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Sample records for host cell lipid

  1. Lipid exchange between Borrelia burgdorferi and host cells.

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    Jameson T Crowley

    2013-01-01

    Full Text Available Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or (3H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

  2. Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome.

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    van der Meer-Janssen, Ynske P M; van Galen, Josse; Batenburg, Joseph J; Helms, J Bernd

    2010-01-01

    Lipids were long believed to have a structural role in biomembranes and a role in energy storage utilizing cellular lipid droplets and plasma lipoproteins. Research over the last decades has identified an additional role of lipids in cellular signaling, membrane microdomain organization and dynamics, and membrane trafficking. These properties make lipids an attractive target for pathogens to modulate host cell processes in order to allow their survival and replication. In this review we will summarize the often ingenious strategies of pathogens to modify the lipid homeostasis of host cells, allowing them to divert cellular processes. To this end pathogens take full advantage of the complexity of the lipidome. The examples are categorized in generalized and emerging principles describing the involvement of lipids in host-pathogen interactions. Several pathogens are described that simultaneously induce multiple changes in the host cell signaling and trafficking mechanisms. Elucidation of these pathogen-induced changes may have important implications for drug development. The emergence of high-throughput lipidomic techniques will allow the description of changes of the host cell lipidome at the level of individual molecular lipid species and the identification of lipid biomarkers.

  3. Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

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    Nagy, Peter D; Pogany, Judit; Xu, Kai

    2016-03-03

    Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

  4. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

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    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  5. Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

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    Carlson Petteri

    2010-10-01

    Full Text Available Abstract Background Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD, which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. Results Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. Conclusions These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

  6. The broad-spectrum antiviral compound ST-669 restricts chlamydial inclusion development and bacterial growth and localizes to host cell lipid droplets within treated cells.

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    Sandoz, Kelsi M; Valiant, William G; Eriksen, Steven G; Hruby, Dennis E; Allen, Robert D; Rockey, Daniel D

    2014-07-01

    Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a lipid droplet-dependent process. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. The role of lipids in host microbe interactions.

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    Lang, Roland; Mattner, Jochen

    2017-06-01

    Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

  8. Avanti lipid tools: connecting lipids, technology, and cell biology.

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    Sims, Kacee H; Tytler, Ewan M; Tipton, John; Hill, Kasey L; Burgess, Stephen W; Shaw, Walter A

    2014-08-01

    Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein-lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions. Copyright © 2014. Published by Elsevier B.V.

  9. Cd1b-Mediated T Cell Recognition of a Glycolipid Antigen Generated from Mycobacterial Lipid and Host Carbohydrate during Infection

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    Moody, D. Branch; Guy, Mark R.; Grant, Ethan; Cheng, Tan-Yun; Brenner, Michael B.; Besra, Gurdyal S.; Porcelli, Steven A.

    2000-01-01

    T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) α and β chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria. PMID:11015438

  10. Lipid Cell Biology: A Focus on Lipids in Cell Division.

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    Storck, Elisabeth M; Özbalci, Cagakan; Eggert, Ulrike S

    2018-06-20

    Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

  11. Differential distribution of lipids in epidermis, gastrodermis and hosted Symbiodinium in the sea anemone Anemonia viridis.

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    Revel, Johana; Massi, Lionel; Mehiri, Mohamed; Boutoute, Marc; Mayzaud, Patrick; Capron, Laure; Sabourault, Cécile

    2016-01-01

    Cnidarian-dinoflagellate symbiosis mainly relies on nutrient recycling, thus providing both partners with a competitive advantage in nutrient-poor waters. Essential processes related to lipid metabolism can be influenced by various factors, including hyperthermal stress. This can affect the lipid content and distribution in both partners, while contributing to symbiosis disruption and bleaching. In order to gain further insight into the role and distribution of lipids in the cnidarian metabolism, we investigated the lipid composition of the sea anemone Anemonia viridis and its photosynthetic dinoflagellate endosymbionts (Symbiodinium). We compared the lipid content and fatty acid profiles of the host cellular layers, non-symbiotic epidermal and symbiont-containing gastrodermal cells, and those of Symbiodinium, in a mass spectrometry-based assessment. Lipids were more concentrated in Symbiodinium cells, and the lipid class distribution was dominated by polar lipids in all tissues. The fatty acid distribution between host cell layers and Symbiodinium cells suggested potential lipid transfers between the partners. The lipid composition and distribution was modified during short-term hyperthermal stress, mainly in Symbiodinium cells and gastrodermis. Exposure to elevated temperature rapidly caused a decrease in polar lipid C18 unsaturated fatty acids and a strong and rapid decrease in the abundance of polar lipid fatty acids relative to sterols. These lipid indicators could therefore be used as sensitive biomarkers to assess the physiology of symbiotic cnidarians, especially the effect of thermal stress at the onset of cnidarian bleaching. Overall, the findings of this study provide some insight on key lipids that may regulate maintenance of the symbiotic interaction. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response

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    Adriano Queiroz

    Full Text Available Abstract: The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

  13. Modification of the Host Cell Lipid Metabolism Induced by Hypolipidemic Drugs Targeting the Acetyl Coenzyme A Carboxylase Impairs West Nile Virus Replication.

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    Merino-Ramos, Teresa; Vázquez-Calvo, Ángela; Casas, Josefina; Sobrino, Francisco; Saiz, Juan-Carlos; Martín-Acebes, Miguel A

    2016-01-01

    West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Polar lipids of Burkholderia pseudomallei induce different host immune responses.

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    Mercedes Gonzalez-Juarrero

    Full Text Available Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4(+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4(+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster.

  15. Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

    Science.gov (United States)

    Gonzalez-Juarrero, Mercedes; Mima, Naoko; Trunck, Lily A.; Schweizer, Herbert P.; Bowen, Richard A.; Dascher, Kyle; Mwangi, Waithaka; Eckstein, Torsten M.

    2013-01-01

    Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster. PMID:24260378

  16. Ebola virus host cell entry.

    Science.gov (United States)

    Sakurai, Yasuteru

    2015-01-01

    Ebola virus is an enveloped virus with filamentous structure and causes a severe hemorrhagic fever in human and nonhuman primates. Host cell entry is the first essential step in the viral life cycle, which has been extensively studied as one of the therapeutic targets. A virus factor of cell entry is a surface glycoprotein (GP), which is an only essential viral protein in the step, as well as the unique particle structure. The virus also interacts with a lot of host factors to successfully enter host cells. Ebola virus at first binds to cell surface proteins and internalizes into cells, followed by trafficking through endosomal vesicles to intracellular acidic compartments. There, host proteases process GPs, which can interact with an intracellular receptor. Then, under an appropriate circumstance, viral and endosomal membranes are fused, which is enhanced by major structural changes of GPs, to complete host cell entry. Recently the basic research of Ebola virus infection mechanism has markedly progressed, largely contributed by identification of host factors and detailed structural analyses of GPs. This article highlights the mechanism of Ebola virus host cell entry, including recent findings.

  17. Host Lipid Mediators in Leprosy: The Hypothesized Contributions to Pathogenesis

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    Carlos A. M. Silva

    2018-02-01

    Full Text Available The spectrum of clinical forms observed in leprosy and its pathogenesis are dictated by the host’s immune response against Mycobacterium leprae, the etiological agent of leprosy. Previous results, based on metabolomics studies, demonstrated a strong relationship between clinical manifestations of leprosy and alterations in the metabolism of ω3 and ω6 polyunsaturated fatty acids (PUFAs, and the diverse set of lipid mediators derived from PUFAs. PUFA-derived lipid mediators provide multiple functions during acute inflammation, and some lipid mediators are able to induce both pro- and anti-inflammatory responses as determined by the cell surface receptors being expressed, as well as the cell type expressing the receptors. However, little is known about how these compounds influence cellular immune activities during chronic granulomatous infectious diseases, such as leprosy. Current evidence suggests that specialized pro-resolving lipid mediators (SPMs are involved in the down-modulation of the innate and adaptive immune response against M. leprae and that alteration in the homeostasis of pro-inflammatory lipid mediators versus SPMs is associated with dramatic shifts in the pathogenesis of leprosy. In this review, we discuss the possible consequences and present new hypotheses for the involvement of ω3 and ω6 PUFA metabolism in the pathogenesis of leprosy. A specific emphasis is placed on developing models of lipid mediator interactions with the innate and adaptive immune responses and the influence of these interactions on the outcome of leprosy.

  18. Defining Lipid Transport Pathways in Animal Cells

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    Pagano, Richard E.; Sleight, Richard G.

    1985-09-01

    A new technique for studying the metabolism and intracellular transport of lipid molecules in living cells based on the use of fluorescent lipid analogs is described. The cellular processing of various intermediates (phosphatidic acid and ceramide) and end products (phosphatidylcholine and phosphatidylethanolamine) in lipid biosynthesis is reviewed and a working model for compartmentalization during lipid biosynthesis is presented.

