasis. • Effects on proliferation and differentiation profiles are LET dependent. • Skin barrier function is not compromised following low dose exposure. - Abstract: Outside the protection of Earth's atmosphere, astronauts are exposed to low doses of high linear energy transfer (LET) radiation. Future NASA plans for deep space missions or a permanent settlement on the moon are limited by the health risks associated with space radiation exposures. There is a paucity of direct epidemiological data for low dose exposures to space radiation-relevant high LET ions. Health risk models are used to estimate the risk for such exposures, though these models are based on high dose experiments. There is increasing evidence, however, that low and high dose exposures result in different signaling events at the molecular level, and may involve different response mechanisms. Further, despite their low abundance, high LET particles have been identified as the major contributor to health risk during manned space flight. The human skin is exposed in every external radiation scenario, making it an ideal epithelial tissue model in which to study radiation induced effects. Here, we exposed an in vitro three dimensional (3-D) human organotypic skin tissue model to low doses of high LET oxygen (O), silicon (Si) and iron (Fe) ions. We measured proliferation and differentiation profiles in the skin tissue and examined the integrity of the skin's barrier function. We discuss the role of secondary particles in changing the proportion of cells receiving a radiation dose, emphasizing the possible impact on radiation-induced health issues in astronauts.

  5. Comparison of Polymer Networks Synthesized by Conventional Free Radical and RAFT Copolymerization Processes in Supercritical Carbon Dioxide

    OpenAIRE

    Patricia Pérez-Salinas; Gabriel Jaramillo-Soto; Alberto Rosas-Aburto; Humberto Vázquez-Torres; María Josefa Bernad-Bernad; Ángel Licea-Claverie; Eduardo Vivaldo-Lima

    2017-01-01

    There is a debate in the literature on whether or not polymer networks synthesized by reversible deactivation radical polymerization (RDRP) processes, such as reversible addition-fragmentation radical transfer (RAFT) copolymerization of vinyl/divinyl monomers, are less heterogeneous than those synthesized by conventional free radical copolymerization (FRP). In this contribution, the syntheses by FRP and RAFT of hydrogels based on 2-hydroxyethylene methacrylate (HEMA) and ethylene glycol dimet...

  6. Novel Tertiary Amino Containing Blinding Composite Membranes via Raft Polymerization and Their Preliminary CO2 Permeation Performance.

    Science.gov (United States)

    Zhu, Lifang; Zhou, Mali; Yang, Shanshan; Shen, Jiangnan

    2015-04-23

    Facile synthesis of poly (N,N-dimethylaminoethyl methacrylate) (PDMAEMA) star polymers on the basis of the prepolymer chains, PDMAEMA as the macro chain transfer agent and divinyl benzene (DVB) as the cross-linking reagent by reversible addition-fragmentation chain transfer (RAFT) polymerization was described. The RAFT polymerizations of DMAEMA at 70 °C using four RAFT agents with different R and Z group were investigated. The RAFT agents used in these polymerizations were dibenzyl trithiocarbonate (DBTTC), s-1-dodecyl-s'-(α,α'-dimethyl-α-acetic acid) trithiocarbonate (MTTCD), s,s'-bis (2-hydroxyethyl-2'-dimethylacrylate) trithiocarbonate (BDATC) and s-(2-cyanoprop-2-yl)-s-dodecyltrithiocarbonate (CPTCD). The results indicated that the structure of the end-group of RAFT agents had significant effects on the ability to control polymerization. Compared with the above-mentioned RAFT agents, CPTCD provides better control over the molecular weight and molecular weight distribution. The polydispersity index (PDI) was determined to be within the scope of 1.26 to 1.36. The yields, molecular weight, and distribution of the star polymers can be tuned by changing the molar ratio of DVB/PDMAEMA-CPTCD. The chemical composition and structure of the linear and star polymers were characterized by GPC, FTIR, 1H NMR, XRD analysis. For the pure Chitosan membrane, a great improvement was observed for both CO₂ permeation rate and ideal selectivity of the blending composite membrane upon increasing the content of SPDMAEMA-8. At a feed gas pressure of 37.5 cmHg and 30 °C, the blinding composite membrane (Cs: SPDMAEMA-8 = 4:4) has a CO₂ permeation rate of 8.54 × 10⁻⁴ cm³ (STP) cm⁻²∙s⁻¹∙cm∙Hg⁻¹ and a N₂ permeation rate of 6.76 × 10⁻⁵ cm³ (STP) cm⁻²∙s⁻¹∙cm∙Hg⁻¹, and an ideal CO₂/N₂ selectivity of 35.2.

  7. 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.

  8. Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells.

    Science.gov (United States)

    Shaikh, Saame Raza; Rockett, Benjamin Drew; Salameh, Muhammad; Carraway, Kristen

    2009-09-01

    An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

  9. Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs.

    Science.gov (United States)

    Gnedeva, Ksenia; Hudspeth, A J; Segil, Neil

    2018-06-01

    The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

  10. 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

  11. Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle

    International Nuclear Information System (INIS)

    Hamilton, Valerie T.; Stone, Diana M.; Cantor, Glenn H.

    2003-01-01

    Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5 + , IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5 + B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis

  12. A novel and generalizable organotypic slice platform to evaluate stem cell potential for targeting pediatric brain tumors

    Directory of Open Access Journals (Sweden)

    Li Shengwen

    2008-05-01

    Full Text Available Abstract Brain tumors are now the leading cause of cancer-related deaths in children under age 15. Malignant gliomas are, for all practical purposes, incurable and new therapeutic approaches are desperately needed. One emerging strategy is to use the tumor tracking capacity inherent in many stem cell populations to deliver therapeutic agents to the brain cancer cells. Current limitations of the stem cell therapy strategy include that stem cells are treated as a single entity and lack of uniform technology is adopted for selection of clinically relevant sub-populations of stem cells. Specifically, therapeutic success relies on the selection of a clinically competent stem cell population based on their capacity of targeting brain tumors. A novel and generalizable organotypic slice platform to evaluate stem cell potential for targeting pediatric brain tumors is proposed to fill the gap in the current work flow of stem cell-based therapy. The organotypic slice platform has advantages of being mimic in vivo model, easier to manipulate to optimize parameters than in vivo models such as rodents and primates. This model serves as a framework to address the discrepancy between anticipated in vivo results and actual in vivo results, a critical barrier to timely progress in the field of the use of stem cells for the treatment of neurological disorders.

  13. 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.

  14. RAFT-mediated synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride] brushes for quantitative DNA immobilization

    International Nuclear Information System (INIS)

    Demirci, Serkan; Caykara, Tuncer

    2013-01-01

    The synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride)] [poly(VBTAC)] brushes was achieved via reversible addition-fragmentation chain transfer (RAFT) polymerization and used for quantitative DNA immobilization. Initially, silicon surfaces were modified with RAFT chain transfer agent by utilizing an amide reaction involving a silicon wafer modified with allylamine and 4-cyanopentanoic acid dithiobenzoate (CPAD). Poly(VBTAC) brushes were then prepared via RAFT-mediated polymerization from the surface immobilized CPAD. Various characterization techniques including ellipsometry, X-ray photoelectron spectroscopy, grazing angle-Fourier transform infrared spectroscopy, atomic force microscopy and contact-angle goniometer were used to characterize the immobilization of CPAD on the silicon wafer and the subsequent polymer formation. The addition of free CPAD was required for the formation of well-defined polymer brushes, which subsequently resulted in the presence of free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. Moreover, from atomic force microscopy and ellipsometry measurements, it was also determined that the density of immobilized DNA on the cationic poly(VBTAC) brushes can be quantitatively controlled by adjusting the solution concentration. Highlights: ► The cationic poly(VBTAC) brushes were prepared by RAFT polymerization. ► Grafting density of cationic poly(VBTAC) brushes was as high as 0.76 chains/nm 2 . ► The cationic poly(VBTAC) brushes were used for quantitative DNA immobilization.

  15. 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.

  16. ELECTROPHYSIOLOGICAL CHARACTERIZATION OF DOPAMINERGIC AND NONDOPAMINERGIC NEURONS IN ORGANOTYPIC SLICE CULTURES OF THE RAT VENTRAL MESENCEPHALON

    DEFF Research Database (Denmark)

    STEENSEN, BH; NEDERGAARD, S; OSTERGAARD, K

    1995-01-01

    -old organotypic slice cultures of the ventral mesencephalon prepared from newborn rats. Dopaminergic neurones were distinguished from non-dopaminergic neurones by staining with the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and briefly viewing the preparation with short exposures to ultraviolet...... 81 M Omega), were silent or fired spontaneously at a low frequency (0-9 Hz), and no spontaneous GABA(A)-ergic inhibitory postsynaptic potentials or inward rectification were present. In contrast, non-dopaminergic neurones had fast action potentials (0.6-3.2 ms), low input resistance (mean 32 M Omega...

  17. Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs.

    Science.gov (United States)

    Tokmakov, Alexander A; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

    2010-05-01

    Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.

  18. Carboxyl-Functionalized Polymeric Microspheres Prepared by One-Stage Photoinitiated RAFT Dispersion Polymerization

    Directory of Open Access Journals (Sweden)

    Jianbo Tan

    2017-12-01

    Full Text Available Herein, we report a photoinitiated reversible addition-fragmentation chain transfer (RAFT dispersion copolymerization of methyl methacrylate (MMA and methyl methacrylic (MAA for the preparation of highly monodisperse carboxyl-functionalized polymeric microspheres. High rates of polymerization were observed, with more than 90% particle yields being achieved within 3 h of UV irradiation. Effects of reaction parameters (e.g., MAA concentration, RAFT agent concentration, photoinitiator concentration, and solvent composition were studied in detail, and highly monodisperse polymeric microspheres were obtained in most cases. Finally, silver (Ag composite microspheres were prepared by in situ reduction of AgNO3 using the carboxyl-functionalized polymeric microspheres as the template. The obtained Ag composite microspheres were able to catalyze the reduction of methylene blue (MB with NaBH4 as a reductant.

  19. Three-dimensional analysis for piled raft machine foundation embedded in sand

    Directory of Open Access Journals (Sweden)

    Mahmood Mahmood

    2018-01-01

    Full Text Available Three-dimensional analysis for the dynamic response of a piled raft foundation subjected to vertical vibration is presented in this study. The analysis considers several factors affecting the amplitude of displacement for deep foundation such as pile cap embedment, pile cap thickness, relative density of the sand and the boundary effect. A validation for an experimental piled raft model depending on a scale factor of (20 using at (Plaxis 3D computer program was performed. The sand is simulated using Mohr-Coloumb model while the concrete is simulated as linear elastic material. It has been found that embedding the pile cap in the soil and increasing its thickness lead to decrease the maximum amplitude of displacement. Furthermore, the predictions showed that increasing the distance between the foundation and the boundaries and increasing the relative density of the sand can significantly minimize the dynamic response of the foundation.

  20. Estrogen Interactions With Lipid Rafts Related to Neuroprotection. Impact of Brain Ageing and Menopause

    Directory of Open Access Journals (Sweden)

    Raquel Marin

    2018-03-01

    Full Text Available Estrogens (E2 exert a plethora of neuroprotective actions against aged-associated brain diseases, including Alzheimer's disease (AD. Part of these actions takes place through binding to estrogen receptors (ER embedded in signalosomes, where numerous signaling proteins are clustered. Signalosomes are preferentially located in lipid rafts which are dynamic membrane microstructures characterized by a peculiar lipid composition enriched in gangliosides, saturated fatty acids, cholesterol, and sphingolipids. Rapid E2 interactions with ER-related signalosomes appear to trigger intracellular signaling ultimately leading to the activation of molecular mechanisms against AD. We have previously observed that the reduction of E2 blood levels occurring during menopause induced disruption of ER-signalosomes at frontal cortical brain areas. These molecular changes may reduce neuronal protection activities, as similar ER signalosome derangements were observed in AD brains. The molecular impairments may be associated with changes in the lipid composition of lipid rafts observed in neurons during menopause and AD. These evidences indicate that the changes in lipid raft structure during aging may be at the basis of alterations in the activity of ER and other neuroprotective proteins integrated in these membrane microstructures. Moreover, E2 is a homeostatic modulator of lipid rafts. Recent work has pointed to this relevant aspect of E2 activity to preserve brain integrity, through mechanisms affecting lipid uptake and local biosynthesis in the brain. Some evidences have demonstrated that estrogens and the docosahexaenoic acid (DHA exert synergistic effects to stabilize brain lipid matrix. DHA is essential to enhance molecular fluidity at the plasma membrane, promoting functional macromolecular interactions in signaling platforms. In support of this, DHA detriment in neuronal lipid rafts has been associated with the most common age-associated neuropathologies

  1. Functional copolymer/organo-MMT nanoarchitectures. VI. Synthesis and characterization of novel nanocomposites by interlamellar controlled/living radical copolymerization via preintercalated RAFT-agent/organoclay complexes.

    Science.gov (United States)

    Rzayev, Zakir M O; Söylemez, A Ernur

    2011-04-01

    We have developed a new approach for the synthesis of polymer nanocomposites using a bifunctional reversible addition-fragmentation chain transfer (RAFT) agent, two types of organo-montmorillonites, such as a non-reactive dimethyldodecyl ammonium (DMDA)-MMT and a reactive octadecylamine (ODA)-MMT organoclays, and a radical initiator. The method includes the following stages: (1) synthesis of RAFT intercalated O-MMTs by a physical or chemical interaction of the RAFT agent having two pendant carboxylic groups [S,S-bis(alpha,alpha'-dimethyl-alpha"-acetic acid)trithiocarbonate] with surface alkyl amines of O-MMT containing tertiary ammonium cation or primary amine groups through strong H-bonding and complexing/amidization reactions, respectively, and (2) utilization of these well-dispersed and intercalated RAFT ... O-MMT complexes and their amide derivatives as new modified RAFT agents in radical-initiated interlamellar controlled/living copolymerization of itaconic acid (IA)-n-butylmethacrylate (BMA) monomer pair. The structure and compositions of the synthesized RAFT ... O-MMT complexes and functional copolymer/O-MMT hybrids were confirmed by FTIR, XRD, thermal (DSC-TGA), SEM and TEM morphology analyses. It was demonstrated that the degree of interaction/exfoliation, morphology and thermal behavior of nanocomposites significantly depended on the type of organoclay and in situ interaction, as well as on the content of flexible butyl-ester linkages as a internal plasticizer. The results of the comparative analysis of the nanocomposites structure-composition-property relations show that the functional copolymer-organoclay hybrids prepared with reactive RAFT ... ODA-MMT complex and containing a combination of partially intercalated and predominantly exfoliated nano-structures exhibit relatively higher thermal stability and fine dispersed morphology. These effects were explained by in situ interfacial chemical reactions through amidization of RAFT with surface alkyl amine

  2. Efficiency of RAFT-synthesized PDMAEMA in gene transfer to the retina.

    Science.gov (United States)

    Bitoque, Diogo B; Simão, Sónia; Oliveira, Ana V; Machado, Susana; Duran, Margarita R; Lopes, Eduardo; da Costa, Ana M Rosa; Silva, Gabriela A

    2017-01-01

    Gene therapy has long been heralded as the new hope to evolve from symptomatic care of genetic pathologies to a full cure. Recent successes in using gene therapy for treating several ocular and haematopoietic pathologies have shown the great potential of this approach that, in the early days, relied on the use of viral vectors, which were considered by many to be undesirable for human treatment. Therefore, there is considerable interest and effort in developing non-viral vectors, with efficiency close to that of viral vectors. The aim of this study was to develop suitable non-viral carriers for gene therapy to treat pathologies affecting the retina. In this study poly(2-(N,N-dimethylamino)ethyl methacrylate), PDMAEMA was synthesized by reversible addition-fragmentation chain transfer (RAFT) and the in vitro cytocompatibility and transfection efficiency of a range of polymer:DNA ratios evaluated using a retinal cell line; in vivo biocompatibility was evaluated by ocular injection in C57BL/6 mice. The results showed that through RAFT, it is possible to produce a defined-size polymer that is compatible with cell viability in vitro and capable of efficiently directing gene expression in a polymer-DNA ratio-dependent manner. When injected into the eyes of mice, these vectors induced a transient, mild inflammation, characteristic of the implantation of medical devices. These results form the basis of future studies where RAFT-synthesized PDMAEMA will be used to deliver gene expression systems to the retina of mouse models of retinal pathologies. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  3. 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

  4. 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

  5. PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of posttraumatic epilepsy

    OpenAIRE

    Berdichevsky, Yevgeny; Dryer, Alexandra M.; Saponjian, Yero; Mahoney, Mark M.; Pimentel, Corrin A.; Lucini, Corrina A.; Usenovic, Marija; Staley, Kevin J.