  19. Host-directed strategies using lipid nanoparticles to reduce mycobacteria survival

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, L. [Faculty of Sciences and Technology of the New University of Lisbon (Portugal); Diogo, J.; Mateus, R.; Pimentel, M.; Videira, M., E-mail: mvideira@ff.ul.pt [iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy of the University of Lisbon, Intracellular Trafficking Modulation for Advanced Drug Delivery InTraCell-ADD Research Group (Portugal)

    2015-02-15

    Antibiotic-resistant infections and the stagnations in the development of new drugs have increased the demand for new therapeutic approaches against Mycobacterium tuberculosis. Innovative systems that are able to target and eradicate the bacteria in the infected host cells may represent a therapeutic breakthrough while avoiding latency. The development of nanosystems aiming a controlled and targeted intracellular drug release, have proved to increase cytosolic therapeutic concentration while reducing undesired side effects. This work’s main goal was to develop a host-directed strategy against mycobacterial infection through the design of a biocompatible nanocarrier for phage-derived protein delivery, using M. smegmatis as model. Since mycobacterial pathogenicity is strongly supported by the presence of lipids in the cell wall, their degradation induces bacterial destruction through cell wall hydrolysis. Phage-based lipolytic enzymes such as, LysB a mycolylarabinogalactan esterase, represent an appealing therapeutic approach. The herein proposed Ms6 LysB-containing lipid nanocarrier (SLN-LysB) explores the known advantages of nanomedicine-based systems for phagocytic cells selectively targeting thus allowing LysB intracellular accumulation and a more pronounced mycobacterial infection eradication. Adsorption efficiency value indicates the potential of this system as a protein nanocarrier. Moreover, promising outcomes were obtained in host-infected macrophages treated with SLN-LysB. The results show that the herein proposed strategy was more effective in inhibiting the growth of M. smegmatis than free LysB, which might be related to the nanocarrier internalization. Acting as effective protein nanocarriers, the protein-guided delivery in the infected phagocytic cells allows it to exert its hydrolytic action on the lipid layer of the Mycobacterium.

  20. Host-directed strategies using lipid nanoparticles to reduce mycobacteria survival

    Science.gov (United States)

    Pereira, L.; Diogo, J.; Mateus, R.; Pimentel, M.; Videira, M.

    2015-02-01

    Antibiotic-resistant infections and the stagnations in the development of new drugs have increased the demand for new therapeutic approaches against Mycobacterium tuberculosis. Innovative systems that are able to target and eradicate the bacteria in the infected host cells may represent a therapeutic breakthrough while avoiding latency. The development of nanosystems aiming a controlled and targeted intracellular drug release, have proved to increase cytosolic therapeutic concentration while reducing undesired side effects. This work's main goal was to develop a host-directed strategy against mycobacterial infection through the design of a biocompatible nanocarrier for phage-derived protein delivery, using M. smegmatis as model. Since mycobacterial pathogenicity is strongly supported by the presence of lipids in the cell wall, their degradation induces bacterial destruction through cell wall hydrolysis. Phage-based lipolytic enzymes such as, LysB a mycolylarabinogalactan esterase, represent an appealing therapeutic approach. The herein proposed Ms6 LysB-containing lipid nanocarrier (SLN_LysB) explores the known advantages of nanomedicine-based systems for phagocytic cells selectively targeting thus allowing LysB intracellular accumulation and a more pronounced mycobacterial infection eradication. Adsorption efficiency value indicates the potential of this system as a protein nanocarrier. Moreover, promising outcomes were obtained in host-infected macrophages treated with SLN_LysB. The results show that the herein proposed strategy was more effective in inhibiting the growth of M. smegmatis than free LysB, which might be related to the nanocarrier internalization. Acting as effective protein nanocarriers, the protein-guided delivery in the infected phagocytic cells allows it to exert its hydrolytic action on the lipid layer of the Mycobacterium.

  1. Host-directed strategies using lipid nanoparticles to reduce mycobacteria survival

    International Nuclear Information System (INIS)

    Pereira, L.; Diogo, J.; Mateus, R.; Pimentel, M.; Videira, M.

    2015-01-01

    Antibiotic-resistant infections and the stagnations in the development of new drugs have increased the demand for new therapeutic approaches against Mycobacterium tuberculosis. Innovative systems that are able to target and eradicate the bacteria in the infected host cells may represent a therapeutic breakthrough while avoiding latency. The development of nanosystems aiming a controlled and targeted intracellular drug release, have proved to increase cytosolic therapeutic concentration while reducing undesired side effects. This work’s main goal was to develop a host-directed strategy against mycobacterial infection through the design of a biocompatible nanocarrier for phage-derived protein delivery, using M. smegmatis as model. Since mycobacterial pathogenicity is strongly supported by the presence of lipids in the cell wall, their degradation induces bacterial destruction through cell wall hydrolysis. Phage-based lipolytic enzymes such as, LysB a mycolylarabinogalactan esterase, represent an appealing therapeutic approach. The herein proposed Ms6 LysB-containing lipid nanocarrier (SLN-LysB) explores the known advantages of nanomedicine-based systems for phagocytic cells selectively targeting thus allowing LysB intracellular accumulation and a more pronounced mycobacterial infection eradication. Adsorption efficiency value indicates the potential of this system as a protein nanocarrier. Moreover, promising outcomes were obtained in host-infected macrophages treated with SLN-LysB. The results show that the herein proposed strategy was more effective in inhibiting the growth of M. smegmatis than free LysB, which might be related to the nanocarrier internalization. Acting as effective protein nanocarriers, the protein-guided delivery in the infected phagocytic cells allows it to exert its hydrolytic action on the lipid layer of the Mycobacterium

  2. Lipids in the cell: organisation regulates function.

    Science.gov (United States)

    Santos, Ana L; Preta, Giulio

    2018-06-01

    Lipids are fundamental building blocks of all cells and play important roles in the pathogenesis of different diseases, including inflammation, autoimmune disease, cancer, and neurodegeneration. The lipid composition of different organelles can vary substantially from cell to cell, but increasing evidence demonstrates that lipids become organised specifically in each compartment, and this organisation is essential for regulating cell function. For example, lipid microdomains in the plasma membrane, known as lipid rafts, are platforms for concentrating protein receptors and can influence intra-cellular signalling. Lipid organisation is tightly regulated and can be observed across different model organisms, including bacteria, yeast, Drosophila, and Caenorhabditis elegans, suggesting that lipid organisation is evolutionarily conserved. In this review, we summarise the importance and function of specific lipid domains in main cellular organelles and discuss recent advances that investigate how these specific and highly regulated structures contribute to diverse biological processes.

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

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    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.

  4. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, s...... flippase activities in the plasma membrane of cells, using yeast as an example.......P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases......, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  5. The Making and Taking of Lipids: The Role of Bacterial Lipid Synthesis and the Harnessing of Host Lipids in Bacterial Pathogenesis.

    Science.gov (United States)

    Fozo, E M; Rucks, E A

    2016-01-01

    In order to survive environmental stressors, including those induced by growth in the human host, bacterial pathogens will adjust their membrane physiology accordingly. These physiological changes also include the use of host-derived lipids to alter their own membranes and feed central metabolic pathways. Within the host, the pathogen is exposed to many stressful stimuli. A resulting adaptation is for pathogens to scavenge the host environment for readily available lipid sources. The pathogen takes advantage of these host-derived lipids to increase or decrease the rigidity of their own membranes, to provide themselves with valuable precursors to feed central metabolic pathways, or to impact host signalling and processes. Within, we review the diverse mechanisms that both extracellular and intracellular pathogens employ to alter their own membranes as well as their use of host-derived lipids in membrane synthesis and modification, in order to increase survival and perpetuate disease within the human host. Furthermore, we discuss how pathogen employed mechanistic utilization of host-derived lipids allows for their persistence, survival and potentiation of disease. A more thorough understanding of all of these mechanisms will have direct consequences for the development of new therapeutics, and specifically, therapeutics that target pathogens, while preserving normal flora. © 2016 Elsevier Ltd All rights reserved.

  6. Host cell reactivation in mammalian cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Benane, S.G.; Stafford, J.E.

    1976-01-01

    The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promoted photereactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7 to 0.8 for ovary cells and 0.5 to 0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more effecient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survival curves of herpes virus in Potoroo cells indicated a high level of 'dark' host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (lambda>600 nm) and human cells with normal repair and with cells deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreactivating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps. (author)

  7. Presentation of lipid antigens to T cells.

    Science.gov (United States)

    Mori, Lucia; De Libero, Gennaro

    2008-04-15

    T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

  8. Lipids as organizers of cell membranes.

    Science.gov (United States)

    Kornmann, Benoît; Roux, Aurélien

    2012-08-01

    The 105th Boehringer Ingelheim Fonds International Titisee Conference 'Lipids as Organizers of Cell Membranes' took place in March 2012, in Germany. Kai Simons and Gisou Van der Goot gathered cell biologists and biophysicists to discuss the interplay between lipids and proteins in biological membranes, with an emphasis on how technological advances could help fill the gap in our understanding of the lipid part of the membrane.

  9. [Germ cell membrane lipids in spermatogenesis].

    Science.gov (United States)

    Wang, Ting; Shi, Xiao; Quan, Song

    2016-05-01

    Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.