    2013-01-01

    mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordi...

  6. Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model

    DEFF Research Database (Denmark)

    Kuntsche, Judith; Bunjes, Heike; Fahr, Alfred

    2008-01-01

    Various lipid nanoparticle formulations were investigated with respect to (trans)dermal drug delivery with special regard to the mechanism of their effects on human and an organotypic cell culture epidermis. Potential alterations of stratum corneum lipid domains were studied using fluorescence...... assays with labeled liposomes and thermal analysis of isolated stratum corneum. Influences on the permeation of corticosterone were investigated and the occlusive properties of the nanoparticles were determined by measurements of the transepidermal water loss (TEWL). The penetration of a fluorescence dye...... studies and thermal analysis of human and cell culture epidermis indicate that surface lipids, which are not present to the same extent in the cell culture model than in human epidermis, seem to play an important role....

  7. The pumice raft-forming 2012 Havre submarine eruption was effusive

    Science.gov (United States)

    Manga, Michael; Fauria, Kristen E.; Lin, Christina; Mitchell, Samuel J.; Jones, Meghan; Conway, Chris E.; Degruyter, Wim; Hosseini, Behnaz; Carey, Rebecca; Cahalan, Ryan; Houghton, Bruce F.; White, James D. L.; Jutzeler, Martin; Soule, S. Adam; Tani, Kenichiro

    2018-05-01

    A long-standing conceptual model for deep submarine eruptions is that high hydrostatic pressure hinders degassing and acceleration, and suppresses magma fragmentation. The 2012 submarine rhyolite eruption of Havre volcano in the Kermadec arc provided constraints on critical parameters to quantitatively test these concepts. This eruption produced a >1 km3 raft of floating pumice and a 0.1 km3 field of giant (>1 m) pumice clasts distributed down-current from the vent. We address the mechanism of creating these clasts using a model for magma ascent in a conduit. We use water ingestion experiments to address why some clasts float and others sink. We show that at the eruption depth of 900 m, the melt retained enough dissolved water, and hence had a low enough viscosity, that strain-rates were too low to cause brittle fragmentation in the conduit, despite mass discharge rates similar to Plinian eruptions on land. There was still, however, enough exsolved vapor at the vent depth to make the magma buoyant relative to seawater. Buoyant magma was thus extruded into the ocean where it rose, quenched, and fragmented to produce clasts up to several meters in diameter. We show that these large clasts would have floated to the sea surface within minutes, where air could enter pore space, and the fate of clasts is then controlled by the ability to trap gas within their pore space. We show that clasts from the raft retain enough gas to remain afloat whereas fragments from giant pumice collected from the seafloor ingest more water and sink. The pumice raft and the giant pumice seafloor deposit were thus produced during a clast-generating effusive submarine eruption, where fragmentation occurred above the vent, and the subsequent fate of clasts was controlled by their ability to ingest water.

  8. 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 ...

  9. 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

  10. 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, ...

  11. The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation.

    Science.gov (United States)

    Wu, Qiong; Wu, Shiyong

    2017-07-01

    Prohibitin (PHB) plays a role in regulation of ultraviolet B light (UVB)-induced apoptosis of human keratinocytes, HaCaT cells. The regulatory function of PHB appears to be associated with its lipid raft translocation. However, the detailed mechanism for PHB-mediated apoptosis of these keratinocytes upon UVB irradiation is not clear. In this report, we determined the role of lipid raft translocation of PHB in regulation of UVB-induced apoptosis. Our data show that upon UVB irradiation PHB is translocated from the non-raft membrane to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid raft translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and increased apoptosis of the cells upon UVB irradiation. These results suggest that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequential lipid raft translocation. © 2017 Wiley Periodicals, Inc.

  12. Acrylonitrile-Butadiene Rubber (NBR) Prepared via Living/Controlled Radical Polymerization (RAFT).

    Science.gov (United States)

    Kaiser, Andreas; Brandau, Sven; Klimpel, Michael; Barner-Kowollik, Christopher

    2010-09-15

    In the current work we present results on the controlled/living radical copolymerization of acrylonitrile (AN) and 1,3-butadiene (BD) via reversible addition fragmentation chain transfer (RAFT) polymerization techniques. For the first time, a solution polymerization process for the synthesis of nitrile butadiene rubber (NBR) via the use of dithioacetate and trithiocarbonate RAFT agents is described. It is demonstrated that the number average molar mass, $\\overline M _{\\rm n} $, of the NBR can be varied between a few thousand and 60 000 g · mol(-1) with polydispersities between 1.2 and 2.0 (depending on the monomer to polymer conversion). Excellent agreement between the experimentally observed and the theoretically expected molar masses is found. Detailed information on the structure of the synthesized polymers is obtained by variable analytical techniques such as infrared spectroscopy (IR), nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and electrospray ionization-mass spectrometry (ESI-MS). Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Synthesis and characterization of functional acrylic copolymers via RAFT mini-emulsion polymerization

    Science.gov (United States)

    Yılmaz, Onur; Özkan, ćiǧdem Kılıçarislan; Yılmaz, Catalina N.; Yorgancıoǧlu, Ali; Özgünay, Hasan; Karavana, Hüseyin Ata

    2017-12-01

    Copolymers bearing reactive functional groups with controlled molecular weights are of importance since they can be used in many fields such as composites, coatings, membranes, catalysis, biology, optoelectronics, pharmaceuticals, etc. In the present study low molecular weight copolymers based on butyl acrylate (BA) and methyl methacrylate (MMA) in combination with reactive functional monomers of vinyl trietoxysilane (VTES), 3-trimetoxysilylpropyl methacrylate (TMSPMA) and glycidyl methacrylate (GMA) were synthesized via RAFT mini-emulsion technique using 2-cyano 2-propyldodecyldithiocarbonate as CTA agent. The results showed that the average molecular weights of copolymers were close to the theoretical values. On the other hand, PDI values were found to be higher than conventional RAFT polymers. The particle sizes of the latexes were small with very homogenous distributions and good stability. FTIR, H-NMR and TGA results verified the success of copolymer syntheses.

  14. 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

  15. Use of social information in seabirds: compass rafts indicate the heading of food patches.

    Science.gov (United States)

    Weimerskirch, Henri; Bertrand, Sophie; Silva, Jaime; Marques, Jose Carlos; Goya, Elisa

    2010-03-29

    Ward and Zahavi suggested in 1973 that colonies could serve as information centres, through a transfer of information on the location of food resources between unrelated individuals (Information Centre Hypothesis). Using GPS tracking and observations on group movements, we studied the search strategy and information transfer in two of the most colonial seabirds, Guanay cormorants (Phalacrocorax bougainvillii) and Peruvian boobies (Sula variegata). Both species breed together and feed on the same prey. They do return to the same feeding zone from one trip to the next indicating high unpredictability in the location of food resources. We found that the Guanay cormorants use social information to select their bearing when departing the colony. They form a raft at the sea surface whose position is continuously adjusted to the bearing of the largest returning columns of cormorants. As such, the raft serves as a compass signal that gives an indication on the location of the food patches. Conversely, Peruvian boobies rely mainly on personal information based on memory to take heading at departure. They search for food patches solitarily or in small groups through network foraging by detecting the white plumage of congeners visible at long distance. Our results show that information transfer does occur and we propose a new mechanism of information transfer based on the use of rafts off colonies. The use of rafts for information transfer may be common in central place foraging colonial seabirds that exploit short lasting and/or unpredictably distributed food patches. Over the past decades Guanay cormorants have declined ten times whereas Peruvian boobies have remained relatively stable. We suggest that the decline of the cormorants could be related to reduced social information opportunities and that social behaviour and search strategies have the potential to play an important role in the population dynamics of colonial animals.

  16. Use of social information in seabirds: compass rafts indicate the heading of food patches.

    Directory of Open Access Journals (Sweden)

    Henri Weimerskirch

    Full Text Available Ward and Zahavi suggested in 1973 that colonies could serve as information centres, through a transfer of information on the location of food resources between unrelated individuals (Information Centre Hypothesis. Using GPS tracking and observations on group movements, we studied the search strategy and information transfer in two of the most colonial seabirds, Guanay cormorants (Phalacrocorax bougainvillii and Peruvian boobies (Sula variegata. Both species breed together and feed on the same prey. They do return to the same feeding zone from one trip to the next indicating high unpredictability in the location of food resources. We found that the Guanay cormorants use social information to select their bearing when departing the colony. They form a raft at the sea surface whose position is continuously adjusted to the bearing of the largest returning columns of cormorants. As such, the raft serves as a compass signal that gives an indication on the location of the food patches. Conversely, Peruvian boobies rely mainly on personal information based on memory to take heading at departure. They search for food patches solitarily or in small groups through network foraging by detecting the white plumage of congeners visible at long distance. Our results show that information transfer does occur and we propose a new mechanism of information transfer based on the use of rafts off colonies. The use of rafts for information transfer may be common in central place foraging colonial seabirds that exploit short lasting and/or unpredictably distributed food patches. Over the past decades Guanay cormorants have declined ten times whereas Peruvian boobies have remained relatively stable. We suggest that the decline of the cormorants could be related to reduced social information opportunities and that social behaviour and search strategies have the potential to play an important role in the population dynamics of colonial animals.

  17. 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

  18. Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

    Science.gov (United States)

    Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

    2013-06-01

    Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

  19. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  20. Effects of chemotherapeutics on organotypic corticostriatal slice cultures identified by a panel of fluorescent and immunohistochemical markers

    DEFF Research Database (Denmark)

    Nørregaard, Annette; Jensen, Stine Skov; Kolenda, Jesper

    2012-01-01

    no toxicity was observed. Corresponding immunostaining showed loss of MAP2 and increased expression of GFAP and p25α for cultures exposed to 1,000 nM VCR. Cultures exposed to high concentrations of ACNU and IM disintegrated, leaving no tissue for histology. In conclusion, corticostriatal slice cultures...... specific neuronal and glial degeneration induced by chemotherapeutics in organotypic rat corticostriatal slice cultures. The slice cultures were exposed to the alkylating agents temozolomide (TMZ) and nimustine (ACNU), the tyrosine kinase inhibitor imatinib mesylate (IM) and the microtubule...

  1. 77 FR 41406 - Evaluation of In Vitro Tests for Identifying Eye Injury Hazard Potential of Chemicals and...

    Science.gov (United States)

    2012-07-13

    ...://www.epa.gov/oppad001/eye-irritation.pdf ). The IRE test is an organotypic test method that evaluates... rabbit corneal epithelial cells following test substance exposure (Takahashi et al., 2008). NICEATM is..., mailing address, phone, fax, email, and sponsoring organization, as applicable). NICEATM prefers that data...

  2. Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

    Science.gov (United States)

    Cizmecioglu, Onur; Ni, Jing; Xie, Shaozhen; Zhao, Jean J; Roberts, Thomas M

    2016-01-01

    We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β. DOI: http://dx.doi.org/10.7554/eLife.17635.001 PMID:27700986

  3. 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.

  4. Gastroesophageal reflux in pregnancy: a systematic review on the benefit of raft forming agents.

    Science.gov (United States)

    Quartarone, G

    2013-10-01

    The prevalence of gastroesophageal reflux disease (GERD) symptoms in pregnancy is very high, up to 80%, with a maximum peak during the third trimester. Together with lifestyle modifications, antacids and antisecretive agents, such as proton pump inhibitors (PPIs) and histamine H2 receptor antagonists (H2RAs), are commonly prescribed in non-pregnant, adult population. In certain Countries these drugs are not allowed in or are allowed only during the late stages of pregnancy. Alginate-based formulations have been used for the symptomatic treatment of heartburn for decades, as they usually contain sodium or potassium bicarbonate. In the presence of gastric acid, a foamy raft is created above the gastric contents. The alginate raft moves into the esophagus in place or ahead of acidic gastric contents during reflux episodes physically preventing reflux of gastric contents into the esophagus. Alginate-based formulations are allowed with no restrictions also in pregnancy: their safety profile make them a very valid option taking into account the risk/benefit ratio for both parturient and unborn baby. This systematic review paper aims to explore the use of medications for treating GERD in pregnancy, including alginate raft-forming-agents, highlighting the benefits for both the mother and the fetus. Electronic search in databases was conducted on databases such as Medline, PubMed, Ovid retrieving data concerning the reflux treatments in pregnancy, with a special focus on alginate raft forming antireflux agents. From the literature on alginate use in pregnancy, no particular risks have been shown to date for both parturient and unborn baby when alginate had been administered during all the pregnancy trimesters. The physical mode of action ensures the maximum esophageal protection by the neutral foam floating in the stomach, maintaining physiological pH values at stomach level, without interfering with the digestive processes. The symptoms' healing has been markedly improved

  5. 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 ℃.

  6. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts

    Science.gov (United States)

    Xu, Congfeng; Zhang, Yanhui H.; Thangavel, Muthusamy; Richardson, Mekel M.; Liu, Li; Zhou, Bin; Zheng, Yi; Ostrom, Rennolds S.; Zhang, Xin A.

    2009-01-01

    Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.—Xu, C., Zhang, Y. H., Thangavel, M., Richardson, M. M., Liu, L., Zhou, B., Zheng, Y., Ostrom, R. S., Zhang, X. A. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts. PMID:19497983

  7. PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of posttraumatic epilepsy

    Science.gov (United States)

    Berdichevsky, Yevgeny; Dryer, Alexandra M.; Saponjian, Yero; Mahoney, Mark M.; Pimentel, Corrin A.; Lucini, Corrina A.; Usenovic, Marija; Staley, Kevin J.