  10. Bartonella entry mechanisms into mammalian host cells.

    Science.gov (United States)

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment. © 2012 Blackwell Publishing Ltd.

  11. Do lipids shape the eukaryotic cell cycle?

    Science.gov (United States)

    Furse, Samuel; Shearman, Gemma C

    2018-01-01

    Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  12. 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.

  13. Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

    Directory of Open Access Journals (Sweden)

    Hervé Lecoeur

    Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

  14. Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

    Science.gov (United States)

    2013-01-01

    Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease. PMID:23899561

  15. Perturbation of host-cell membrane is a primary mechanism of HIV cytopathology.

    Science.gov (United States)

    Cloyd, M W; Lynn, W S

    1991-04-01

    Cytopathic viruses injure cells by a number of different mechanisms. The mechanism by which HIV-1 injures T cells was studied by temporally examining host-cell macromolecular syntheses, stages of the cell cycle, and membrane permeability following acute infection. T cells cytopathically infected at an m.o.i. of 1-5 grew normally for 24-72 hr, depending on the cell line, followed by the first manifestation of cell injury, slowing of cell division. At that time significant amounts of unintegrated HIV DNA and p24 core protein became detectable, and acridine orange flow cytometric cell cycle studies demonstrated the presence of fewer cells in the G2/M stage of the cell cycle. There was no change in the frequency of cells in the S-stage, and metabolic pulsing with radioactive precursors demonstrated that host-cell DNA, RNA, and protein syntheses were normal at that time and normal up to the time cells started to die (approximately 24 hr later), when all three decreased. Cellular lipid synthesis, however, was perturbed when cell multiplication slowed, with phospholipid synthesis reduced and neutral lipid synthesis enhanced. Permeability of the host-cell membrane to small molecules, such as Ca2+ and sucrose, was slightly enhanced early postinfection, and by the time of slowing of cell division, host membrane permeability was greatly increased to both Ca2+ and sucrose (Stokes radius 5.2 A) but not to inulin (Stokes radium 20 A). These changes in host-cell membrane permeability and phospholipid synthesis were not observed in acutely infected H9 cells, which are not susceptible to HIV cytopathology. Thus, HIV-1 appeared to predominantly injure T cells by perturbing host-cell membrane permeability and lipid synthesis, which is similar to the cytopathic mechanisms of paramyxoviruses.

  16. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

    Science.gov (United States)

    Ouimet, Mireille; Koster, Stefan; Sakowski, Erik; Ramkhelawon, Bhama; van Solingen, Coen; Oldebeken, Scott; Karunakaran, Denuja; Portal-Celhay, Cynthia; Sheedy, Frederick J; Ray, Tathagat Dutta; Cecchini, Katharine; Zamore, Philip D; Rayner, Katey J; Marcel, Yves L; Philips, Jennifer A; Moore, Kathryn J

    2016-06-01

    Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

  17. Lipid map of the mammalian cell

    NARCIS (Netherlands)

    van Meer, G.; de Kroon, A.I.P.M.

    2010-01-01

    Technological developments, especially in mass spectrometry and bioinformatics, have revealed that living cells contain thousands rather than dozens of different lipids [for classification and nomenclature, see Fahy et al. (Fahy et al., 2009)]. Now, the resulting questions are what is the relevance

  18. Insect Cells as Hosts for Recombinat Proteins

    OpenAIRE

    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  19. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    Science.gov (United States)

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  20. Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

    Directory of Open Access Journals (Sweden)

    Pirson Chris

    2012-06-01

    Full Text Available Abstract Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM and trehalose monomycolate (TMM, the apolar phthiocerol dimycocersates (PDIMs, triacyl glycerol (TAG, pentacyl trehalose (PAT, phenolic glycolipid (PGL, and mono-mycolyl glycerol (MMG. Polar lipids identified included glucose monomycolate (GMM, diphosphatidyl glycerol (DPG, phenylethanolamine (PE and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs. These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

  1. Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

    Directory of Open Access Journals (Sweden)

    Ivan Milovanović

    2009-03-01

    Full Text Available Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2 at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1, or sacrificed at days zero, 14 and 42 (model 2 for the measurement of total cholesterol (Chl, high density lipoproteins (HDL, low density lipoproteins (LDL and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02 in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013. Conversely, LDL was unaltered until day 42, when it increased (p = 0.043. Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice, while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041 and cyst counts above 300 (p = 0.044. Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

  2. Counting Legionella cells within single amoeba host cells

    Science.gov (United States)

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  3. Disruption of cell walls for enhanced lipid recovery

    Science.gov (United States)

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  4. Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

    Science.gov (United States)

    Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

    2017-01-01

    The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

  5. The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism.

    Science.gov (United States)

    Hang, Saiyu; Purdy, Alexandra E; Robins, William P; Wang, Zhipeng; Mandal, Manabendra; Chang, Sarah; Mekalanos, John J; Watnick, Paula I

    2014-11-12

    Vibrio cholerae is lethal to the model host Drosophila melanogaster through mechanisms not solely attributable to cholera toxin. To examine additional virulence determinants, we performed a genetic screen in V. cholerae-infected Drosophila and identified the two-component system CrbRS. CrbRS controls transcriptional activation of acetyl-CoA synthase-1 (ACS-1) and thus regulates the acetate switch, in which bacteria transition from excretion to assimilation of environmental acetate. The resultant loss of intestinal acetate leads to deactivation of host insulin signaling and lipid accumulation in enterocytes, resulting in host lethality. These metabolic effects are not observed upon infection with ΔcrbS or Δacs1 V. cholerae mutants. Additionally, uninfected flies lacking intestinal commensals, which supply short chain fatty acids (SCFAs) such as acetate, also exhibit altered insulin signaling and intestinal steatosis, which is reversed upon acetate supplementation. Thus, acetate consumption by V. cholerae alters host metabolism, and dietary acetate supplementation may ameliorate some sequelae of cholera. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Hepatitis C virus host cell interactions uncovered

    DEFF Research Database (Denmark)

    Gottwein, Judith; Bukh, Jens

    2007-01-01

      Insights into virus-host cell interactions as uncovered by Randall et al. (1) in a recent issue of PNAS further our understanding of the hepatitis C virus (HCV) life cycle, persistence, and pathogenesis and might lead to the identification of new therapeutic targets. HCV persistently infects 180...... million individuals worldwide, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The only approved treatment, combination therapy with IFN- and ribavirin, targets cellular pathways (2); however, a sustained virologic response is achieved only in approximately half of the patients...... treated. Therefore, there is a pressing need for the identification of novel drugs against hepatitis C. Although most research focuses on the development of HCV-specific antivirals, such as protease and polymerase inhibitors (3), cellular targets could be pursued and might allow the development of broad...

  7. Enforcing host cell polarity: an apicomplexan parasite strategy towards dissemination.

    Science.gov (United States)

    Baumgartner, Martin

    2011-08-01

    The propagation of apicomplexan parasites through transmitting vectors is dependent on effective dissemination of parasites inside the mammalian host. Intracellular Toxoplasma and Theileria parasites face the challenge that their spread inside the host depends in part on the motile capacities of their host cells. In response, these parasites influence the efficiency of dissemination by altering adhesive and/or motile properties of their host cells. Theileria parasites do so by targeting signalling pathways that control host cell actin dynamics. The resulting enforced polar host cell morphology facilitates motility and invasiveness, by establishing focal adhesion and invasion structures at the leading edge of the infected cell. This parasite strategy highlights mechanisms of motility regulation that are also likely relevant for immune or cancer cell motility. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

    Science.gov (United States)

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

    2015-09-01

    Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

  9. Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Duelund, Lars; Pakkanen, Kirsi Inkeri

    2010-01-01

    triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid...... aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model...

  10. 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.

  11. 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.

  12. Host cells and methods for production of isobutanol

    Science.gov (United States)

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2017-10-17

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  13. Methods for production of proteins in host cells

    Science.gov (United States)

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  14. Genetic reprogramming of host cells by bacterial pathogens.

    Science.gov (United States)

    Tran Van Nhieu, Guy; Arbibe, Laurence

    2009-10-29

    During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

  15. Lipid and Phylogenetic Analysis of a Gypsum-hosted Endoevaporitic Microbial Community

    Science.gov (United States)

    Turk, K. A.; Jahnke, L. L.; Green, S. J.; Kubo, M. D.; Vogel, M. B.; Des Marais, D. J.

    2007-12-01

    Gypsum evaporites host diverse, productive and volumetrically significant microbial communities and are relevant modern-day analogs to both Precambrian sabkha deposits and, potentially, Martian evaporites. Extensive evaporites form in subaqueous environments of high salinity ponds (>150 permil) maintained by the Exportadora de Sal, S. A. (ESSA) in Guerrero Negro, B.C.S., Mexico. A gypsarenite (reworked clastic gypsum) crust found along the southeast margin of ESSA's Pond 9 was collected in February 2004 and each vibrantly colored layer in the top centimeter was sampled. Extant microbial communities from each layer were characterized using complementary culture-independent molecular techniques, lipid biomarker analysis, and compound specific isotopic analysis. Coupling molecular analysis with lipid biomarker analysis revealed that oxygenic photosynthetic organisms dominate the surface layers (top 3 mm). Polar lipids from the surface layers consisted predominantly of glycolipids, which are characteristic of algae, cyanobacteria and green anoxygenic photosynthetic bacteria. Consistent with prior analyses of gypsum evaporites, 16S rRNA gene clone libraries indicate that cyanobacterial populations belong primarily to the genus Cyanothece. The bacterial community below the surface layers is more diverse and dominated by anaerobic organisms. Phototrophic purple sulfur bacteria, sulfate-reducing bacteria (SRB), and Bacteroidetes were particularly abundant. The relative abundances of SRB increased with depth; Desulfobacteraceae clones were distributed throughout the crust, but not at the surface, while Desulfovibrionaceae clones were found predominantly in the deepest layers. These molecular results are consistent with fatty acid biomarker analysis. δ13C values of major lipid classes in the crust and sediment range from 14 to 36‰, which is considerably lower than corresponding values for benthic Microcoleus-dominated cyanobacterial mats found at lower salinities at ESSA

  16. How pathogens use linear motifs to perturb host cell networks

    KAUST Repository

    Via, Allegra; Uyar, Bora; Brun, Christine; Zanzoni, Andreas

    2015-01-01

    Molecular mimicry is one of the powerful stratagems that pathogens employ to colonise their hosts and take advantage of host cell functions to guarantee their replication and dissemination. In particular, several viruses have evolved the ability to interact with host cell components through protein short linear motifs (SLiMs) that mimic host SLiMs, thus facilitating their internalisation and the manipulation of a wide range of cellular networks. Here we present convincing evidence from the literature that motif mimicry also represents an effective, widespread hijacking strategy in prokaryotic and eukaryotic parasites. Further insights into host motif mimicry would be of great help in the elucidation of the molecular mechanisms behind host cell invasion and the development of anti-infective therapeutic strategies.

  17. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Energy Technology Data Exchange (ETDEWEB)

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.

    2017-05-23

    ABSTRACT

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

    IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

  18. The changes of oil palm roots cell wall lipids during pathogenesis of Ganoderma boninense

    Science.gov (United States)

    Alexander, A.; Dayou, J.; Abdullah, S.; Chong, K. P.

    2017-07-01

    One of the first physical defences of plants against fungal infection is their cell wall. Interaction between combinations of metabolism enzymes known as the “weapons” of pathogen and the host cell wall probably determines the fate of possible invasion of the pathogen in the host. The present work aims to study the biochemical changes of cell wall lipids of oil palm roots and to determine novel information on root cell wall composition during pathogenesis of Ganoderma boninense by using Gas Chromatography Mass Spectrometry. Based on Total Ion Chromatogram analysis, 67 compounds were found more abundant in the roots infected with G. boninense compared to the healthy roots (60 compounds). Interestingly, nine new compounds were identified from the cell wall lipids of roots infected with G. boninense. These includes Cyclohexane, 1,2-dimethyl-, Methyl 2-hydroxy 16-methyl-heptadecanoate, 2-Propenoic acid, methyl ester, Methyl 9-oxohexacosanoate, 5-[(3,7,11,15-Tetramethylhexadecyl)oxy]thiophene-2carboxylic acid, Ergosta-5,7,22,24(28)-tetraen-3beta-ol, 7-Hydroxy-3',4'-methylenedioxyflavan, Glycine and (S)-4'-Hydroxy-4-methoxydalbergione, this may involve as response to pathogen invasion. This paper provides an original comparative lipidomic analysis of oil palm roots cell wall lipids in plant defence during pathogenesis of G. boninense.

  19. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    Science.gov (United States)

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  20. Simultaneous transcriptional profiling of bacteria and their host cells.

    Directory of Open Access Journals (Sweden)

    Michael S Humphrys

    Full Text Available We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness. Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

  1. Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

    Directory of Open Access Journals (Sweden)

    Shaw Edward I

    2010-09-01

    Full Text Available Abstract Background Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection. Results We have identified 36 host cell genes that are specifically regulated when de novo C. burnetii protein synthesis occurs during infection using comparative microarray analysis. Two parallel sets of infected and uninfected THP-1 cells were grown for 48 h followed by the addition of chloramphenicol (CAM to 10 μg/ml in one set. Total RNA was harvested at 72 hpi from all conditions, and microarrays performed using Phalanx Human OneArray™ slides. A total of 784 (mock treated and 901 (CAM treated THP-1 genes were up or down regulated ≥2 fold in the C. burnetii infected vs. uninfected cell sets, respectively. Comparisons between the complementary data sets (using >0 fold, eliminated the common gene expression changes. A stringent comparison (≥2 fold between the separate microarrays revealed 36 host cell genes modulated by C. burnetii protein synthesis. Ontological analysis of these genes identified the innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization as predominant cellular functions modulated by C. burnetii protein synthesis. Conclusions Collectively, these data indicate that C. burnetii proteins actively regulate the expression of specific host cell genes and pathways. This is in addition to host cell genes that respond to the presence of the

  2. Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

    Science.gov (United States)

    Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

    2016-03-01

    Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research. © 2016 WILEY Periodicals, Inc.

  3. Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Carvalho TMU

    1999-01-01

    Full Text Available Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV. In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

  4. Kupffer cell complement receptor clearance function and host defense.

    Science.gov (United States)

    Loegering, D J

    1986-01-01

    Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

  5. Fungal invasion of normally non-phagocytic host cells.

    Directory of Open Access Journals (Sweden)

    Scott G Filler

    2006-12-01

    Full Text Available Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

  6. Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes.

    Directory of Open Access Journals (Sweden)

    Himanshu Khandelia

    Full Text Available Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.

  7. Host cells and methods for producing isoprenyl alkanoates

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  8. CD1 and mycobacterial lipids activate human T cells.

    Science.gov (United States)

    Van Rhijn, Ildiko; Moody, D Branch

    2015-03-01

    For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

    Science.gov (United States)

    Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

    2011-06-01

    Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

  10. Light exposure at night disrupts host/cancer circadian regulatory dynamics: impact on the Warburg effect, lipid signaling and tumor growth prevention.

    Directory of Open Access Journals (Sweden)

    David E Blask

    Full Text Available The central circadian clock within the suprachiasmatic nucleus (SCN plays an important role in temporally organizing and coordinating many of the processes governing cancer cell proliferation and tumor growth in synchrony with the daily light/dark cycle which may contribute to endogenous cancer prevention. Bioenergetic substrates and molecular intermediates required for building tumor biomass each day are derived from both aerobic glycolysis (Warburg effect and lipid metabolism. Using tissue-isolated human breast cancer xenografts grown in nude rats, we determined that circulating systemic factors in the host and the Warburg effect, linoleic acid uptake/metabolism and growth signaling activities in the tumor are dynamically regulated, coordinated and integrated within circadian time structure over a 24-hour light/dark cycle by SCN-driven nocturnal pineal production of the anticancer hormone melatonin. Dim light at night (LAN-induced melatonin suppression disrupts this circadian-regulated host/cancer balance among several important cancer preventative signaling mechanisms, leading to hyperglycemia and hyperinsulinemia in the host and runaway aerobic glycolysis, lipid signaling and proliferative activity in the tumor.

  11. Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees.

    Science.gov (United States)

    Li, Wenfeng; Chen, Yanping; Cook, Steven C

    2018-05-01

    Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21 days post inoculation (21 days pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host's whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6 days pi. At 7 days pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14 days pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14 days pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse. Published by Elsevier Ltd.

  12. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

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    Archana Shrestha

    2016-12-01

    Full Text Available Clostridium perfringens enterotoxin (CPE binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

  13. Transcriptome and microRNome of Theileria annulata Host Cells

    KAUST Repository

    Rchiad, Zineb

    2016-06-01

    Tropical Theileriosis is a parasitic disease of calves with a profound economic impact caused by Theileria annulata, an apicomplexan parasite of the genus Theileria. Transmitted by Hyalomma ticks, T. annulata infects and transforms bovine lymphocytes and macrophages into a cancer-like phenotype characterized by all six hallmarks of cancer. In the current study we investigate the transcriptional landscape of T. annulata-infected lymphocytes to define genes and miRNAs regulated by host cell transformation using next generation sequencing. We also define genes and miRNAs differentially expressed as a result of the attenuation of a T.annulata-infected macrophage cell line used as a vaccine. By comparing the transcriptional landscape of one attenuated and two transformed cell lines we identify four genes that we propose as key factors in transformation and virulence of the T. annulata host cells. We also identify miR- 126-5p as a key regulator of infected cells proliferation, adhesion, survival and invasiveness. In addition to the host cell trascriptome we studied T. annulata transcriptome and identified the role of ROS and TGF-β2 in controlling parasite gene expression. Moreover, we have used the deep parasite ssRNA-seq data to refine the available T. annulata annotation. Taken together, this study provides the full list of host cell’s genes and miRNAs transcriptionally perturbed after infection with T. annulata and after attenuation and describes genes and miRNAs never identified before as players in this type of host cell transformation. Moreover, this study provides the first database for the transcriptome of T. annulata and its host cells using next generation sequencing.