    2013-01-01

    mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with LDH release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well-correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are: (1), the organotypic hippocampal culture model of posttraumatic epilepsy comprises a rapid assay of antiepileptogenic and neuroprotective activities and, in this model (2), mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy. PMID:23699517

  8. A Self-Reporting Photocatalyst for Online Fluorescence Monitoring of High Throughput RAFT Polymerization.

    Science.gov (United States)

    Yeow, Jonathan; Joshi, Sanket; Chapman, Robert; Boyer, Cyrille Andre Jean Marie

    2018-04-25

    Translating controlled/living radical polymerization (CLRP) from batch to the high throughput production of polymer libraries presents several challenges in terms of both polymer synthesis and characterization. Although recently there have been significant advances in the field of low volume, high throughput CLRP, techniques able to simultaneously monitor multiple polymerizations in an "online" manner have not yet been developed. Here, we report our discovery that 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) is a self-reporting photocatalyst that can mediate PET-RAFT polymerization as well as report on monomer conversion via changes in its fluorescence properties. This enables the use of a microplate reader to conduct high throughput "online" monitoring of PET-RAFT polymerizations performed directly in 384-well, low volume microtiter plates. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Ab-Initio-Based Kinetic Modeling to Understand RAFT Exchange: The Case of 2-Cyano-2-Propyl Dodecyl Trithiocarbonate and Styrene.

    Science.gov (United States)

    Desmet, Gilles B; De Rybel, Nils; Van Steenberge, Paul H M; D'hooge, Dagmar R; Reyniers, Marie-Françoise; Marin, Guy B

    2018-01-01

    Ab-initio-calculated rate coefficients for addition and fragmentation in reversible-addition fragmentation chain transfer (RAFT) polymerization of styrene with 2-cyano-2-propyl dodecyl trithiocarbonate initiated by azobisisobutyronitrile allow the reliable simulation of the experimentally observed conversion, number average chain length, and dispersity. The rate coefficient for addition of a macroradical R i to the macroRAFT agent R i X at 333 K (6.8 10 4 L mol -1 s -1 ) is significantly lower than to the initial RAFT agent R 0 X (3.2 10 6 L mol -1 s -1 ), mainly due to a difference in activation energy (15.4 vs 3.0 kJ mol -1 ), which causes the dispersity to spike in the beginning of the polymerization. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 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...

  11. Fabrication of high-capacity polyelectrolyte brush-grafted porous AAO-silica composite membrane via RAFT polymerization.

    Science.gov (United States)

    Song, Cunfeng; Wang, Meijie; Liu, Xin; Wang, He; Chen, Xiaoling; Dai, Lizong

    2017-09-01

    Surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization has been utilized to fabricate high-capacity strong anion-exchange (AEX) membrane for the separation of protein. By means of RAFT polymerization, quaternized poly(3-(methacrylamidomethyl)-pyridine) brushes formed 3-dimensional nanolayers on the surface of porous anodic aluminum oxide (AAO)-silica composite membrane. The surface properties of the membranes were analyzed by SEM, water contact angle, ATR-FTIR, XPS and TGA. To investigate the adsorption performance, the new AEX membranes were applied to recover a model protein, ovalbumin (OVA). High adsorption capacities of 95.8mg/g membranes (static) and 65.3mg/g membranes (dynamic) were obtained at ambient temperature. In the further studies, up to 90% of the adsorbed OVA was efficiently eluted by using phosphate buffer-1M NaCl as elution medium. The successful separation of OVA with high purity from a mixture protein solution was also achieved by using the AEX membranes. The present study demonstrated that under mild reaction condition, RAFT polymerization can be used to fabricate ion-exchange membrane which has many remarkable features, such as high capacity and selectivity, easy elution and so on. Copyright © 2017. Published by Elsevier B.V.

  12. Self-catalyzed photo-initiated RAFT polymerization for fabrication of fluorescent polymeric nanoparticles with aggregation-induced emission feature.

    Science.gov (United States)

    Zeng, Guangjian; Liu, Meiying; Jiang, Ruming; Huang, Qiang; Huang, Long; Wan, Qing; Dai, Yanfeng; Wen, Yuanqing; Zhang, Xiaoyong; Wei, Yen

    2018-02-01

    In recent years, the fluorescent polymeric nanoparticles (FPNs) with aggregation-induced emission (AIE) feature have been extensively exploited in various biomedical fields owing to their advantages, such as low toxicity, biodegradation, excellent biocompatibility, good designability and optical properties. Therefore, development of a facile, efficient and well designable strategy should be of great importance for the biomedical applications of these AIE-active FPNs. In this work, a novel method for the fabrication of AIE-active FPNs has been developed through the self-catalyzed photo-initiated reversible addition fragmentation chain transfer (RAFT) polymerization using an AIE dye containing chain transfer agent (CTA), which could initiate the RAFT polymerization under light irradiation. The results suggested that the final AIE-active FPNs (named as TPE-poly(St-PEGMA)) showed great potential for biomedical applications owing to their optical and biological properties. More importantly, the method described in the work is rather simple and effective and can be further extended to prepare many other different AIE-active FPNs owing to the good monomer adoptability of RAFT polymerization. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

    Directory of Open Access Journals (Sweden)

    George A. Robinson

    2017-11-01

    Full Text Available It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β, and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed.

  14. Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms

    DEFF Research Database (Denmark)

    Phillips, Wiktor S; Herly, Mikkel; Del Negro, Christopher A

    2016-01-01

    containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brainstem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of 7-43 days in vitro......Acute brainstem slice preparations in vitro have advanced understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. Here, we developed an organotypic slice culture preparation...... of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared to acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended...

  15. Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1α-induced invasion of human esophageal carcinoma cells.

    Science.gov (United States)

    Lin, Meng-Liang; Lu, Yao-Cheng; Chen, Hung-Yi; Lee, Chuan-Chun; Chung, Jing-Gung; Chen, Shih-Shun

    2014-05-01

    Stromal cell-derived factor-1α (SDF-1α) is a ligand for C-X-C chemokine receptor type 4 (CXCR4), which contributes to the metastasis of cancer cells by promoting cell migration. Here, we show that the SDF-1α/CXCR4 axis can significantly increase invasion of esophageal carcinoma (EC) cells. We accomplished this by examining the effects of CXCR4 knockdown as well as treatment with a CXCR4-neutralizing antibody and the CXCR4-specific inhibitor AMD3100. Curcumin suppressed SDF-1α-induced cell invasion and matrix metalloproteinase-2 (MMP-2) promoter activity, cell surface localization of CXCR4 at lipid rafts, and lipid raft-associated ras-related C3 botulinum toxin substrate 1 (Rac1)/phosphatidylinositol 3-kinase (PI3K) p85α/Akt signaling. Curcumin inhibited SDF-1α-induced cell invasion by suppressing the Rac1-PI3K signaling complex at lipid rafts but did not abrogate lipid raft formation. We further demonstrate that the attenuation of lipid raft-associated Rac1 activity by curcumin was critical for the inhibition of SDF-1α-induced PI3K/Akt/NF-κB activation, cell surface localization of CXCR4 at lipid rafts, MMP-2 promoter activity, and cell invasion. Collectively, our results indicate that curcumin inhibits SDF-1α-induced EC cell invasion by suppressing the formation of the lipid raft-associated Rac1-PI3K-Akt signaling complex, the localization of CXCR4 with lipid rafts at the cell surface, and MMP-2 promoter activity, likely through the inhibition of Rac1 activity. © 2012 Wiley Periodicals, Inc.

  16. Preparation of thermo-responsive graft copolymer by using a novel macro-RAFT agent and its application for drug delivery

    International Nuclear Information System (INIS)

    Song, Cunfeng; Yu, Shirong; Liu, Cheng; Deng, Yuanming; Xu, Yiting; Chen, Xiaoling; Dai, Lizong

    2016-01-01

    A methodology to prepare thermo-responsive graft copolymer by using a novel macro-RAFT agent was proposed. The macro-RAFT agent with pendant dithioester (ZC(S)SR) was facilely prepared via the combination of RAFT polymerization and esterification reaction. By means of ZC(S)SR-initiated RAFT polymerization, the thermo-responsive graft copolymer consisting of poly(methyl methacrylate-co-hydroxylethyl methacrylate) (P(MMA-co-HEMA)) backbone and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) side chains was constructed through the “grafting from” approach. The chemical compositions and molecular weight distributions of the synthesized polymers were respectively characterized by "1H nuclear magnetic resonance ("1H NMR) and gel permeation chromatography (GPC). Self-assembly behavior of the amphiphilic graft copolymers (P(MMA-co-HEMA)-g-PNIPAAm) was studied by transmission electron microscopy (TEM), dynamic light scattering (DLS) and spectrofluorimeter. The critical micelle concentration (CMC) value was 0.052 mg mL"−"1. These micelles have thermo-responsibility and a low critical solution temperature (LCST) of 33.5 °C. Further investigation indicated that the guest molecule release property of these micelles, which can be well described by a first-order kinetic model, was significantly affected by temperature. Besides, the micelles exhibited excellent biocompatibility and cellular uptake property. Hence, these micelles are considered to have potential application in controlled drug delivery. - Highlights: • A novel macro-RAFT agent with ZC(S)SR was used for preparing graft copolymer. • P(MMA-co-HEMA)-g-PNIPAAm was successful prepared via the “grafting from” approach. • Thermo-responsibility of the P(MMA-co-HEMA)-g-PNIPAAm micelles was investigated. • The drug release behavior of the P(MMA-co-HEMA)-g-PNIPAAm micelles was studied. • These micelles exhibited excellent biocompatibility and cellular uptake property.

  17. Preparation of thermo-responsive graft copolymer by using a novel macro-RAFT agent and its application for drug delivery

    Energy Technology Data Exchange (ETDEWEB)

    Song, Cunfeng; Yu, Shirong [Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005 (China); Liu, Cheng; Deng, Yuanming; Xu, Yiting [Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005 (China); Fujian Provincial Key Laboratory of Fire Retardant Materials, Xiamen University, Xiamen 361005 (China); Chen, Xiaoling, E-mail: tinachen0628@163.com [Department of Endodontics, Xiamen Stomatology Hospital, Teaching Hospital of Fujian Medical University, Xiamen 361003 (China); Dai, Lizong, E-mail: lzdai@xmu.edu.cn [Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005 (China); Fujian Provincial Key Laboratory of Fire Retardant Materials, Xiamen University, Xiamen 361005 (China)

    2016-05-01

    A methodology to prepare thermo-responsive graft copolymer by using a novel macro-RAFT agent was proposed. The macro-RAFT agent with pendant dithioester (ZC(S)SR) was facilely prepared via the combination of RAFT polymerization and esterification reaction. By means of ZC(S)SR-initiated RAFT polymerization, the thermo-responsive graft copolymer consisting of poly(methyl methacrylate-co-hydroxylethyl methacrylate) (P(MMA-co-HEMA)) backbone and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) side chains was constructed through the “grafting from” approach. The chemical compositions and molecular weight distributions of the synthesized polymers were respectively characterized by {sup 1}H nuclear magnetic resonance ({sup 1}H NMR) and gel permeation chromatography (GPC). Self-assembly behavior of the amphiphilic graft copolymers (P(MMA-co-HEMA)-g-PNIPAAm) was studied by transmission electron microscopy (TEM), dynamic light scattering (DLS) and spectrofluorimeter. The critical micelle concentration (CMC) value was 0.052 mg mL{sup −1}. These micelles have thermo-responsibility and a low critical solution temperature (LCST) of 33.5 °C. Further investigation indicated that the guest molecule release property of these micelles, which can be well described by a first-order kinetic model, was significantly affected by temperature. Besides, the micelles exhibited excellent biocompatibility and cellular uptake property. Hence, these micelles are considered to have potential application in controlled drug delivery. - Highlights: • A novel macro-RAFT agent with ZC(S)SR was used for preparing graft copolymer. • P(MMA-co-HEMA)-g-PNIPAAm was successful prepared via the “grafting from” approach. • Thermo-responsibility of the P(MMA-co-HEMA)-g-PNIPAAm micelles was investigated. • The drug release behavior of the P(MMA-co-HEMA)-g-PNIPAAm micelles was studied. • These micelles exhibited excellent biocompatibility and cellular uptake property.

  18. 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...

  19. Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvβ3 targeting using single photon emission computed tomography and optical imaging.

    Science.gov (United States)

    Briat, Arnaud; Wenk, Christiane H F; Ahmadi, Mitra; Claron, Michael; Boturyn, Didier; Josserand, Véronique; Dumy, Pascal; Fagret, Daniel; Coll, Jean-Luc; Ghezzi, Catherine; Sancey, Lucie; Vuillez, Jean-Philippe

    2012-06-01

    Integrin α(v)β(3) expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin α(v)β(3) in vitro and in vivo. When labeled with indium-111, the RAFT-RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT-RGD renal retention in tumor-bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT-RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of (111)In-DOTA-RAFT-RGD and A700-RAFT-RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. © 2012 Japanese Cancer Association.

  20. Preparation of thermo-responsive graft copolymer by using a novel macro-RAFT agent and its application for drug delivery.

    Science.gov (United States)

    Song, Cunfeng; Yu, Shirong; Liu, Cheng; Deng, Yuanming; Xu, Yiting; Chen, Xiaoling; Dai, Lizong

    2016-05-01

    A methodology to prepare thermo-responsive graft copolymer by using a novel macro-RAFT agent was proposed. The macro-RAFT agent with pendant dithioester (ZC(S)SR) was facilely prepared via the combination of RAFT polymerization and esterification reaction. By means of ZC(S)SR-initiated RAFT polymerization, the thermo-responsive graft copolymer consisting of poly(methyl methacrylate-co-hydroxylethyl methacrylate) (P(MMA-co-HEMA)) backbone and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) side chains was constructed through the "grafting from" approach. The chemical compositions and molecular weight distributions of the synthesized polymers were respectively characterized by (1)H nuclear magnetic resonance ((1)H NMR) and gel permeation chromatography (GPC). Self-assembly behavior of the amphiphilic graft copolymers (P(MMA-co-HEMA)-g-PNIPAAm) was studied by transmission electron microscopy (TEM), dynamic light scattering (DLS) and spectrofluorimeter. The critical micelle concentration (CMC) value was 0.052 mg mL(-1). These micelles have thermo-responsibility and a low critical solution temperature (LCST) of 33.5°C. Further investigation indicated that the guest molecule release property of these micelles, which can be well described by a first-order kinetic model, was significantly affected by temperature. Besides, the micelles exhibited excellent biocompatibility and cellular uptake property. Hence, these micelles are considered to have potential application in controlled drug delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.

    Science.gov (United States)

    Berdichevsky, Yevgeny; Dryer, Alexandra M; Saponjian, Yero; Mahoney, Mark M; Pimentel, Corrin A; Lucini, Corrina A; Usenovic, Marija; Staley, Kevin J

    2013-05-22

    mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an anti-epileptogenic, or merely anticonvulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long-term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with lactate dehydrogenase (LDH) release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are as follows: (1) the organotypic hippocampal culture model of post-traumatic epilepsy comprises a rapid assay of anti-epileptogenic and neuroprotective activities and, in this model (2) mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.

  2. Organotypic cultures as tools for testing neuroactive drugs - link between in-vitro and in-vivo experiments.

    Science.gov (United States)

    Drexler, B; Hentschke, H; Antkowiak, B; Grasshoff, C

    2010-01-01

    The development of neuroactive drugs is a time consuming procedure. Candidate drugs must be run through a battery of tests, including receptor studies and behavioural tests on animals. As a rule, numerous substances with promising properties as assessed in receptor studies must be eliminated from the development pipeline in advanced test phases because of unforeseen problems like intolerable side-effects or unsatisfactory performance in the whole organism. Clearly, test systems of intermediate complexity would alleviate this inefficiency. In this review, we propose cultured organotypic brain slices as model systems that could bridge the 'interpolation gap' between receptors and the brain, with a focus on the development of new general anaesthetics with lesser side effects. General anaesthesia is based on the modulation of neurotransmitter receptors and other conductances located on neurons in diverse brain regions, including cerebral cortex and spinal cord. It is well known that different components of general anaesthesia, e.g. hypnosis and immobility, are produced by the depression of neuronal activity in distinct brain regions. The ventral horn of the spinal cord is an important structure for the induction of immobility. Thus, the potentially immobilizing effects of a newly designed drug can be estimated from its depressant effect on neuronal network activity in cultured spinal slices. A drug's sedative and hypnotic potential can be examined in cortical cultures. Combined with genetically engineered mice, this approach can point to receptor subtypes most relevant to the drug's intended net effect and in return can help in the design of more selective drugs. In conclusion, the use of organotypic cultures permits predictions of neuroactive properties of newly designed drugs on an intermediate level, and should therefore open up avenues for a more creative and economic drug development process.

  3. Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

    DEFF Research Database (Denmark)

    Lambert, S.; Ameels, H.; Gniadecki, R.

    2008-01-01

    The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internal......The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo...... internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests...... the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption Udgivelsesdato: 2008/12...

  4. Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.

    Science.gov (United States)

    Noël, Geoffroy; Tham, Daniel Kai Long; Moukhles, Hakima

    2009-07-17

    Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

  5. 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 ...

  6. Amine functionalization of cellulose surface grafted with glycidyl methacrylate by γ-initiated RAFT polymerization

    International Nuclear Information System (INIS)

    Barsbay, Murat; Güven, Olgun; Kodama, Yasko

    2016-01-01

    This study presents the functionalization of poly(glycidyl methacrylate) (PGMA) grafted cellulose filter paper by a model compound, ethylenediamine (EDA), through the epoxy groups of PGMA. Cellulose based copolymers were prepared via the radiation-induced and RAFT-mediated graft polymerization. The samples were characterized by ATR–FTIR spectroscopy, X-ray photoelectron spectroscopy (XPS), elemental analysis, contact angle measurements and scanning electron microscopy (SEM). An efficient modification density of around 1 mmol EDA/mg copolymer was attained within ca. 8 h, indicating that chemical composition of well-defined copolymers may further be tuned by appropriately selecting the reactive agents for use in many emerging fields. - Highlights: • Ethylenediamine (EDA) was immobilized to cellulose-g-PGMA copolymers. • FTIR, XPS, SEM, EA and CA measurements were used for characterization. • The useful qualities of the RAFT were combined with the versatility of PGMA.

  7. Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

    Science.gov (United States)

    Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

    2014-05-02

    The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

  8. 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

  9. Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts

    Directory of Open Access Journals (Sweden)

    Marco Emanuele

    2016-05-01

    Full Text Available Alpha-synuclein (αSyn interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

  10. Helium isotopes in geothermal systems: Iceland, The Geysers, Raft River and Steamboat Springs

    International Nuclear Information System (INIS)

    Torgersen, T.

    1982-01-01

    Helium isotope ratios have been measured in geothermal fluids from Iceland, The Geysers, Raft River, Steamboat Springs and Hawaii. These ratios have been interpreted in terms of the processes which supply He in distinct isotopic ratios and in terms of the processes which can alter the isotopic ratio. Using this interpretational scheme, Iceland is found to be an area of hot-spot magmatic He implying an active volcanic source although the data are suggestive of high-temperature weathering release of crustal He incorporated in the geothermal fluids. By comparison to fumarolic gases from Hawaii and Juan De Fuca and Cayman Trench basaltic glass samples, The Geysers contains MOR type magmatic He again implying an active volcanic source possibly a 'leaky' transform related to the San Andreas Fault System. Raft River contains only crustal He indicating no active volcanic sources. Steamboat Springs He isotope ratios are distinctly less than typical plate margin volcanics but must still have a magmatic source. (author)

  11. Controlled radical copolymerization of styrene and maleic anhydride and the synthesis of novel polyolefin-based block copolymers by reversible addition-fragmentation chain-transfer (RAFT) polymerization

    NARCIS (Netherlands)

    Brouwer, de J.A.M.; Schellekens, M.A.J.; Klumperman, B.; Monteiro, M.J.; German, A.L.

    2000-01-01

    Reversible addn.-fragmentation chain transfer (RAFT) was applied to the copolymn. of styrene and maleic anhydride. The product had a low polydispersity and a predetd. molar mass. Novel, well-defined polyolefin-based block copolymers were prepd. with a macromol. RAFT agent prepd. from a com.

  12. Reactor building with internal structure of which the movements are independent of those of the general raft and process for building these internal structures

    International Nuclear Information System (INIS)

    Hista, J.C.

    1982-01-01

    This reactor building includes a containment enclosure for the internal structures composed of a slab wedged on its periphery against the containment enclosure gusset and resting on the general raft by means of a peripheral bearing ring, a compressible layer being provided between the general raft and the slab [fr

  13. The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

    Science.gov (United States)

    Petruk, Ariel Alcides; Varriale, Sonia; Coscia, Maria Rosaria; Mazzarella, Lelio; Merlino, Antonello; Oreste, Umberto

    2013-11-01

    Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction. © 2013.

  14. In Situ Monitoring of RAFT Polymerization by Tetraphenylethylene-Containing Agents with Aggregation-Induced Emission Characteristics.

    Science.gov (United States)

    Liu, Shunjie; Cheng, Yanhua; Zhang, Haoke; Qiu, Zijie; Kwok, Ryan T K; Lam, Jacky W Y; Tang, Ben Zhong

    2018-05-22

    A facile and efficient approach is demonstrated to visualize the polymerization in situ. A group of tetraphenylethylene (TPE)-containing dithiocarbamates were synthesized and screened as agents for reversible addition fragmentation chain transfer (RAFT) polymerizations. The spatial-temporal control characteristics of photochemistry enabled the RAFT polymerizations to be ON and OFF on demand under alternating visible light irradiation. The emission of TPE is sensitive to the local viscosity change owing to its aggregation-induced emission characteristic. Quantitative information could be easily acquired by the naked eye without destroying the reaction system. Furthermore, the versatility of such a technique was well demonstrated by 12 different polymerization systems. The present approach thus demonstrated a powerful platform for understanding the controlled living radical polymerization process. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A novel method for pulmonary research: Assessment of bioenergetic function at the air–liquid interface

    Directory of Open Access Journals (Sweden)

    Weiling Xu

    2014-01-01

    Full Text Available Air–liquid interface cell culture is an organotypic model for study of differentiated functional airway epithelium in vitro. Dysregulation of cellular energy metabolism and mitochondrial function have been suggested to contribute to airway diseases. However, there is currently no established method to determine oxygen consumption and glycolysis in airway epithelium in air–liquid interface. In order to study metabolism in differentiated airway epithelial cells, we engineered an insert for the Seahorse XF24 Analyzer that enabled the measure of respiration by oxygen consumption rate (OCR and glycolysis by extracellular acidification rate (ECAR. Oxidative metabolism and glycolysis in airway epithelial cells cultured on the inserts were successfully measured. The inserts did not affect the measures of OCR or ECAR. Cells under media with apical and basolateral feeding had less oxidative metabolism as compared to cells on the inserts at air-interface with basolateral feeding. The design of inserts that can be used in the measure of bioenergetics in small numbers of cells in an organotypic state may be useful for evaluation of new drugs and metabolic mechanisms that underlie airway diseases.

  16. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

    Science.gov (United States)

    Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

    2015-07-07

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Fluidic system for long-term in vitro culturing and monitoring of organotypic brain slices

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Troels-Smith, Ane R.; Dimaki, Maria

    2015-01-01

    Brain slice preparations cultured in vitro have long been used as a simplified model for studying brain development, electrophysiology, neurodegeneration and neuroprotection. In this paper an open fluidic system developed for improved long term culturing of organotypic brain slices is presented....... The positive effect of continuous flow of growth medium, and thus stability of the glucose concentration and waste removal, is simulated and compared to the effect of stagnant medium that is most often used in tissue culturing. Furthermore, placement of the tissue slices in the developed device was studied...... by numerical simulations in order to optimize the nutrient distribution. The device was tested by culturing transverse hippocampal slices from 7 days old NMRI mice for a duration of 14 days. The slices were inspected visually and the slices cultured in the fluidic system appeared to have preserved...

  18. RAFT Aqueous Dispersion Polymerization of N-(2-(Methacryloyloxy)ethyl)pyrrolidone: A Convenient Low Viscosity Route to High Molecular Weight Water-Soluble Copolymers.

    Science.gov (United States)

    Cunningham, Victoria J; Derry, Matthew J; Fielding, Lee A; Musa, Osama M; Armes, Steven P

    2016-06-28

    RAFT solution polymerization of N -(2-(methacryoyloxy)ethyl)pyrrolidone (NMEP) in ethanol at 70 °C was conducted to produce a series of PNMEP homopolymers with mean degrees of polymerization (DP) varying from 31 to 467. Turbidimetry was used to assess their inverse temperature solubility behavior in dilute aqueous solution, with an LCST of approximately 55 °C being observed in the high molecular weight limit. Then a poly(glycerol monomethacylate) (PGMA) macro-CTA with a mean DP of 63 was chain-extended with NMEP using a RAFT aqueous dispersion polymerization formulation at 70 °C. The target PNMEP DP was systematically varied from 100 up to 6000 to generate a series of PGMA 63 -PNMEP x diblock copolymers. High conversions (≥92%) could be achieved when targeting up to x = 5000. GPC analysis confirmed high blocking efficiencies and a linear evolution in M n with increasing PNMEP DP. A gradual increase in M w / M n was also observed when targeting higher DPs. However, this problem could be minimized ( M w / M n RAFT aqueous dispersion polymerization of NMEP was approximately four times faster than the RAFT solution polymerization of NMEP in ethanol when targeting the same DP in each case. This is perhaps surprising because both 1 H NMR and SAXS studies indicate that the core-forming PNMEP chains remain relatively solvated at 70 °C in the latter formulation. Moreover, dissolution of the initial PGMA 63 -PNMEP x particles occurs on cooling from 70 to 20 °C as the PNMEP block passes through its LCST. Hence this RAFT aqueous dispersion polymerization formulation offers an efficient route to a high molecular weight water-soluble polymer in a rather convenient low-viscosity form. Finally, the relatively expensive PGMA macro-CTA was replaced with a poly(methacrylic acid) (PMAA) macro-CTA. High conversions were also achieved for PMAA 85 -PNMEP x diblock copolymers prepared via RAFT aqueous dispersion polymerization for x ≤ 4000. Again, better control was achieved when

  19. 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

  20. The RAFT Telemedicine Network: Lessons Learnt and Perspectives from a Decade of Educational and Clinical Services in Low- and Middle-Incomes Countries.

    Science.gov (United States)

    Bediang, Georges; Perrin, Caroline; Ruiz de Castañeda, Rafael; Kamga, Yannick; Sawadogo, Alexandre; Bagayoko, Cheick Oumar; Geissbuhler, Antoine

    2014-01-01

    The objectives of this paper are to (i) provide an overview of the educational and clinical experiences of the Réseau en Afrique Francophone pour la Télémédecine (RAFT) network, (ii) analyze key challenges and lessons learnt throughout a decade of activity, and (iii) draw a vision and perspectives of its sustainability. The study was carried out following three main stages: (i) a literature review, (ii) the analysis of key documents, and (iii) discussions with key collaborators of the RAFT. Réseau en Afrique Francophone pour la Télémédecine has been offering an important quantity of educational, clinical, and public health activities during the last decade. The educational activities include the weekly delivery of video-lectures for continuing and post-graduate medical education, the use of virtual patients for training in clinical decision making, research training activities using ICTs and other e-learning activities. The clinical and public health activities include tele-expertise to support health professionals in the management of difficult clinical cases, the implementation of clinical information systems in African hospitals, the deployment of mHealth projects, etc. Since 2010, the RAFT has been extended to the Altiplano in Bolivia and Nepal (in progress). Lessons Learnt and Perspectives: Important lessons have been learnt from the accumulated experiences throughout these years. These lessons concern: social and organization, human resources, technologies and data security, policy and legislation, and economy and financing. Also, given the increase of the activities and the integration of eHealth and telemedicine in the health system of most of the countries, the RAFT network faces many other challenges and perspectives such as learning throughout life, recognition, and valorization of teaching or learning activities, the impact evaluation of interventions, and the scaling up and transferability out of Africa of RAFT activities. Based on the RAFT

  1. 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

  2. Fluorescent Labeling and Biodistribution of Latex Nanoparticles Formed by Surfactant-Free RAFT Emulsion Polymerization.

    Science.gov (United States)

    Poon, Cheuk Ka; Tang, Owen; Chen, Xin-Ming; Kim, Byung; Hartlieb, Matthias; Pollock, Carol A; Hawkett, Brian S; Perrier, Sébastien

    2017-10-01

    The authors report the preparation of a novel range of functional polyacrylamide stabilized polystyrene nanoparticles, obtained by surfactant-free reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization, their fluorescent tagging, cellular uptake, and biodistribution. The authors show the versatility of the RAFT emulsion process for the design of functional nanoparticles of well-defined size that can be used as drug delivery vectors. Functionalization with a fluorescent tag offers a useful visualization tool for tracing, localization, and clearance studies of these carriers in biological models. The studies are carried out by labeling the sterically stabilized latex particles chemically with rhodamine B. The fluorescent particles are incubated in a healthy human renal proximal tubular cell line model, and intravenously injected into a mouse model. Cellular localization and biodistribution of these particles on the biological models are explored. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

    Directory of Open Access Journals (Sweden)

    Naoki Takizawa

    2016-09-01

    Full Text Available The influenza glycoproteins, hemagglutinin (HA and neuraminidase (NA, which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1 in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

  4. Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

    Science.gov (United States)

    Takizawa, Naoki; Momose, Fumitaka; Morikawa, Yuko; Nomoto, Akio

    2016-09-10

    The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

  5. Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase

    DEFF Research Database (Denmark)

    Bhartia, Tripta; Cornelius, Flemming; Ipsen, J. H.