  14. Insights into Host Cell Modulation and Induction of New Cells by the Corn Smut Ustilago maydis

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    Amey Redkar

    2017-05-01

    Full Text Available Many filamentous fungal pathogens induce drastic modulation of host cells causing abnormal infectious structures such as galls, or tumors that arise as a result of re-programming in the original developmental cell fate of a colonized host cell. Developmental consequences occur predominantly with biotrophic phytopathogens. This suggests that these host structures result as an outcome of efficient defense suppression and intimate fungal–host interaction to suit the pathogen’s needs for completion of its infection cycle. This mini-review mainly summarizes host cell re-programming that occurs in the Ustilago maydis – maize interaction, in which the pathogen deploys cell-type specific effector proteins with varying activities. The fungus senses the physiological status and identity of colonized host cells and re-directs the endogenous developmental program of its host. The disturbance of host cell physiology and cell fate leads to novel cell shapes, increased cell size, and/or the number of host cells. We particularly highlight the strategies of U. maydis to induce physiologically varied host organs to form the characteristic tumors in both vegetative and floral parts of maize.

  15. Cell lipids: from isolation to functional dynamics

    NARCIS (Netherlands)

    Veldman, R.JJ; Pecheur, EL; van Ijzendoorn, Sven; Kok, Jan Willem; Hoekstra, Dirk

    2003-01-01

    81. Veldman RJ, Pécheur EI., Van IJzendoorn SCD., Kok JW. and Hoekstra D. (2003) . In: Essential Cell Biology. Cell Structure (Davey, J. and Lord, M. eds.) Oxford University Press, Oxford. Vol. 1, pp.

  16. Altered lipid homeostasis in Sertoli cells stressed by mild hyperthermia.

    Directory of Open Access Journals (Sweden)

    Ana S Vallés

    Full Text Available Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC. Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days of rat testes to 43 °C led to accumulation of neutral lipids. This SC-specific lipid function took 1-2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43 °C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin and microfilament (f-actin organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster.

  17. Host cells and methods for producing diacid compounds

    Energy Technology Data Exchange (ETDEWEB)

    Steen, Eric J.; Fortman, Jeffrey L.; Dietrich, Jeffrey A.; Keasling, Jay D.

    2018-04-24

    The present invention provides for a method of producing one or more fatty acid derived dicarboxylic acids in a genetically modified host cell which does not naturally produce the one or more derived fatty acid derived dicarboxylic acids. The invention provides for the biosynthesis of dicarboxylic acid ranging in length from C3 to C26. The host cell can be further modified to increase fatty acid production or export of the desired fatty acid derived compound, and/or decrease fatty acid storage or metabolism.

  18. Role of Gag and lipids during HIV-1 assembly in CD4 T cells and Macrophages

    Directory of Open Access Journals (Sweden)

    Charlotte eMariani

    2014-06-01

    Full Text Available HIV-1 is an RNA enveloped virus that preferentiallyinfects CD4+ T lymphocytes andalso macrophages. In CD4+ T cells, HIV-1mainly buds from the host cell plasma membrane.The viral Gag polyprotein targets theplasma membrane and is the orchestrator ofthe HIV assembly as its expression is sufficientto promote the formation of virus-likeparticles particles carrying a lipidic envelopederiving from the host cell membrane. Certainlipids are enriched in the viral membraneand are thought to play a key role in theassembly process and the envelop composition.A large body of work performed oninfected CD4+ T cells has provided importantknowledge about the assembly process andthe membrane virus lipid composition. WhileHIV assembly and budding in macrophages isthought to follow the same general Gag-drivenmechanism as in T-lymphocytes, the HIV cyclein macrophage exhibits specific features.In these cells, new virions bud from the limitingmembrane of seemingly intracellular compartments,where they accumulate while remaininginfectious. These structures are now oftenreferred to as Virus Containing Compartments(VCCs. Recent studies suggest that VCCsrepresent intracellularly sequestered regionsof the plasma membrane, but their precisenature remains elusive. The proteomic andlipidomic characterization of virions producedby T cells or macrophages has highlightedthe similarity between their composition andthat of the plasma membrane of producercells, as well as their enrichment in acidiclipids, some components of raft lipids andin tetraspanin-enriched microdomains. Greatchances are that Gag promotes the coalescenceof these components into an assemblyplatform from which viral budding takesplace. How Gag exactly interacts with membranelipids and what are the mechanisms involvedin the interaction between the differentmembrane nanodomains within the assemblyplatform remains unclear. Here we review recentliterature regarding the role of Gag andlipids

  19. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    International Nuclear Information System (INIS)

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-01

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  20. Interaction of Dendritic Polymers with Synthetic Lipid and Cell Membranes

    Science.gov (United States)

    Mecke, Almut; Hong, Seungpyo; Bielinska, Anna U.; Banaszak Holl, Mark M.; Orr, Bradford G.; Baker, James R., Jr.

    2004-03-01

    Polyamidoamine (PAMAM) dendrimers are promising candidates for the development of nanoscale therapeutic transport agents. Here we present studies on dendrimer-membrane interactions leading to a better understanding of possible uptake mechanisms into cells. Using synthetic lipid and natural cell membranes as model systems it is shown that the effect of PAMAM dendrimers on a membrane strongly depends on the dendrimer generation, architecture and chemical properties of the branch end groups. Atomic force microscopy data indicates that generation 7 dendrimers have the ability to form small ( 10-100 nm) holes in a lipid bilayer. When dendrimers with otherwise identical chemical properties are arranged in a covalently linked cluster, no hole formation occurs. Dendrimer-lipid micelle formation is proposed and investigated as a possible mechanism for this behavior. Smaller dendrimers (generation 5) have a greatly reduced ability to remove lipid molecules from a bilayer. In addition to the size of the dendrimer, the charge of the branch end groups plays a significant role for dendrimer-membrane interactions. These results agree well with biological studies using cultured cells and point to a new mechanism of specific targeting and uptake into cells.

  1. The Size And Localisation Of Yellow Pigmented Lipid Cells 6 ...

    African Journals Online (AJOL)

    The size and distribution of the main pungent principle (6-gingerol) in two ginger varieties “ Tafin giwa” (the yellow variety) and “Yatsum biri” (the dark variety) at 4, 5, 6, and 8 months stages of maturity at harvest were studied empirically by the determination of the mean number of yellow pigmented lipid cells per unit area ...

  2. 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

  3. The lipid organisation of the cell membrane

    Directory of Open Access Journals (Sweden)

    Ladha, S.

    2000-04-01

    Full Text Available Lipids and proteins in biological membranes are arranged in a mosaic of domains in the membrane. These domains represent small-scale heterogeneities in composition, shape and fluidity within the plane of the membrane, over the range of hundreds of nanometers to a few micrometers. They arise from the complex interactions of the heterogeneous mixtures of phospholipids, sterols, and proteins that make up all biological membranes.Los lípidos y las proteínas en las membranas biológicas están dispuestos en un mosaico de campos en la membrana. Estos campos representan heterogeneidades a pequeña escala en la composición, forma y fluidez dentro del plano de la membrana, en un rango que va de los cientos de nanómetros a los pocos micrómetros. Estos campos se originan de las complejas interacciones de las mezclas heterogéneas de fosfolípidos, esteroles y proteínas de las que están hechas todas y cada una de las membranas biológicas.

  4. Cell disruption and lipid extraction for microalgal biorefineries: A review.

    Science.gov (United States)

    Lee, Soo Youn; Cho, Jun Muk; Chang, Yong Keun; Oh, You-Kwan

    2017-11-01

    The microalgae-based biorefinement process has attracted much attention from academic and industrial researchers attracted to its biofuel, food and nutraceutical applications. In this paper, recent developments in cell-disruption and lipid-extraction methods, focusing on four biotechnologically important microalgal species (namely, Chlamydomonas, Haematococcus, Chlorella, and Nannochloropsis spp.), are reviewed. The structural diversity and rigidity of microalgal cell walls complicate the development of efficient downstream processing methods for cell-disruption and subsequent recovery of intracellular lipid and pigment components. Various mechanical, chemical and biological cell-disruption methods are discussed in detail and compared based on microalgal species and status (wet/dried), scale, energy consumption, efficiency, solvent extraction, and synergistic combinations. The challenges and prospects of the downstream processes for the future development of eco-friendly and economical microalgal biorefineries also are outlined herein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Host defense, dendritic cells and the human lung

    NARCIS (Netherlands)

    J.M.W. van Haarst (Jan Maarten)

    1995-01-01

    textabstractHost defense mechanisms protect the body against microorganisms and other foreign structures. These mechanisms can be divided in nonspecific, or innate, and specific, or acquired, immunity. In both branches of immunity the several types of leukocytes (white blood cells) play a dominant

  6. Regulatory T Cells and Host Anti-CML Responses

    National Research Council Canada - National Science Library

    Wong, Jr, K. K

    2008-01-01

    CD4+CD25+FoxP-3+ regulatory T-cells (Tregs) suppress immune responses to "self" antigens, but also have been shown to suppress host anti-tumor responses in several human malignancies, including breast, gastrointestinal, and ovarian cancer...