    2016-01-01

    The structure of functional lipid domains (rafts) in biological membranes has for long time been unresolved due to their small length scales and transient nature. These cooperative properties of the lipid bilayer matrix are modelled by free-standing giant unilammellar vesicles (GUVs) with well...... mixtures of DOPC/DPPC/cholesterol with and without Na,K-ATPase (NKA), a transmembrane protein known to be associated with rafts. Two mechanisms of domain formation are revealed: 1) close to lo/ld phase coexistence, domains in size up to 100 nm appear as thermally induced droplet fluctuations, 2) NKA shows...... interfacial activity and cluster in lo/ld micro-emulsion droplets. Some perspectives for the application of the techniques and the understanding of the nature of raft domains are outlined....

  6. Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi

    Czech Academy of Sciences Publication Activity Database

    Malínský, Jan; Opekarová, Miroslava; Grossmann, G.; Tanner, W.

    2013-01-01

    Roč. 64, April (2013), s. 501-529 ISSN 1543-5008 R&D Projects: GA ČR(CZ) GAP302/11/0146; GA ČR GAP205/12/0720 Institutional research plan: CEZ:AV0Z50390703 Institutional support: RVO:61388971 ; RVO:68378041 Keywords : membrane microdomain * lipid raft * detergent resistant membranes Subject RIV: EB - Genetics ; Molecular Biology; EA - Cell Biology (MBU-M) Impact factor: 18.900, year: 2013

  7. Mural cell associated VEGF is required for organotypic vessel formation.

    Directory of Open Access Journals (Sweden)

    Lasse Evensen

    Full Text Available BACKGROUND: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. METHODS AND FINDINGS: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. CONCLUSIONS: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.

  8. Ototoxicity of paclitaxel in rat cochlear organotypic cultures

    International Nuclear Information System (INIS)

    Dong, Yang; Ding, Dalian; Jiang, Haiyan; Shi, Jian-rong; Salvi, Richard; Roth, Jerome A.

    2014-01-01

    Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24 h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea. - Highlights: • Paclitaxel was toxic to cochlear hair cells and spiral ganglion neurons. • Paclitaxel-induced spiral ganglion degeneration was apoptotic. • Paclitaxel activated caspase-6, -8 and -8 in spiral ganglion neurons

  9. Ototoxicity of paclitaxel in rat cochlear organotypic cultures

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yang [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Ding, Dalian; Jiang, Haiyan [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Shi, Jian-rong [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Salvi, Richard [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Roth, Jerome A., E-mail: jaroth@buffalo.edu [Department of Pharmacology and Toxicology, University at Buffalo, NY 14214 (United States)

    2014-11-01

    Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24 h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea. - Highlights: • Paclitaxel was toxic to cochlear hair cells and spiral ganglion neurons. • Paclitaxel-induced spiral ganglion degeneration was apoptotic. • Paclitaxel activated caspase-6, -8 and -8 in spiral ganglion neurons.

  10. Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice.

    Science.gov (United States)

    Staal, Jerome A; Alexander, Samuel R; Liu, Yao; Dickson, Tracey D; Vickers, James C

    2011-01-01

    Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented. In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4-6), adolescent animals (P25-28) and mature adults (P50+). Cultures were prepared using the membrane interface method. Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (pglial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers. In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.

  11. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Directory of Open Access Journals (Sweden)

    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  12. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Science.gov (United States)

    Martín, César; Uribe, Kepa B; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2011-02-23

    Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  13. Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-α

    Directory of Open Access Journals (Sweden)

    Scuderi Caterina

    2012-03-01

    Full Text Available Abstract Background In addition to cytotoxic mechanisms directly impacting neurons, β-amyloid (Aβ-induced glial activation also promotes release of proinflammatory molecules that may self-perpetuate reactive gliosis and damage neighbouring neurons, thus amplifying neuropathological lesions occurring in Alzheimer's disease (AD. Palmitoylethanolamide (PEA has been studied extensively for its anti-inflammatory, analgesic, antiepileptic and neuroprotective effects. PEA is a lipid messenger isolated from mammalian and vegetable tissues that mimics several endocannabinoid-driven actions, even though it does not bind to cannabinoid receptors. Some of its pharmacological properties are considered to be dependent on the expression of peroxisome proliferator-activated receptors-α (PPARα. Findings In the present study, we evaluated the effect of PEA on astrocyte activation and neuronal loss in models of Aβ neurotoxicity. To this purpose, primary rat mixed neuroglial co-cultures and organotypic hippocampal slices were challenged with Aβ1-42 and treated with PEA in the presence or absence of MK886 or GW9662, which are selective PPARα and PPARγ antagonists, respectively. The results indicate that PEA is able to blunt Aβ-induced astrocyte activation and, subsequently, to improve neuronal survival through selective PPARα activation. The data from organotypic cultures confirm that PEA anti-inflammatory properties implicate PPARα mediation and reveal that the reduction of reactive gliosis subsequently induces a marked rebound neuroprotective effect on neurons. Conclusions In line with our previous observations, the results of this study show that PEA treatment results in decreased numbers of infiltrating astrocytes during Aβ challenge, resulting in significant neuroprotection. PEA could thus represent a promising pharmacological tool because it is able to reduce Aβ-evoked neuroinflammation and attenuate its neurodegenerative consequences.

  14. Cox2 and β-Catenin/T-cell Factor Signaling Intestinalize Human Esophageal Keratinocytes When Cultured under Organotypic Conditions

    Directory of Open Access Journals (Sweden)

    Jianping Kong

    2011-09-01

    Full Text Available The incidence of esophageal adenocarcinoma (EAC is rising in the United States. An important risk factor for EAC is the presence of Barrett esophagus (BE. BE is the replacement of normal squamous esophageal epithelium with a specialized columnar epithelium in response to chronic acid and bile reflux. However, the emergence of BE from squamous keratinocytes has not yet been demonstrated. Our research has focused on this. Wnt and cyclooxygenase 2 (Cox2 are two pathways whose activation has been associated with BE and progression to EAC, but their role has not been tested experimentally. To explore their contribution, we engineered a human esophageal keratinocyte cell line to express either a dominant-active Wnt effector CatCLef or a Cox2 complementary DNA. In a two-dimensional culture environment, Cox2 expression increases cell proliferation and migration, but neither transgene induces known BE markers. In contrast, when these cells were placed into three-dimensional organotypic culture conditions, we observed more profound effects. CatCLef-expressing cells were more proliferative, developed a thicker epithelium, and upregulated Notch signaling and several BE markers including NHE2. Cox2 expression also increased cell proliferation and induced a thicker epithelium. More importantly, we observed cysts form within the epithelium, filled with intestinal mucins including Muc5B and Muc17. This suggests that Cox2 expression in a three-dimensional culture environment induces a lineage of mucin-secreting cells and supports an important causal role for Cox2 in BE pathogenesis. We conclude that in vitro modeling of BE pathogenesis can be improved by enhancing Wnt signaling and Cox2 activity and using three-dimensional organotypic culture conditions.

  15. Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture.

    Directory of Open Access Journals (Sweden)

    Qian Wu

    Full Text Available Tumor necrosis factor (TNF-α is a cytokine involved in acute inflammatory phase reactions, and is the primary upstream mediator in the cochlear inflammatory response. Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters' and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an in vitro model of inflammatory-induced ototoxicity.

  16. Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures

    Directory of Open Access Journals (Sweden)

    Liu Bob Y

    2007-02-01

    Full Text Available Abstract Background Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues. Results Here, we test the effect of ectopic Wnt1 expression on the behavior of interfollicular progenitor cells in an organotypic culture model, and find that Wnt1 signaling inhibits their growth and promotes terminal differentiation. Conclusion These results are consistent with the phenotypes reported for transgenic mice engineered to have gain or loss of function of Wnt signaling in skin, which would recommend our culture model as an accurate one for molecular analysis. Since it is known that canonical ligands are expressed in skin, it is likely that this pathway normally regulates the balance of growth and differentiation, and suggests it could be important to pathogenesis.

  17. Oxygenation and response to irradiation of organotypic multicellular spheroids of human glioma.

    Science.gov (United States)

    Sminia, Peter; Acker, Helmut; Eikesdal, Hans Petter; Kaaijk, Patricia; Enger, Per øvind; Slotman, Ben; Bjerkvig, Rolf

    2003-01-01

    Investigation of the oxygenation status of organotypic multicellular spheroids (OMS) and their response to irradiation. Tumour specimens of glioblastoma multiforme patients (n = 16) were initiated as OMS. Following 20 Gy gamma-irradiation, the cell migratory capacity was evaluated. Spheroid oxygenation was determined by micro-electrode pO2 measurements and pimonidazole immunostaining. Spheroids prepared from established human glioma cell lines were used as a reference. Irradiation inhibited spheroid outgrowth by 12 to 88% relative to the non-irradiated controls. A large interpatient variation was noticed. Oxygen measurements revealed a gradual decrease in pO2 level from the periphery to the core of the spheroids, but the pO2 values remained within an oxygenated range. However, in the cell line spheroids an intermediate layer of hypoxia surrounding the central core was observed. Cell line spheroids with a hypoxic cell fraction and well-oxygenated OMS both show high resistance to irradiation, indicating that hypoxia may not be the biological factor determining the radioresistance of glioma spheroids in vitro.

  18. Experimental study on the shear behavior of the interface between cushion materials and the concrete raft

    Science.gov (United States)

    Li, Yaokun; Han, Xiaolei; Galal, Khaled; Ji, Jing

    2018-01-01

    Cushion is a layer of granular materials between the raft and the ground. The shear behavior of the interface between the cushion and the raft may influence the seismic performance of the superstructure. In order to quantify such influences, horizontal shear tests on the interfaces between different cushion materials and concrete raft under monotonic and cyclic loading were carried out. The vertical pressure P v, material type and cushion thickness h c were taken as variables. Conclusions include: 1) under monotonic loading, P v is the most significant factor; the shear resistance P hmax increases as P v increases, but the normalized factor of resistance μ n has an opposite tendency; 2) for the materials used in this study, μ n varies from 0.40 to 0.70, the interface friction angle δ s varies from 20° to 35°, while u max varies from 3 mm to 15 mm; 3) under cyclic loading, the interface behavior can be abstracted as a "three-segment" back-bone curve, the main parameters include μ n, the displacement u 1 and stiffness K 1 of the elastic stage, the displacement u 2 and stiffness K 2 of the plastic stage; 4) by observation and statistical analysis, the significance of different factors, together with values of K 1, K 2 and μ n have been obtained.

  19. Preparation of Thermo-Responsive and Cross-Linked Fluorinated Nanoparticles via RAFT-Mediated Aqueous Polymerization in Nanoreactors.

    Science.gov (United States)

    Ma, Jiachen; Zhang, Luqing; Geng, Bing; Azhar, Umair; Xu, Anhou; Zhang, Shuxiang

    2017-01-25

    In this work, a thermo-responsive and cross-linked fluoropolymer poly(2,2,2-Trifluoroethyl) methacrylate (PTFEMA) was successfully prepared by reversible addition-fragmentation chain transfer (RAFT) mediated aqueous polymerization with a thermo-responsive diblock poly(dimethylacrylamide- b - N -isopropylacrylamide) (PDMA- b -PNIPAM) that performed a dual function as both a nanoreactor and macro-RAFT agent. The cross-linked polymer particles proved to be in a spherical-like structure of about 50 nm in diameter and with a relatively narrow particle size distribution. ¹H-NMR and 19 F-NMR spectra showed that thermo-responsive diblock P(DMA- b -NIPAM) and cross-linked PTFEMA particles were successfully synthesized. Influence of the amount of ammonium persulfate (APS), the molar ratio of monomers to RAFT agent, influence of the amount of cross-linker on aqueous polymerization and thermo-responsive characterization of the particles are investigated. Monomer conversion increased from 44% to 94% with increasing the molar ratio of APS and P(DMA- b -NIPAM) from 1:9 to1:3. As the reaction proceeded, the particle size increased from 29 to 49 nm due to the consumption of TFEMA monomer. The size of cross-linked nanoparticles sharply decreased from 50.3 to 40.5 nm over the temperature range 14-44 °C, suggesting good temperature sensitivity for these nanoparticles.

  20. 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...

  1. A grafting from approach to graft polystyrene chains at the surface of graphene nanolayers by RAFT polymerization: Various graft densities from hydroxyl groups

    Energy Technology Data Exchange (ETDEWEB)

    Roghani-Mamaqani, Hossein, E-mail: r.mamaghani@sut.ac.ir [Department of Polymer Engineering, Sahand University of Technology, PO Box 51335-1996, Tabriz (Iran, Islamic Republic of); Khezri, Khezrollah [School of Chemistry, University College of Science, University of Tehran, PO Box 14155-6455, Tehran (Iran, Islamic Republic of)

    2016-01-01

    Graphical abstract: (3-Aminopropyl) triethoxysilane was grafted at the surface of GO in low and high different graft densities to yield GOHAL and GOHAH, respectively. Subsequently, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (RA) was attached at the surface of GOHAL and GOHAH by an amidation reaction to yield GOHRL and GOHRH, respectively. Then, GOHRL and GOHRH were used in grafting from RAFT polymerization of styrene. - Highlights: • A RAFT agent grafted GO was used in grafting from RAFT polymerization of styrene. • The efficiency of RAFT agent attachment at the surface of GO is 41.12% for high density sample. • Polystyrene molecular weight is decreased by the addition of graphene content and also graft density of RAFT agent. - Abstract: (3-Aminopropyl) triethoxysilane was grafted at the surface of GO in low and high different graft densities to yield GOHAL and GOHAH, respectively. Subsequently, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (RA) was attached at the surface of GOHAL and GOHAH by an amidation reaction to yield GOHRL and GOHRH, respectively. Then, GOHRL and GOHRH were used in grafting from RAFT polymerization of styrene. Grafting of APTES and RA was approved by Fourier transform infrared spectroscopy, X-ray photo electron spectroscopy, and Raman spectroscopy. Expansion of graphene interlayer by oxidation and functionalization processes was evaluated by X-ray diffraction. Conversion values of styrene were calculated using gas chromatography. Molecular weight and PDI values of attached polystyrene (PS) chains were studied by size exclusion chromatography. Thermogravimetric analysis was also used to investigate the degradation temperatures, char contents, and graft contents of modifiers and PS chains. GOHRH and GOHRL reach to char content of 55.3 and 45.2% at 600 °C, which shows that weight ratio of modifier (APTES and RA moieties) is 15.3 and 5.2%, respectively. Scanning and transmission electron microscopies show that

  2. Phototherapeutic LASEK for a persistent epithelial defect and a recurrent epithelial erosion.

    Science.gov (United States)

    Hondur, Ahmet; Bilgihan, Kamil; Hasanreisoglu, Berati

    2005-01-01

    To present two patients, one with persistent epithelial defect and one with recurrent epithelial erosion, unresponsive to conventional therapy treated with phototherapeutic keratectomy (PTK) with the laser subepithelial keratomileusis (LASEK) technique (phototherapeutic LASEK). The epithelial flap was created following 18% ethanol application for 20 seconds. A 10-microm deep ablation was performed in the central 7.0-mm zone. A contact lens was placed and the patient examined daily until epithelial closure. Upon epithelial closure, the contact lens was removed. A mild topical steroid and artificial tears were applied for 2 weeks. The epithelium healed in 4 days in both patients. Patients reported only mild pain until epithelial closure. The manifest refraction and uncorrected visual acuity remained unchanged in both eyes. No haze was noted. The first patient has remained asymptomatic without any recurrence for 12 months, and the second for 9 months. Phototherapeutic LASEK provides a therapeutic option for refractory recurrent erosions and persistent epithelial defects, with the additional benefit of being less painful and less risky for haze development than conventional PTK.