  7. Models of lipid droplets growth and fission in adipocyte cells

    Energy Technology Data Exchange (ETDEWEB)

    Boschi, Federico, E-mail: federico.boschi@univr.it [Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona (Italy); Rizzatti, Vanni; Zamboni, Mauro [Department of Medicine, Geriatric Section, University of Verona, Piazzale Stefani 1, 37126 Verona (Italy); Sbarbati, Andrea [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy)

    2015-08-15

    Lipid droplets (LD) are spherical cellular inclusion devoted to lipids storage. It is well known that excessive accumulation of lipids leads to several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis and atherosclerosis. LDs' size range from fraction to one hundred of micrometers in adipocytes and is related to the lipid content, but their growth is still a puzzling question. It has been suggested that LDs can grow in size due to the fusion process by which a larger LD is obtained by the merging of two smaller LDs, but these events seems to be rare and difficult to be observed. Many other processes are thought to be involved in the number and growth of LDs, like the de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets. Moreover the number and size of LDs are influenced by the catabolism and the absorption or interaction with other organelles. The comprehension of these processes could help in the confinement of the pathologies related to lipid accumulation. In this study the LDs' size distribution, number and the total volume of immature (n=12), mature (n=12, 10-days differentiated) and lipolytic (n=12) 3T3-L1 adipocytes were considered. More than 11,000 LDs were measured in the 36 cells after Oil Red O staining. In a previous work Monte Carlo simulations were used to mimic the fusion process alone between LDs. We found that, considering the fusion as the only process acting on the LDs, the size distribution in mature adipocytes can be obtained with numerical simulation starting from the size distribution in immature cells provided a very high rate of fusion events. In this paper Monte Carlo simulations were developed to mimic the interaction between LDs taking into account many other processes in addition to fusion (de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets) in order to reproduce the LDs growth and we also simulated the

  8. Models of lipid droplets growth and fission in adipocyte cells

    International Nuclear Information System (INIS)

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2015-01-01

    Lipid droplets (LD) are spherical cellular inclusion devoted to lipids storage. It is well known that excessive accumulation of lipids leads to several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis and atherosclerosis. LDs' size range from fraction to one hundred of micrometers in adipocytes and is related to the lipid content, but their growth is still a puzzling question. It has been suggested that LDs can grow in size due to the fusion process by which a larger LD is obtained by the merging of two smaller LDs, but these events seems to be rare and difficult to be observed. Many other processes are thought to be involved in the number and growth of LDs, like the de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets. Moreover the number and size of LDs are influenced by the catabolism and the absorption or interaction with other organelles. The comprehension of these processes could help in the confinement of the pathologies related to lipid accumulation. In this study the LDs' size distribution, number and the total volume of immature (n=12), mature (n=12, 10-days differentiated) and lipolytic (n=12) 3T3-L1 adipocytes were considered. More than 11,000 LDs were measured in the 36 cells after Oil Red O staining. In a previous work Monte Carlo simulations were used to mimic the fusion process alone between LDs. We found that, considering the fusion as the only process acting on the LDs, the size distribution in mature adipocytes can be obtained with numerical simulation starting from the size distribution in immature cells provided a very high rate of fusion events. In this paper Monte Carlo simulations were developed to mimic the interaction between LDs taking into account many other processes in addition to fusion (de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets) in order to reproduce the LDs growth and we also simulated the

  9. Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

    Science.gov (United States)

    Bouklas, Tejas; Alonso-Crisóstomo, Luz; Székely, Tamás; Diago-Navarro, Elizabeth; Orner, Erika P; Smith, Kalie; Munshi, Mansa A; Del Poeta, Maurizio; Balázsi, Gábor; Fries, Bettina C

    2017-05-01

    Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an

  10. 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.

  11. 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.

  12. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

    2015-10-30

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  13. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    International Nuclear Information System (INIS)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-01-01

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  14. Perspectives on the Trypanosoma cruzi–host cell receptor interactions

    Science.gov (United States)

    Villalta, Fernando; Scharfstein, Julio; Ashton, Anthony W.; Tyler, Kevin M.; Guan, Fangxia; Mukherjee, Shankar; Lima, Maria F.; Alvarez, Sandra; Weiss, Louis M.; Huang, Huan; Machado, Fabiana S.

    2009-01-01

    Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets. PMID:19283409

  15. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Directory of Open Access Journals (Sweden)

    Tongfang Tang

    Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  16. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Science.gov (United States)

    Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

    2013-01-01

    Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  17. Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Samual C. Allgood

    2017-09-01

    Full Text Available The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3P and PI(4P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I, a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella

  18. [NKT cells and graft-versus-host disease-review].

    Science.gov (United States)

    Zhao, Lei; Hao, Sha; Yuan, Wei-Ping; Cheng, Tao

    2013-10-01

    NKT cells (nature killer T cells), as a regulatory cellular compartment in the immune system, express cell surface markers of T cells and NK cells. It secretes a variety of cytokines that stimulate specific antigens. Through regulating the balance of Th1/Th2, the NKT cells play an important role in prevention and treatment of graft-versus-host disease (GVHD). Its antitumor and anti-infectious effects serve as a basis of its application in allogeneic hematopoietic stem cell transplantation. A better understanding of the biological and immunological features of NKT cell, as well as its specific immune regulatory mechanisms, will further justify the rationales of using NKT cells in the management of GVHD for patients. In this review, the biologic properties, classification, differentiation and development, immune activation of NKT cells as well as the NKT cells and GVHD including the related mechanisms of prevention and treatment of GVHD with NKT cells, NKT cells and tumors, NKT cells and infection, and NKT cells and clinical GVHD are summarized.

  19. The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

    International Nuclear Information System (INIS)

    Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.; Santos, Nuno C.

    2010-01-01

    Research highlights: → Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. → Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. → This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

  20. 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

  1. Effects of host nutrition on virulence and fitness of entomopathogenic nematodes: Lipid- and protein-based supplements in Tenebrio molitor diets

    Science.gov (United States)

    Shapiro-Ilan, David; Rojas, M. Guadalupe; Morales-Ramos, Juan A.; Lewis, Edwin E.; Tedders, W. Louis

    2008-01-01

    Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency. PMID:19259513

  2. Dietary Lipids, Cell Adhesion and Breast Cancer Metastasis

    Science.gov (United States)

    2003-10-01

    Mustela vison ): a light and electron microscopic study. APMIS 106. 785-799.caspase activity and apoptosis in neuronal cells (Francois et suy PI...N.Y. Acad. Sci. 919, 97-105. tic lesions in the mink ( Mustela vison ): A light and electron microscopic Krause, T., and Turner, G. A. (1999). Are...lipid composition is the most critical factor determining HUVEC with a plasmid regulated by a strong respiratory the efficiency of liposome-mediated

  3. Fatty Acids, Lipid Mediators, and T-Cell Function

    Science.gov (United States)

    de Jong, Anja J.; Kloppenburg, Margreet; Toes, René E. M.; Ioan-Facsinay, Andreea

    2014-01-01

    Research toward the mechanisms underlying obesity-linked complications has intensified during the last years. As a consequence, it has become clear that metabolism and immunity are intimately linked. Free fatty acids and other lipids acquired in excess by current feeding patterns have been proposed to mediate this link due to their immune modulatory capacity. The functional differences between saturated and unsaturated fatty acids, in combination with their dietary intake are believed to modulate the outcome of immune responses. Moreover, unsaturated fatty acids can be oxidized in a tightly regulated and specific manner to generate either potent pro-inflammatory or pro-resolving lipid mediators. These oxidative derivatives of fatty acids have received detailed attention during the last years, as they have proven to have strong immune modulatory capacity, even in pM ranges. Both fatty acids and oxidized fatty acids have been studied especially in relation to macrophage and T-cells functions. In this review, we propose to focus on the effect of fatty acids and their oxidative derivatives on T-cells, as it is an active area of research during the past 5 years. The effect of fatty acids and their derivatives on activation and proliferation of T-cells, as well as the delicate balance between stimulation and lipotoxicity will be discussed. Moreover, the receptors involved in the interaction between free fatty acids and their derivatives with T-cells will be summarized. Finally, the mechanisms involved in modulation of T-cells by fatty acids will be addressed, including cellular signaling and metabolism of T-cells. The in vitro results will be placed in context of in vivo studies both in humans and mice. In this review, we summarize the latest findings on the immune modulatory function of lipids on T-cells and will point out novel directions for future research. PMID:25352844

  4. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

    Science.gov (United States)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-10-30

    Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  6. Dual analysis of the murine cytomegalovirus and host cell transcriptomes reveal new aspects of the virus-host cell interface.

    Directory of Open Access Journals (Sweden)

    Vanda Juranic Lisnic

    Full Text Available Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq. We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus

  7. Lipid Bilayer Formation on Organic Electronic Materials

    KAUST Repository

    Zhang, Yi; Wustoni, Shofarul; Savva, Achilleas; Giovannitti, Alexander; McCulloch, Iain; Inal, Sahika

    2018-01-01

    The lipid bilayer is the elemental structure of cell membrane, forming a stable barrier between the interior and exterior of the cell while hosting membrane proteins that enable selective transport of biologically important compounds and cellular

  8. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  9. Correlation between cationic lipid-based transfection and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael, E-mail: michael@elbaum.ac.il

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

  10. Cryptosporidia: Epicellular parasites embraced by the host cell membrane

    Czech Academy of Sciences Publication Activity Database

    Valigurová, A.; Jirků, Miloslav; Koudela, Břetislav; Gelnar, M.; Modrý, David; Šlapeta, J.