  3. Absence of death receptor translocation into lipid rafts in acquired TRAIL-resistant NSCLC cells.

    Science.gov (United States)

    Ouyang, Wen; Yang, Chunxu; Zhang, Simin; Liu, Yu; Yang, Bo; Zhang, Junhong; Zhou, Fuxiang; Zhou, Yunfeng; Xie, Conghua

    2013-02-01

    Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a major limitation for its clinical use. The mechanisms of TRAIL resistance have been mostly studied in the context of cell lines that are intrinsically resistant to TRAIL. However, little is known about the molecular alterations that contribute to the development of acquired resistance during treatment with TRAIL. In this study, we established H460R, an isogenic cell line with acquired TRAIL resistance, from the TRAIL‑sensitive human lung cancer cell line H460 to investigate the mechanisms of acquired resistance. The acquired TRAIL‑resistant H460R cells remained sensitive to cisplatin. The mRNA and protein expression levels of death receptor 4 (DR4) and death receptor 5 (DR5) were not altered in either of the TRAIL-treated cell lines. Nevertheless, tests in which the DR4 or DR5 gene was overexpressed or silenced suggest that death receptor expression is necessary but not sufficient for TRAIL‑induced apoptosis. Compared with parental TRAIL-sensitive H460 cells, H460R cells showed a decreased TRAIL-induced translocation of DR4/DR5 into lipid rafts. Further studies showed that nystatin partially prevented lipid raft aggregation and DR4 and DR5 clustering and reduced apoptosis in H460 cells again. Analysis of apoptotic molecules showed that more pro-caspase-8, FADD, caspase-3 and Bid, but less cFLIP in H460 cells than in H460R cells. Our findings suggest that the lack of death receptor redistribution negatively impacts DISC assembly in lipid rafts, which at least partially leads to the development of acquired resistance to TRAIL in H460R cells.

  4. 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

  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. Formulation, release characteristics, and bioavailability study of gastroretentive floating matrix tablet and floating raft system of Mebeverine HCl.

    Science.gov (United States)

    El Nabarawi, Mohamed A; Teaima, Mahmoud H; Abd El-Monem, Rehab A; El Nabarawy, Nagla A; Gaber, Dalia A

    2017-01-01

    To prolong the residence time of dosage forms within the gastrointestinal tract until all drug is released at the desired rate is one of the real challenges for oral controlled-release drug delivery systems. This study was designed to develop a controlled-release floating matrix tablet and floating raft system of Mebeverine HCl (MbH) and evaluate different excipients for their floating behavior and in vitro controlled-release profiles. Oral pharmacokinetics of the optimum matrix tablet, raft system formula, and marketed Duspatalin ® 200 mg retard as reference were studied in beagle dogs. The optimized tablet formula (FT-10) and raft system formula (FRS-11) were found to float within 34±5 sec and 15±7 sec, respectively, and both remain buoyant over a period of 12 h in simulated gastric fluid. FT-10 (Compritol/HPMC K100M 1:1) showed the slowest drug release among all prepared tablet formulations, releasing about 80.2% of MbH over 8 h. In contrast, FRS-11 (Sodium alginate 3%/HPMC K100M 1%/Precirol 2%) had the greatest retardation, providing sustained release of 82.1% within 8 h. Compared with the marketed MbH product, the C max of FT-10 was almost the same, while FRS-11 maximum concentration was higher. The t max was 3.33, 2.167, and 3.0 h for marketed MbH product, FT-10, and FRS-11, respectively. In addition, the oral bioavailability experiment showed that the relative bioavailability of the MbH was 104.76 and 116.01% after oral administration of FT-10 and FRS-11, respectively, compared to marketed product. These results demonstrated that both controlled-released floating matrix tablet and raft system would be promising gastroretentive delivery systems for prolonging drug action.

  7. Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Adebiyi, Adebowale, E-mail: aadebiyi@uthsc.edu; Soni, Hitesh; John, Theresa A.; Yang, Fen

    2014-05-15

    Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub i}) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca{sup 2+}]{sub i} elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca{sup 2+}]{sub i} chelator; KN-93, a Ca{sup 2+}/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca{sup 2+}]{sub i}-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs. - Highlights: • AGTR1 is the functional AGTR subtype expressed in neonatal mesangial cells. • Endogenous AGTR1 associates with CAV-1 in neonatal mesangial cells. • Lipid raft disruption attenuates cell surface AGTR1 protein expression. • Lipid raft disruption reduces ANG-II-induced [Ca{sup 2+}]{sub i} elevation in neonatal mesangial cells. • Lipid raft disruption inhibits ANG-II-induced neonatal mesangial cell growth.

  8. Porphyromonas gingivalis Outer Membrane Vesicles Enter Human Epithelial Cells via an Endocytic Pathway and Are Sorted to Lysosomal Compartments ▿

    Science.gov (United States)

    Furuta, Nobumichi; Tsuda, Kayoko; Omori, Hiroko; Yoshimori, Tamotsu; Yoshimura, Fuminobu; Amano, Atsuo

    2009-01-01

    Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis. PMID:19651865

  9. 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…

  10. Saltatory conduction in unmyelinated axons: Clustering of Na+ channels on lipid rafts allows micro-saltatory conduction in C-fibers

    Directory of Open Access Journals (Sweden)

    Ali eNeishabouri

    2014-10-01

    Full Text Available The action potential (AP, the fundamental signal of the nervous system, is carried by two types of axons: unmyelinated and myelinated fibers. In the former the action potential propagates continuously along the axon as established in large-diameter fibers. In the latter axons the AP jumps along the Nodes of Ranvier – discrete, anatomically specialized regions which contain very high densities of sodium ion (Na + channels. Therefore saltatory conduction is thought as the hallmark of myelinated axons, which enables faster and more reliable propagation of signals than in unmyelinated axons of same outer diameter.Recent molecular anatomy showed that in C-fibers, the very thin (0.1 μm diameter axons of the peripheral nervous system, Nav1.8 channels are clustered together on lipid rafts that float in the cell membrane. This localized concentration of Na+ channels resembles in structure the ion channel organization at the Nodes of Ranvier, yet it is currently unknown whether this translates into equivalent phenomenon of saltatory conduction or related-functional benefits and efficiencies. Therefore, we modeled biophysically realistic unmyelinated axons with both conventional and lipid-raft based organization of Na+ channels. We find that action potentials are reliably conducted in a micro-saltatory fashion along lipid rafts.Comparing APs in unmyelinated fibers with and without lipid rafts did not reveal any significant difference in either the metabolic cost or AP propagation velocity. By investigating the efficiency of AP propagation over Nav1.8 channels, we find however that the specific inactivation properties of these channels significantly increase the metabolic cost of signaling in C-fibers.

  11. A simplified algorithm for the design of piled raft foundations applied for the case study of a building erected within Żoliborz-Szczęśliwice glacial tunnel valley

    Directory of Open Access Journals (Sweden)

    Kacprzak Grzegorz

    2015-06-01

    Full Text Available A common engineering solution for excessive settlement with raft foundation (s is the use of piles in order to reduce the vertical displacements, in this method, the whole structural load is transferred to the piles. This is an overly cautious approach, and there remains a need to find an optimal design method for a building’s foundations. Such a solution may be the piled raft foundation, which allows a reduction of the number of piles due to the integration of the raft in the bearing capacity of the foundation. The aim of the article is to estimate the contribution of foundation elements such as the raft and the piles in the bearing capacity of a residential building located in Warsaw, where the geological conditions are characterized by organic soil layers, principally of gyttja.

  12. The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

    Science.gov (United States)

    Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

    2016-01-01

    Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

  13. Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

    Science.gov (United States)

    Marunaka, Yoshinori; Niisato, Naomi; Taruno, Akiyuki; Ohta, Mariko; Miyazaki, Hiroaki; Hosogi, Shigekuni; Nakajima, Ken-ichi; Kusuzaki, Katsuyuki; Ashihara, Eishi; Nishio, Kyosuke; Iwasaki, Yoshinobu; Nakahari, Takashi; Kubota, Takahiro

    2011-01-01

    Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane. PMID:22028593

  14. Micro–Positron Emission Tomography/Contrast-Enhanced Computed Tomography Imaging of Orthotopic Pancreatic Tumor–Bearing Mice Using the αvβ3 Integrin Tracer 64Cu-Labeled Cyclam-RAFT-c(-RGDfK-4

    Directory of Open Access Journals (Sweden)

    Winn Aung

    2013-09-01

    Full Text Available The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET imaging probe, 64Cu-labeled cyclam-RAFT-c(-RGDfK-4 peptide (64Cu-RAFT-RGD, using this model. Varying degrees of αvβ3 integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP, was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT comparing 64Cu-RAFT-RGD and 18F-fluoro-2-deoxy-D-glucose (18F-FDG accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ3 integrin specific. 64Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of 64Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, 64Cu-RAFT-RGD accumulation provided better tumor contrast to background than 18F-FDG. Our results suggest that 64Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ3 integrin–expressing pancreatic tumors.

  15. PENGARUH CITRA MEREK (BRAND IMAGE DAN KEPUASAN WISATAWAN TERHADAP LOYALITAS WISATAWAN PADA BALI ADVENTURE RAFTING

    Directory of Open Access Journals (Sweden)

    Ardilla Nathaurisia

    2017-01-01

    Full Text Available ith loyalty and high power of buying. Research impact of brand image and guest satisfaction on customer loyalty is important to do in order to obtain an explanation of the relationship between brand image and customer satisfaction on customer loyalty. The purpose of this study is 1 to identify the circumstance brand image on customer loyalty. 2 to identify the circumstance customer satisfaction on customer loyalty. 3 to determine the circumstance brand image and customer satisfaction on customer loyalty. This research was conducted at Bali Adventure Rafting. Result shows that branding and customer satisfaction has a significant effect on customer loyalty in Bali Adventure Rafting with result 41,7%. This result is obtained from the value of determination D x 100% = 0,417 x 100% = 41,7%. This means that the brand image and customer satisfaction contributing positively to customer loyalty of rating of 41,7% and the remaining 58,3% is influenced by others factors such as costumers satisfaction, marketing strategy.

  16. Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src.

    Directory of Open Access Journals (Sweden)

    Maoyong Fu

    Full Text Available Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2, a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

  17. The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts

    Directory of Open Access Journals (Sweden)

    Mandy J. Ludford-Menting

    2011-01-01

    Full Text Available Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation.

  18. Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna

    Science.gov (United States)

    Borrell Pichs, Yaisel J.; García-Vazquez, Eva

    2018-01-01

    Anthropogenic plastic pollution is a global problem. In the marine environment, one of its less studied effects is the transport of attached biota, which might lead to introductions of non-native species in new areas or aid in habitat expansions of invasive species. The goal of the present work was to assess if the material composition of beached anthropogenic litter is indicative of the rafting fauna in a coastal area and could thus be used as a simple and cost-efficient tool for risk assessment in the future. Beached anthropogenic litter and attached biota along the 200 km coastline of Asturias, central Bay of Biscay, Spain, were analysed. The macrobiotic community attached to fouled litter items was identified using genetic barcoding combined with visual taxonomic analysis, and compared between hard plastics, foams, other plastics and non-plastic items. On the other hand, the material composition of beached litter was analysed in a standardized area on each beach. From these two datasets, the expected frequency of several rafting taxa was calculated for the coastal area and compared to the actually observed frequencies. The results showed that plastics were the most abundant type of beached litter. Litter accumulation was likely driven by coastal sources (industry, ports) and river/sewage inputs and transported by near-shore currents. Rafting vectors were almost exclusively made up of plastics and could mainly be attributed to fishing activity and leisure/ household. We identified a variety of rafting biota, including species of goose barnacles, acorn barnacles, bivalves, gastropods, polychaetes and bryozoan, and hydrozoan colonies attached to stranded litter. Several of these species were non-native and invasive, such as the giant Pacific oyster (Crassostrea gigas) and the Australian barnacle (Austrominius modestus). The composition of attached fauna varied strongly between litter items of different materials. Plastics, except for foam, had a much more diverse

  19. Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna.

    Directory of Open Access Journals (Sweden)

    Sabine Rech

    Full Text Available Anthropogenic plastic pollution is a global problem. In the marine environment, one of its less studied effects is the transport of attached biota, which might lead to introductions of non-native species in new areas or aid in habitat expansions of invasive species. The goal of the present work was to assess if the material composition of beached anthropogenic litter is indicative of the rafting fauna in a coastal area and could thus be used as a simple and cost-efficient tool for risk assessment in the future. Beached anthropogenic litter and attached biota along the 200 km coastline of Asturias, central Bay of Biscay, Spain, were analysed. The macrobiotic community attached to fouled litter items was identified using genetic barcoding combined with visual taxonomic analysis, and compared between hard plastics, foams, other plastics and non-plastic items. On the other hand, the material composition of beached litter was analysed in a standardized area on each beach. From these two datasets, the expected frequency of several rafting taxa was calculated for the coastal area and compared to the actually observed frequencies. The results showed that plastics were the most abundant type of beached litter. Litter accumulation was likely driven by coastal sources (industry, ports and river/sewage inputs and transported by near-shore currents. Rafting vectors were almost exclusively made up of plastics and could mainly be attributed to fishing activity and leisure/ household. We identified a variety of rafting biota, including species of goose barnacles, acorn barnacles, bivalves, gastropods, polychaetes and bryozoan, and hydrozoan colonies attached to stranded litter. Several of these species were non-native and invasive, such as the giant Pacific oyster (Crassostrea gigas and the Australian barnacle (Austrominius modestus. The composition of attached fauna varied strongly between litter items of different materials. Plastics, except for foam, had a

  20. Anthropogenic marine litter composition in coastal areas may be a predictor of potentially invasive rafting fauna.

    Science.gov (United States)

    Rech, Sabine; Borrell Pichs, Yaisel J; García-Vazquez, Eva

    2018-01-01

    Anthropogenic plastic pollution is a global problem. In the marine environment, one of its less studied effects is the transport of attached biota, which might lead to introductions of non-native species in new areas or aid in habitat expansions of invasive species. The goal of the present work was to assess if the material composition of beached anthropogenic litter is indicative of the rafting fauna in a coastal area and could thus be used as a simple and cost-efficient tool for risk assessment in the future. Beached anthropogenic litter and attached biota along the 200 km coastline of Asturias, central Bay of Biscay, Spain, were analysed. The macrobiotic community attached to fouled litter items was identified using genetic barcoding combined with visual taxonomic analysis, and compared between hard plastics, foams, other plastics and non-plastic items. On the other hand, the material composition of beached litter was analysed in a standardized area on each beach. From these two datasets, the expected frequency of several rafting taxa was calculated for the coastal area and compared to the actually observed frequencies. The results showed that plastics were the most abundant type of beached litter. Litter accumulation was likely driven by coastal sources (industry, ports) and river/sewage inputs and transported by near-shore currents. Rafting vectors were almost exclusively made up of plastics and could mainly be attributed to fishing activity and leisure/ household. We identified a variety of rafting biota, including species of goose barnacles, acorn barnacles, bivalves, gastropods, polychaetes and bryozoan, and hydrozoan colonies attached to stranded litter. Several of these species were non-native and invasive, such as the giant Pacific oyster (Crassostrea gigas) and the Australian barnacle (Austrominius modestus). The composition of attached fauna varied strongly between litter items of different materials. Plastics, except for foam, had a much more diverse

  1. 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.