    2008-01-01

    Roč. 38, 8/9 (2008), s. 913-922 ISSN 0020-7519 R&D Projects: GA ČR GD524/03/H133; GA ČR GA524/05/0992; GA MŠk LC522 Institutional research plan: CEZ:AV0Z60220518 Keywords : Cryptosporidium * host cell invasion * epicellular * parasitophorous sac * ultrastructure Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 3.752, year: 2008

  11. Effects of actonomycin D and ultraviolet irradiation on multiplication of brome mosaic virus in host and non-host cells

    International Nuclear Information System (INIS)

    Maekawa, K.; Furusawa, I.; Okuno, T.

    1981-01-01

    The modes of multiplication of brome mosaic virus (BMV) were compared in protoplasts isolated from host and non-host plants. BMV actively multiplied in the leaves and isolated mesophyll protoplasts of barley, a host of BMV. BMV multiplication in barley protoplasts was inhibited by addition of actinomycin D immediately after inoculation or by u.v. irradiation of the protoplasts before inoculation. In contrast, although BMV could not multiply in leaves of radish and turnip (non-hosts for BMV) it multiplied at a low level in protoplasts isolated from these two plant species. Moreover, u.v. irradiation, or the addition of actinomycin D, enhanced multiplication of BMV in radish and turnip protoplasts. These results suggest that (i) in the host cells replication of BMV is dependent on cellular metabolism of nucleic acid and protein, and (ii) in the non-host cells a substance(s) inhibitory to replication of BMV is synthesized. (author)

  12. Gut-derived short-chain fatty acids are vividly assimilated into host carbohydrates and lipids

    NARCIS (Netherlands)

    den Besten, Gijs; Lange, Katja; Havinga, Rick; van Dijk, Theo H.; Gerding, Albert; van Eunen, Karen; Muller, Michael; Groen, Albert K.; Hooiveld, Guido J.; Bakker, Barbara M.; Reijngoud, Dirk-Jan

    2013-01-01

    Acetate, propionate, and butyrate are the main short-chain fatty acids (SCFAs) that arise from the fermentation of fibers by the colonic microbiota. While many studies focus on the regulatory role of SCFAs, their quantitative role as a catabolic or anabolic substrate for the host has received

  13. Differential proteome analysis of chikungunya virus infection on host cells.

    Directory of Open Access Journals (Sweden)

    Christina Li-Ping Thio

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

  14. Variation in RNA virus mutation rates across host cells.

    Directory of Open Access Journals (Sweden)

    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  15. Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Dylan Myers Owen

    2013-12-01

    Full Text Available The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

  16. Molecular imaging of lipids in cells and tissues

    Science.gov (United States)

    Borner, Katrin; Malmberg, Per; Mansson, Jan-Eric; Nygren, Hakan

    2007-02-01

    The distribution pattern of lipid species in biological tissues was analyzed with imaging mass spectrometry (TOF-SIMS; time-of-flight secondary ion mass spectrometry). The first application shows distribution of a glycosphingolipid, the galactosylceramide-sulfate (sulfatide) with different hydrocarbon chain lengths and the fatty acids palmitate and oleate in rat cerebellum. Sulfatides were seen localized in regions suggested as paranodal areas of rat cerebellar white matter as well as in the granular layer, with highest concentrations at the borders of the white matter. Different distribution patterns could be shown for the fatty acid C16:0 palmitate and C18:1 oleate in rat cerebellum, which seem to origin partly from the hydrocarbon chains of phosphatidylcholine. Results were shown for two different tissue preparation methods, which were plunge-freezing and cryostat sectioning as well as high-pressure freezing, freeze-fracturing and freeze-drying. The second application shows TOF-SIMS analysis on a biological trial of choleratoxin treatment in mouse intestine. The effect of cholera toxin on lipids in the intestinal epithelium was shown by comparing control and cholera toxin treated mouse intestine samples. A significant increase of the cholesterol concentration was seen after treatment. Cholesterol was mainly localized to the brush border of enterocytes of the intestinal villi, which could be explained by the presence of cholesterol-rich lipid rafts present on the microvilli or by relations to cholesterol uptake. After cholera toxin exposure, cholesterol was seen increased in the nuclei of enterocytes and apparently in the interstitium of the villi. We find that imaging TOF-SIMS is a powerful tool for studies of lipid distributions in cells and tissues, enabling the elucidation of their role in cell function and biology.

  17. Some Lipid Droplets Are More Equal Than Others: Different Metabolic Lipid Droplet Pools in Hepatic Stellate Cells.

    Science.gov (United States)

    Molenaar, Martijn R; Vaandrager, Arie B; Helms, J Bernd

    2017-01-01

    Hepatic stellate cells (HSCs) are professional lipid-storing cells and are unique in their property to store most of the retinol (vitamin A) as retinyl esters in large-sized lipid droplets. Hepatic stellate cell activation is a critical step in the development of chronic liver disease, as activated HSCs cause fibrosis. During activation, HSCs lose their lipid droplets containing triacylglycerols, cholesteryl esters, and retinyl esters. Lipidomic analysis revealed that the dynamics of disappearance of these different classes of neutral lipids are, however, very different from each other. Although retinyl esters steadily decrease during HSC activation, triacylglycerols have multiple pools one of which becomes transiently enriched in polyunsaturated fatty acids before disappearing. These observations are consistent with the existence of preexisting "original" lipid droplets with relatively slow turnover and rapidly recycling lipid droplets that transiently appear during activation of HSCs. Elucidation of the molecular machinery involved in the regulation of these distinct lipid droplet pools may open new avenues for the treatment of liver fibrosis.

  18. Ureaplasma parvum infection alters filamin a dynamics in host cells

    Directory of Open Access Journals (Sweden)

    Brown Mary B

    2011-04-01

    Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

  19. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  20. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  1. Host-Polarized Cell Growth in Animal Symbionts.

    Science.gov (United States)

    Pende, Nika; Wang, Jinglan; Weber, Philipp M; Verheul, Jolanda; Kuru, Erkin; Rittmann, Simon K-M R; Leisch, Nikolaus; VanNieuwenhze, Michael S; Brun, Yves V; den Blaauwen, Tanneke; Bulgheresi, Silvia

    2018-04-02

    To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  2. Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET)

    Science.gov (United States)

    Ho, James C. S.; Storm, Petter; Rydström, Anna; Bowen, Ben; Alsin, Fredrik; Sullivan, Louise; Ambite, Inès; Mok, K. H.; Northen, Trent; Svanborg, Catharina

    2013-01-01

    Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance 13C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein. PMID:23629662

  3. 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

  4. Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

    Science.gov (United States)

    Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

    2018-02-28

    Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

  5. Host cell reactivation and UV-enhanced reactivation in synchronized mammalian cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Schmidt, B.J.

    1981-01-01

    Does host cell reactivation (HCR) or UV-enhanced reactivation (UVER) of UV-irradiated Herpes simplex virus (UV-HSV) vary during the host mammalian cell cycle. The answer could be useful for interpreting UVER and or the two-component nature of the UV-HSV survival curve. Procedures were developed for infection of mitotically-synchronized CV-l monkey kidney cells. All virus survival curves determined at different cell cycle stages had two components with similar D 0 's and intercepts of the second components. Thus, no single stage of the host cell cycle was responsible for the second component of the virus survival curve. When the cells were UV-irradiated immediately prior to infection, enhanced survival of UV-HSV occurred for cell irradiation and virus infection initiated during late G 1 early S phase or late S early G 2 phase but not during early G 1 phase. For infection delayed by 24 h after cell irradiation, UVER was found at all investigated times. These results indicate that: (1) HCR is similar at all stages of the host cell cycle: and (2) the ''induction'' of UVER is not as rapid for cell-irradiation in early G 1 phase. This latter observation may be one reason why normal, contact-inhibited cells do not express UVER as rapidly as faster growing, less contact-inhibited cells. (author)

  6. Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application

    Science.gov (United States)

    2018-01-01

    Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids. PMID:29503972

  7. Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

    Energy Technology Data Exchange (ETDEWEB)

    Flitter, Becca A.; Hvorecny, Kelli L.; Ono, Emiko; Eddens, Taylor; Yang, Jun; Kwak, Daniel H.; Bahl, Christopher D.; Hampton, Thomas H.; Morisseau, Christophe; Hammock, Bruce D.; Liu, Xinyu; Lee, Janet S.; Kolls, Jay K.; Levy, Bruce D.; Madden, Dean R.; Bomberger, Jennifer M.

    2016-12-15

    Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8–driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

  8. Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

    Directory of Open Access Journals (Sweden)

    Joanne K Gardner

    Full Text Available Dendritic cells (DCs play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay, upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+ CD8α- DCs, CD4- CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

  9. Rotavirus RRV associates with lipid membrane microdomains during cell entry

    International Nuclear Information System (INIS)

    Isa, Pavel; Realpe, Mauricio; Romero, Pedro; Lopez, Susana; Arias, Carlos F.