  2. Oxygen isotope calibration of the onset of ice-rafting and history of glaciation in the North Atlantic region

    Science.gov (United States)

    Shackleton, N.J.; Backman, J.; Zimmerman, H.; Kent, D.V.; Hall, M.A.; Roberts, David G.; Schnitker, D.; Baldauf, J.G.; Desprairies, A.; Homrighausen, R.; Huddlestun, P.; Keene, J.B.; Kaltenback, A.J.; Krumsiek, K.A.O.; Morton, A.C.; Murray, J.W.; Westberg-Smith, J.

    1984-01-01

    We report here that DSDP Site 552A, cored with the hydraulic piston corer on the west flank of Rockall Bank, recovered an undisturbed sequence of alternating white deep-sea carbonate oozes and dark-coloured layers that are rich in glacial debris. Oxygen isotope analysis of the sequence together with detailed nannofossil and palaeomagnetic stratigraphy shows that the first major horizon of ice-rafting occurred at about 2.4 Myr, and was preceded by a minor pulse of ice-rafting at about 2.5 Myr. The carbon isotope record shows that the site has been bathed by a water mass of similar characteristics to present-day North Atlantic deep water at least since 3.5 Myr. ?? 1984 Nature Publishing Group.

  3. Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

    Science.gov (United States)

    Richards, Mark J; Daniel, Susan

    2017-02-07

    The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

  4. An overview of recent projects to study thermal protection in life rafts, lifeboats and immersion suits

    Energy Technology Data Exchange (ETDEWEB)

    Mak, L.; DuCharme, M. B.; Farnworth, B.; Wissler, E. H.; Brown, R.; Kuczora, A. [Maritime and Arctic Survival Scientific and Engineering Ressearch Team (Canada)

    2011-07-01

    Survival during a marine evacuation in cold regions is very challenging. However international regulations do not require specific thermal protection or ventilation performance criteria for lifeboats. In the same way, the testing methods for approval testing of immersion suits are not standardised. This paper investigated recent projects completed or on-going to study thermal protection in life rafts, lifeboats and immersion suits. An overview of several projects from the Maritime and Arctic Survival Scientific and Engineering Research Team (MASSERT) was conducted. This review provided the necessary knowledge to advance international standards and develop the thermal protection requirements for survival in the Arctic. The results showed the MASSERT correlated thermal insulation values between human subjects and thermal manikins in life rafts and in immersion suits. It was found that the manikins are a valuable evaluation tool, as well as the computerised models used as prediction tools.

  5. Radiation induced, raft mediated grafting of styrene onto poly(ethylene-alt-tetrafluoroethylene) (ETFE)

    International Nuclear Information System (INIS)

    Celik, G.; Barsbay, M.; Gueven, O.

    2011-01-01

    Complete text of publication follows. The development of cost-effective proton exchange membranes to replace the state-of-the-art and expensive perfluorinated membranes such as Nafion, Flemion, and Aciplex is one of the main challenges for commercialization of polymer electrolyte fuel cell technology. Recently, partially fluorinated poly(ethlyene-alt-tetrafluoroethylene) (ETFE) has been identified as a promising alternative base polymer film to prepare low-cost polymer electrolyte membranes because of its advantageous characters like superior mechanical properties and high resistance to radiation-induced damage. The radiation-induced grafting technique, based on the utilization of a polymer material such as ETFE in combination with further chemical modification steps (sulfonation) allows the functionalization of the base material and the introduction of the desired property (proton conductivity) for preparing a fuel cell membrane. However this simple conventional method suffers from one simple flaw: The molecular weight and the polydispersity of the grafted chains cannot be controlled. Predetermined molecular weights and low dispersities as well as homogeneous composition and desired architecture can be achieved by grafting of monomer onto base polymer under living/controlled free radical polymerization (CRP) conditions. Among the CRP methods, Reversible Addition Fragmentation-Chain Transfer (RAFT) is of particular interest as a very wide range of functional monomers can be polymerized in a controlled manner under non-demanding reaction conditions (e.g., tolerance to oxygen and low temperatures). The present study deals with the RAFT mediated radiation-induced (0.032 kGyh -1 , 60 Co) grafting of styrene on ETFE films followed by the sulfonation of the polystyrene grafts. The effect of monomer concentration, absorbed dose and RAFT agent concentration on the grafting were investigated. The synthesized films were characterized by ATR-FTIR, XPS, DSC and TGA methods

  6. 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.

  7. 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...

  8. Novel Anti-Biofouling Soft Contact Lens: l-Cysteine Conjugated Amphiphilic Conetworks via RAFT and Thiol-Ene Click Chemistry.

    Science.gov (United States)

    Zhang, Chengfeng; Liu, Ziyuan; Wang, Haiye; Feng, Xiaofeng; He, Chunju

    2017-07-01

    A unique l-cysteine conjugated antifouling amphiphilic conetwork (APCN) is synthesized through end-crosslinking of well-defined triblock copolymers poly(allyl methacrylate)-b-poly(ethylene glycol)-b-poly(allyl methacrylate) via a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and thiol-ene "click" chemistry. The synthesized poly(ethylene glycol) macro-RAFT agent initiates the polymerization of allyl methacrylate in a controlled manner. The vinyl pendant groups of the precursor partially conjugate with l-cysteine and the rest fully crosslink with mercaptopropyl-containing siloxane via thiol-ene click chemistry under UV irradiation into APCNs, which show distinguished properties, that is, excellent biocompatibility, more than 39.6% water content, 101 barrers oxygen permeability, optimized mechanical properties, and more than 93% visible light transmittance. What's more, the resultant APCNs exhibit eminent resistance to protein adsorption, where the bovine serum albumin and lysozyme adsorption are decreased to 12 and 21 µg cm -2 , respectively. The outstanding properties of APCNs depend on the RAFT controlled method, which precisely designs the hydrophilic/hydrophobic segments and eventually greatly improves the crosslinking efficiency and homogeneity. Meantime, the l-cysteine monolayer can effectively reduce the surface hydrophobicity and prevent protein adsorption, which exhibits the viability for antifouling surface over and under ophthalmic devices, suggesting a promising soft contact lens. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. A novel method for oxygen glucose deprivation model in organotypic spinal cord slices.

    Science.gov (United States)

    Liu, Jing-Jie; Ding, Xiao-Yan; Xiang, Li; Zhao, Feng; Huang, Sheng-Li

    2017-10-01

    This study aimed to establish a model to closely mimic spinal cord hypoxic-ischemic injury with high production and high reproducibility. Fourteen-day cultured organotypic spinal cord slices were divided into 4 groups: control (Ctrl), oxygen glucose deprived for 30min (OGD 30min), OGD 60min, and OGD 120min. The Ctrl slices were incubated with 1ml propidium iodide (PI) solution (5μg/ml) for 30min. The OGD groups were incubated with 1ml glucose-free DMEM/F12 medium and 5μl PI solution (1mg/ml) for 30min, 60min and 120min, respectively. Positive control slice was fixed by 4% paraformaldehyde for 20min. The culture medium in each group was then collected and the Lactate Dehydrogenase (LDH) level in the medium was tested using Multi-Analyte ELISArray kits. Structure and refraction of the spinal cord slices were observed by light microscope. Fluorescence intensity of PI was examined by fluorescence microscopy and was tested by IPP Software. Morphology of astrocytes was observed by immunofluorescence histochemistry. Caspase 3 and caspase 3 active in different groups were tested by Western blot. In the OGD groups, the refraction of spinal cord slices decreased and the structure was unclear. The changes of refraction and structure in the OGD 120min group were similar to that in the positive control slice. Astrocyte morphology changed significantly. With the increase of OGD time, processes became thick and twisted, and nuclear condensations became more apparent. Obvious changes in morphology were observed in the OGD 60min group, and normal morphology disappeared in the OGD 120min group. Fluorescence intensity of PI increased along with the extension of OGD time. The difference was significant between 30min and 60min, but not significant between 60min and 120min. The intensity at OGD 120min was close to that in the positive control. Compare with the Ctrl group, the OGD groups had significantly higher LDH levels and caspase 3 active/caspase 3 ratios. The values increased

  10. 3-Nitropropionic acid neurotoxicity in organotypic striatal and corticostriatal slice cultures is dependent on glucose and glutamate

    DEFF Research Database (Denmark)

    Storgaard, J; Kornblit, B T; Zimmer, J

    2000-01-01

    of lactate dehydrogenase in the medium and glutamic acid decarboxylase in tissue homogenates. 3-NPA toxicity (25-100 microM in 5 mM glucose, 24-48 h) appeared to be highly dependent on culture medium glucose levels. 3-NPA treatment caused also a dose-dependent lactate increase, reaching a maximum......Mitochondrial inhibition by 3-nitropropionic acid (3-NPA) causes striatal degeneration reminiscent of Huntington's disease. We studied 3-NPA neurotoxicity and possible indirect excitotoxicity in organotypic striatal and corticostriatal slice cultures. Neurotoxicity was quantified by assay...... of threefold increase above control at 100 microM. Both a high dose of glutamate (5 mM) and glutamate uptake blockade by dl-threo-beta-hydroxyaspartate potentiated 3-NPA neurotoxicity in corticostriatal slice cultures. Furthermore, striatum from corticostriatal cocultures was more sensitive to 3-NPA than...

  11. Expression of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid rafts and its enhanced lysosomal degradation.

    Directory of Open Access Journals (Sweden)

    Ruth Rollason

    Full Text Available CD317/tetherin (aka BST2 or HM1.24 antigen is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts. It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii internalised tetherin is present in non-raft fractions, iv expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.

  12. Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the αvβ₃ integrin tracer ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄.

    Science.gov (United States)

    Aung, Winn; Jin, Zhao-Hui; Furukawa, Takako; Claron, Michael; Boturyn, Didier; Sogawa, Chizuru; Tsuji, Atsushi B; Wakizaka, Hidekatsu; Fukumura, Toshimitsu; Fujibayashi, Yasuhisa; Dumy, Pascal; Saga, Tsuneo

    2013-09-01

    The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄ peptide (⁶⁴Cu-RAFT-RGD), using this model. Varying degrees of αvβ₃ integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing ⁶⁴Cu-RAFT-RGD and ¹⁸F-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ₃ integrin specific. ⁶⁴Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of ⁶⁴Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, ⁶⁴Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹⁸F-FDG. Our results suggest that ⁶⁴Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ₃ integrin-expressing pancreatic tumors.

  13. Modulation of epithelial sodium channel in human alveolar epithelial ...

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng1,2, Li-Wei Pan2, Sheng-Rong Zhang3, Bin-Yu Ying2, Ben-Ji. Wang2, Guo-Liang Lin2 and Shi-Fang Ding1*. 1Department of Critical Care Medicine, Qilu Hospital of Shandong ...

  14. Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain.

    Science.gov (United States)

    Zhao, LiYing; Sakagami, Hiroyuki; Suzuki, Tatsuo

    2014-10-01

    We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl β-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX-100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD-PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX-100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft-associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level. We systematically investigated the purification process of post-synaptic density (PSD) and synaptic membrane rafts by examining the structures obtained after treatment of the SPMs with TX-100, n-octyl β-d-glucoside or CHAPSO. Differential distribution of type I and type II PSD, synaptic membrane rafts, and other novel subdomains in the SPM give clues to understand the structural organization of synapses at the molecular level. © 2014 International Society for Neurochemistry.

  15. The variable routes of rafting: stranding dynamics of floating bull kelp Durvillaea antarctica (Fucales, Phaeophyceae) on beaches in the SE Pacific.

    Science.gov (United States)

    López, Boris A; Macaya, Erasmo C; Tala, Fadia; Tellier, Florence; Thiel, Martin

    2017-02-01

    Dispersal on floating seaweeds depends on availability, viability, and trajectories of the rafts. In the southern hemisphere, the bull kelp Durvillaea antarctica is one of the most common floating seaweeds, but phylogeographic studies had shown low connectivity between populations from continental Chile, which could be due to limitations in local supply and dispersal of floating kelps. To test this hypothesis, the spatiotemporal dynamics of kelp strandings were examined in four biogeographic districts along the Chilean coast (28°-42°S). We determined the biomass and demography of stranded individuals on 33 beaches for three subsequent years (2013, 2014, 2015) to examine whether rafting is restricted to certain districts and seasons (winter or summer). Stranded kelps were found on all beaches. Most kelps had only one stipe (one individual), although we also frequently found coalesced holdfasts with mature males and females, which would facilitate successful rafting dispersal, gamete release, and reproduction upon arrival. High biomasses of stranded kelps occurred in the northern-central (30°S-33°S) and southernmost districts (37°S-42°S), and lower biomasses in the northernmost (28°S-30°S) and southern-central districts (33°S-37°S). The highest percentages and sizes of epibionts (Lepas spp.), indicative of prolonged floating periods, were found on stranded kelps in the northernmost and southernmost districts. Based on these results, we conclude that rafting dispersal can vary regionally, being more common in the northernmost and southernmost districts, depending on intrinsic (seaweed biology) and extrinsic factors (shore morphology and oceanography) that affect local supply of kelps and regional hydrodynamics. © 2016 Phycological Society of America.

  16. Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells.

    Science.gov (United States)

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-06-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50-70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

  17. Recent progress on lipid lateral heterogeneity in plasma membranes: from rafts to submicrometric domains

    Science.gov (United States)

    Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R.; Tyteca, Donatienne

    2016-01-01

    The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution. PMID:26738447

  18. 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...

  19. Visualizing form and function in organotypic slices of the adult mouse parotid gland.

    Science.gov (United States)

    Warner, Jennifer D; Peters, Christian G; Saunders, Rudel; Won, Jong Hak; Betzenhauser, Matthew J; Gunning, William T; Yule, David I; Giovannucci, David R

    2008-09-01

    An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.

  20. Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line.

    Science.gov (United States)

    Zaman, Sabiha N; Resek, Mary E; Robbins, Stephen M

    2008-10-01

    Chemokines play pivotal roles in regulating a wide variety of biological processes by modulating cell migration and recruitment. Deregulation of chemokine signaling can alter cell recruitment, contributing to the pathogenic states associated with autoimmune disease, inflammatory disorders, and sepsis. During chemotaxis, lipid rafts and their resident signaling molecules have been demonstrated to partition to different parts of the cell. Herein, we investigated the role of lipid raft resident Src-family kinases (SFK) in stromal cell-derived factor 1/CXCL12-mediated chemotaxis. We have shown that Lck-deficient J.CaM 1.6 cells are defective in CXCL12-mediated chemotaxis in contrast to their parental counterpart, Jurkat cells. Ectopic expression of the SFK hematopoietic cell kinase (Hck) in J.CaM 1.6 cells reconstituted CXCL12 responsiveness. The requirement of lipid raft association of SFK was assessed using both isoforms of Hck: the dually acylated p59(Hck) isoform that is targeted to lipid rafts and the monoacylated p61(Hck) isoform that is nonraft-associated. We have shown using several gain and loss of acylation alleles that dual acylation of Hck was required for CXCL12-mediated chemotaxis in J.CaM 1.6 cells. These results highlight the importance of the unique microenvironment provided by lipid rafts and their specific contribution in providing specificity to CXCL12 signaling.

  1. Molecular nuclear imaging of tumoral angio genesis using a rgd-containing tracer, Raft-RGD, targeted at the neo vessel-specific integrin αvβ3

    International Nuclear Information System (INIS)

    Sancey, L.

    2006-06-01

    Tumoral neo-angio genesis targeting is currently a major field of research for the diagnostic and treatment of solid tumors. Endothelial cells from neo vessels over express several specific markers such as the α v β 3 integrin, which binds RGD (-Arg-Gly-Asp-)- containing peptides. We evaluated the potential of a novel radiotracer - RAFT-RGD - for the molecular nuclear imaging of neo vessels. In vitro, the coupling of 4 c(RGDfK) to the RAFT platform resulted in an increased cellular uptake of the tracer by α v β 3 positive cells when compared to c(RGDfK). Furthermore, RAFTRGD has a higher affinity than c(RGDfK) and similar properties for angio genesis inhibition. In vivo, both α v β 3 positive and negative tumors were visible by non invasive whole body planar and tomographic imaging from 30 min to 24 h post-injection, using a gamma camera dedicated to small animal imaging. Despite a lack of significant contrast improvement compare with c(RGDfK), RAFT-RGD could represent a promising tracer for tumoral angio genesis since it could provide invaluable information about tumor development and treatment efficacy in Nuclear Medicine departments. Furthermore, thanks to its chemical structure, RAFT-RGD can be labelled with a variety of radioisotopes including γ and β - emitters, allowing interesting therapeutical applications such as internal targeted radiotherapy. (author)

  2. 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...

  3. 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

  4. Suppression of phospho-p85α-GTP-Rac1 lipid raft interaction by bichalcone analog attenuates cancer cell invasion.

    Science.gov (United States)

    Lu, Hui-Li; Chen, Shih-Shun; Hsu, Wen-Tung; Lu, Yao-Cheng; Lee, Chuan-Chun; Wu, Tian-Shung; Lin, Meng-Liang

    2016-12-01

    The p85α subunit of phosphatidylinositol 3-kinase (PI3K) acts as a key regulator of cell proliferation and motility, which mediates signals that confer chemoresistance to many human cancer cells. Using small interfering RNAs against matrix metalloproteinase-2 (MMP-2) and the MMP-2 promoter-driven luciferase assay, we showed that the new synthetic bichalcone analog TSWU-CD4 inhibits the invasion of human cancer cells by down-regulating MMP-2 expression. Treatment with TSWU-CD4 inhibited MMP-2 expression and cell invasion, which were restored by ectopic wild type (wt) p85α or a constitutively active form of MAPK kinase 3 (CA MKK3), CA MKK6, or CA p38α mitogen-activated protein kinase (MAPK). The attenuated formation of lipid raft-associated phospho (p)-p85α-GTP-Rac1 complexes, protein kinase B (Akt) Ser 473 phosphorylation, and cell invasion by TSWU-CD4 was reversed by overexpression of wt p85α or the p85α Brc-homology (BH) domain. The ectopic expression of CA Rac1 L61 (but not wt Rac1) could overcome the suppression of Ser 473 phosphorylation, lipid raft association of Akt, the interaction between GTP-bound Rac1 and p85α in lipid rafts, and cell invasion by TSWU-CD4. The involvement of Akt activity in the functions of NF-κB-mediated MMP-2 was further confirmed through the attenuation of Akt phosphorylation signaling using the Akt-specific inhibitor MK-2206 and ectopic expression of NF-κB p65. Collectively, the inhibitory effect of TSWU-CD4 on cancer cell invasion was likely to suppress the p-p85α-GTP-Rac1 interaction in lipid rafts by targeting the p85α BH domain, which resulted in the suppression of MMP-2 expression via the PI3K-Akt-mediated ERK-MKK3/MKK6-p38 MAPK-NF-κB signaling pathway. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Histamine-imprinted microspheres: Comparison between conventional and raft-mediated polymerization techniques

    International Nuclear Information System (INIS)

    Romano, Edwin F. Jr.; So, Regina C.; Holdsworth, Clovia I.

    2015-01-01

    Molecularly imprinted microspheres (MIM) were synthesized via conventional free radical polymerization (CTP) and RAFT-mediated controlled radical polymerization (CRP) method using histamine as the template molecule. Optimal polymerization conditions were achieved using 4%(w/w) monomer feed concentration with 80=90% EGDMA as crosslinker, and histamine: MAA ratio of 1:4 in acetonitrile at 60°C for 24 hours. The size of CTP-M90 and CTP-M80 imprinted microspheres are comparable with that of RAFT polymer CRP-M80 at 264.5 ±12 nm in the swollen (DLS-DMSO) and collapsed state (SEM). For the CTP method, the presence of the template allows for a bigger particle size compared to the non-imprinted counterpart (NIM). Further, controlled growth was observed for the CRP technique, where the size of the imprinted microsphere, CRP-M80, is comparable to CRP-N80. The binding studies of CTP and CRP microspheres toward histamine were studied at concentrations well below biding with buffer concentration of 25mM at pH7. Results showed that the binding isotherms were found to conform to the Freundlich model. Moreover, results revealed that the difference in binding capacity (N) between MIM and NIM imparted by the imprinting process is significantly higher in CTP-80 (26 μmol/g) than both CTP-90 and CRP-80 (9 μmol/g). Non-competitive and competitive binding assays with L-histidine, imidazole, and tryptamine using CTP-80 and CRP-80 were also carried out. MIMs were shown to exhibit binding preference towards the template. (author)

  6. The world of epithelial sheets.

    Science.gov (United States)

    Honda, Hisao

    2017-06-01

    An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

  7. Dating of major normal fault systems using thermochronology: An example from the Raft River detachment, Basin and Range, western United States

    Science.gov (United States)

    Wells, M.L.; Snee, L.W.; Blythe, A.E.

    2000-01-01

    Application of thermochronological techniques to major normal fault systems can resolve the timing of initiation and duration of extension, rates of motion on detachment faults, timing of ductile mylonite formation and passage of rocks through the crystal-plastic to brittle transition, and multiple events of extensional unroofing. Here we determine the above for the top-to-the-east Raft River detachment fault and shear zone by study of spatial gradients in 40Ar/39Ar and fission track cooling ages of footwall rocks and cooling histories and by comparison of cooling histories with deformation temperatures. Mica 40Ar/39Ar cooling ages indicate that extension-related cooling began at ???25-20 Ma, and apatite fission track ages show that motion on the Raft River detachment proceeded until ???7.4 Ma. Collective cooling curves show acceleration of cooling rates during extension, from 5-10??C/m.y. to rates in excess of 70-100??C/m.y. The apparent slip rate along the Raft River detachment, recorded in spatial gradients of apatite fission track ages, is 7 mm/yr between 13.5 and 7.4 Ma and is interpreted to record the rate of migration of a rolling hinge. Microstructural study of footwall mylonite indicates that deformation conditions were no higher than middle greenschist facies and that deformation occurred during cooling to cataclastic conditions. These data show that the shear zone and detachment fault represent a continuum produced by progressive exhumation and shearing during Miocene extension and preclude the possibility of a Mesozoic age for the ductile shear zone. Moderately rapid cooling in middle Eocene time likely records exhumation resulting from an older, oppositely rooted, extensional shear zone along the west side of the Grouse Creek, Raft River, and Albion Mountains. Copyright 2000 by the American Geophysical Union.

  8. Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells123

    Science.gov (United States)

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-01-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering. PMID:21525263

  9. Efficient Homodifunctional Bimolecular Ring-Closure Method for Cyclic Polymers by Combining RAFT and Self-Accelerating Click Reaction.

    Science.gov (United States)

    Qu, Lin; Sun, Peng; Wu, Ying; Zhang, Ke; Liu, Zhengping

    2017-08-01

    An efficient metal-free homodifunctional bimolecular ring-closure method is developed for the formation of cyclic polymers by combining reversible addition-fragmentation chain transfer (RAFT) polymerization and self-accelerating click reaction. In this approach, α,ω-homodifunctional linear polymers with azide terminals are prepared by RAFT polymerization and postmodification of polymer chain end groups. By virtue of sym-dibenzo-1,5-cyclooctadiene-3,7-diyne (DBA) as small linkers, well-defined cyclic polymers are then prepared using the self-accelerating double strain-promoted azide-alkyne click (DSPAAC) reaction to ring-close the azide end-functionalized homodifunctional linear polymer precursors. Due to the self-accelerating property of DSPAAC ring-closing reaction, this novel method eliminates the requirement of equimolar amounts of telechelic polymers and small linkers in traditional bimolecular ring-closure methods. It facilitates this method to efficiently and conveniently produce varied pure cyclic polymers by employing an excess molar amount of DBA small linkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Prion protein accumulation in lipid rafts of mouse aging brain.

    Directory of Open Access Journals (Sweden)

    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.

  11. Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A.

    Science.gov (United States)

    Yabuuchi, Satomi; Endo, Satoshi; Baek, KeangOk; Hoshino, Kunihide; Tsujino, Yoshio; Vestergaard, Mun'delanji C; Takagi, Masahiro

    2017-12-01

    Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Molecular nuclear imaging of tumoral angio genesis using a rgd-containing tracer, Raft-RGD, targeted at the neo vessel-specific integrin {alpha}{sub v}{beta}{sub 3}; Evaluation d'un radioligand de l'integrine {alpha}{sub v}{beta}{sub 3} (RAFT-RGD) pour l'imagerie moleculaire de l'angiogenese tumorale

    Energy Technology Data Exchange (ETDEWEB)

    Sancey, L

    2006-06-15

    Tumoral neo-angio genesis targeting is currently a major field of research for the diagnostic and treatment of solid tumors. Endothelial cells from neo vessels over express several specific markers such as the {alpha}{sub v}{beta}{sub 3} integrin, which binds RGD (-Arg-Gly-Asp-)- containing peptides. We evaluated the potential of a novel radiotracer - RAFT-RGD - for the molecular nuclear imaging of neo vessels. In vitro, the coupling of 4 c(RGDfK) to the RAFT platform resulted in an increased cellular uptake of the tracer by {alpha}{sub v}{beta}{sub 3} positive cells when compared to c(RGDfK). Furthermore, RAFTRGD has a higher affinity than c(RGDfK) and similar properties for angio genesis inhibition. In vivo, both {alpha}{sub v}{beta}{sub 3} positive and negative tumors were visible by non invasive whole body planar and tomographic imaging from 30 min to 24 h post-injection, using a gamma camera dedicated to small animal imaging. Despite a lack of significant contrast improvement compare with c(RGDfK), RAFT-RGD could represent a promising tracer for tumoral angio genesis since it could provide invaluable information about tumor development and treatment efficacy in Nuclear Medicine departments. Furthermore, thanks to its chemical structure, RAFT-RGD can be labelled with a variety of radioisotopes including {gamma} and {beta}{sup -} emitters, allowing interesting therapeutical applications such as internal targeted radiotherapy. (author)

  13. Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors

    Czech Academy of Sciences Publication Activity Database

    Svobodová, Irena; Bhattacharya, Anirban; Ivetic, Milorad; Bendová, Z.; Zemková, Hana

    2018-01-01

    Roč. 9, Mar 6 (2018), č. článku 192. ISSN 1663-9812 R&D Projects: GA ČR(CZ) GA16-12695S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : suprachiasmatic nucleus * organotypic cultures * astrocytes * P2X7 receptor * P2Y1 receptor * P2Y2 receptor * pannexin-1 hemichannel * ATP release Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.400, year: 2016

  14. CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity.

    Science.gov (United States)

    Cardaba, Clara Moyano; Kerr, Jason S; Mueller, Anja

    2008-09-01

    The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-beta-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca(2+) and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Galphai G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Galphai G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Galphai G-proteins.

  15. Fabrication of luminescent hydroxyapatite nanorods through surface-initiated RAFT polymerization: Characterization, biological imaging and drug delivery applications

    Energy Technology Data Exchange (ETDEWEB)

    Heng, Chunning [Shaanxi Key Laboratory of Degradable Biomedical Materials, Shaanxi R& D Center of Biomaterials and Fermentation Engineering, School of Chemical and Engineering, Northwest University, Xi’an, 710069 (China); Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Zheng, Xiaoyan [Shaanxi Key Laboratory of Degradable Biomedical Materials, Shaanxi R& D Center of Biomaterials and Fermentation Engineering, School of Chemical and Engineering, Northwest University, Xi’an, 710069 (China); Liu, Meiying; Xu, Dazhuang; Huang, Hongye; Deng, Fengjie [Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Hui, Junfeng, E-mail: huijunfeng@126.com [Shaanxi Key Laboratory of Degradable Biomedical Materials, Shaanxi R& D Center of Biomaterials and Fermentation Engineering, School of Chemical and Engineering, Northwest University, Xi’an, 710069 (China); Zhang, Xiaoyong, E-mail: xiaoyongzhang1980@gmail.com [Department of 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)

    2016-11-15

    Highlights: • Hydrophobic hydroxyapatite nanorods were obtained from hydrothermal synthesis. • Surface initiated RAFT polymerization was adopted to surface modification of hydroxyapatite nanorods. • These modified hydroxyapatite nanorods showed high water dispersibility and biocompatibility. • These modified hydroxyapatite nanorods can be used for controlled drug delivery. - Abstract: Hydroxyapatite nanomaterials as an important class of nanomaterials, have been widely applied for different biomedical applications for their excellent biocompatibility, biodegradation potential and low cost. In this work, hydroxyapatite nanorods with uniform size and morphology were prepared through hydrothermal synthesis. The surfaces of these hydroxyapatite nanorods are covered with hydrophobic oleic acid, making them poor dispersibility in aqueous solution and difficult for biomedical applications. To overcome this issue, a simple surface initiated polymerization strategy has been developed via combination of the surface ligand exchange and reversible addition fragmentation chain transfer (RAFT) polymerization. Hydroxyapatite nanorods were first modified with Riboflavin-5-phosphate sodium (RPSSD) via ligand exchange reaction between the phosphate group of RPSSD and oleic acid. Then hydroxyl group of nHAp-RPSSD was used to immobilize chain transfer agent, which was used as the initiator for surface-initiated RAFT polymerization. The nHAp-RPSSD-poly(IA-PEGMA) nanocomposites were c