    2004-01-01

    Rotavirus cell entry is a multistep process, not completely understood, which requires at least four interactions between the virus and cell surface molecules. In this work, we investigated the role of the sphingolipid- and cholesterol-enriched lipid microdomains (rafts) in the entry of rotavirus strain RRV to MA104 cells. We found that ganglioside GM1, integrin subunits α2 and β3, and the heat shock cognate protein 70 (hsc70), all of which have been implicated as rotavirus receptors, are associated with TX-100 and Lubrol WX detergent-resistant membranes (DRMs). Integrin subunits α2 and β3 were found to be particularly enriched in DRMs resistant to lysis by Lubrol WX. When purified RRV particles were incubated with cells at 4 deg. C, about 10% of the total infectious virus was found associated with DRMs, and the DRM-associated virus increased to 37% in Lubrol-resistant membrane domains after 60-min incubation at 37 deg. C. The virus was excluded from DRMs if the cells were treated with methyl-β-cyclodextrin (MβCD). Immunoblot analysis of the viral proteins showed that the virus surface proteins became enriched in DRMs upon incubation at 37 deg. C, being almost exclusively localized in Lubrol-resistant DRMs after 60 min. These data suggest that detergent-resistant membrane domains play an important role in the cell entry of rotaviruses, which could provide a platform to facilitate the efficient interaction of the rotavirus receptors with the virus particle

  10. Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

    Science.gov (United States)

    Nair, Shiny; Boddupalli, Chandra Sekhar; Verma, Rakesh; Liu, Jun; Yang, Ruhua; Pastores, Gregory M; Mistry, Pramod K; Dhodapkar, Madhav V

    2015-02-19

    Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders. © 2015 by The American Society of Hematology.

  11. Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

    Science.gov (United States)

    Romano, Julia D; de Beaumont, Catherine; Carrasco, Jose A; Ehrenman, Karen; Bavoil, Patrik M; Coppens, Isabelle

    2013-02-01

    The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

  12. 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.

  13. Intravital imaging of donor allogeneic effector and regulatory T cells with host dendritic cells during GVHD.

    Science.gov (United States)

    Lin, Kaifeng Lisa; Fulton, LeShara M; Berginski, Matthew; West, Michelle L; Taylor, Nicholas A; Moran, Timothy P; Coghill, James M; Blazar, Bruce R; Bear, James E; Serody, Jonathan S

    2014-03-06

    Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon-DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.

  14. The effect of MLS laser radiation on cell lipid membrane.

    Science.gov (United States)

    Pasternak, Kamila; Wróbel, Dominika; Nowacka, Olga; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

    2018-03-14

    Authors of numerous publications have proved the therapeutic effect of laser irradiation on biological material, but the mechanisms at cellular and subcellular level are not yet well understood. The aim of this study was to assess the effect of laser radiation emitted by the MLS M1 system (Multiwave Locked System) at two wavelengths (808 nm continuous and 905 nm pulsed) on the stability and fluidity of liposomes with a lipid composition similar to that of human erythrocyte membrane or made of phosphatidylocholine. Liposomes were exposed to low-energy laser radiation at surface densities 195 mW/cm2 (frequency 1,000 Hz) and 230 mW/cm2 (frequency 2,000 Hz). Different doses of radiation energy in the range 0-15 J were applied. The surface energy density was within the range 0.46 - 4.9 J/cm 2. The fluidity and stability of liposomes subjected to such irradiation changed depending on the parameters of radiation used. Since MLS M1 laser radiation, depending on the parameters used, affects fluidity and stability of liposomes with the lipid content similar to erythrocyte membrane, it may also cause structural and functional changes in cell membranes.

  15. Mechanical cell disruption of Parachlorella kessleri microalgae: Impact on lipid fraction composition.

    Science.gov (United States)

    Clavijo Rivera, E; Montalescot, V; Viau, M; Drouin, D; Bourseau, P; Frappart, M; Monteux, C; Couallier, E

    2018-05-01

    Samples of nitrogen-starved Parachlorella kessleri containing intact cells (IC), cells ground by bead milling (BM), and cells subjected to high-pressure cell disruption (HPD), together with their supernatants after centrifugation, were compared for granulometry and lipid profiles. The effects of disruption on the lipid profile and organisation were evaluated. The quantity of lipids available for extraction increased with disruption, and up to 81% could be recovered in supernatants after centrifugation, but a marked reorganization occurred. The proportion of amphiphilic free fatty acids and lysophosphatidylcholine increased during disruption due to their release or owing to lipid degradation by enzymes or physical conditions. This effect was more marked in HPD than in BM. Lipids contained in the aqueous phase, after disruption and centrifugation, were enriched in unsaturated fatty acids, BM leading to larger droplets than HPD. The larger liquid lipid droplet would be easier to recover in the following downstream processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

    Science.gov (United States)

    Bieberich, Erhard

    2011-04-26

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

  17. Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes

    Directory of Open Access Journals (Sweden)

    T.R. Oliveira

    2009-09-01

    Full Text Available Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR and normotensive control rat strains (WKY and NWR. Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

  18. Sustained Cytotoxicity of Wogonin on Breast Cancer Cells by Encapsulation in Solid Lipid Nanoparticles

    Directory of Open Access Journals (Sweden)

    Jong-Suep Baek

    2018-03-01

    Full Text Available While wogonin has been known to have cytotoxicity against various cancer cells, its bioavailability and cytotoxicity are low due to its low water solubility. Therefore, wogonin-loaded solid lipid nanoparticles were fabricated using a hot-melted evaporation technique. The highest solubility of wogonin was observed in stearic acid. Hence, wogonin-loaded solid lipid nanoparticles were composed of stearic acid as the lipid matrix. The physicochemical properties of the wogonin-loaded solid lipid nanoparticles were evaluated by dynamic laser scattering and scanning electron microscopy. The wogonin-loaded solid lipid nanoparticles exhibited sustained and controlled release up to 72 h. In addition, it was observed that the wogonin-loaded solid lipid nanoparticles exhibited enhanced cytotoxicity and inhibited poly (ADP-ribose polymerase in MCF-7 breast cancer cells. Overall, the results indicate that wogonin-loaded solid lipid nanoparticles could be an efficient delivery system for the treatment of breast cancer.

  19. Lipid profiling of in vitro cell models of adipogenic differentiation: relationships with mouse adipose tissues

    OpenAIRE

    Liaw, Lucy; Prudovsky, Igor; Koza, Robert A.; Anunciado-Koza, Rea V.; Siviski, Matthew E.; Lindner, Volkhard; Friesel, Robert E.; Rosen, Clifford J.; Baker, Paul R.S.; Simons, Brigitte; Vary, Calvin P.H.

    2016-01-01

    Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MSALL. Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-...

  20. Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins.

    Science.gov (United States)

    Weir, Dawn L; Smith, Ina L; Bossart, Katharine N; Wang, Lin-Fa; Broder, Christopher C

    2013-09-01

    Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s). Published by Elsevier Inc.

  1. Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2014-10-01

    Full Text Available Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

  2. Regulation of stem-cell mediated host immunity by the sphingolipid ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Regulation of stem-cell mediated host immunity by the sphingolipid pathway ... in the generation of mature immune cells and the functioning of the surrounding ... methods with human cells and genetically engineered mice to examine how the ...

  3. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    Science.gov (United States)

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  4. Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

    Science.gov (United States)

    Pollock, Abigail H; Tedla, Nicodemus; Hancock, Sarah E; Cornely, Rhea; Mitchell, Todd W; Yang, Zhengmin; Kockx, Maaike; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-05-15

    Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. The perfect host: a mouse host embryo facilitating more efficient germ line transmission of genetically modified embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Robert A Taft

    Full Text Available There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs. We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium. ESC germline transmission was observed in 9/11 (82% lines using PH blastocysts, compared to 6/11 (55% when conventional host blastocysts were used. Furthermore, less than 35% (83/240 of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137 of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the

  6. Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

    Science.gov (United States)

    Kamachi, Yasuharu; Omasa, Takeshi

    2018-04-01

    Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Preparation of labelled lipids by the use of plant cell cultures

    International Nuclear Information System (INIS)

    Mangold, H.K.

    1978-01-01

    The preparation of some radioacitvely labelled lipids by the use of plant cell cultures is discussed and further applications of the new method are suggested. Cell suspension cultures of rape (Brassica napus) and soya (Glycine max) have been used for the preparation of lipids labelled with radioisotopes. Radioactive acetic acid as well as various long-chain fatty acids are readily incorporated into the neutral and ionic lipids of plant cell cultures. In addition, 14 C-labelled glycerol, ethanolamine and choline are well utilized by the cells. Randomly labelled lipids have been obtained by incubating cell suspension cultures of rape and soya with [1- 14 C] acetic acid, and uniformly labelled lipids have been isolated from cultures that had been incubated with a mixture of [1- 14 C] acetic acid plus [2- 14 C] acetic acid. The use of techniques of plant cell cultures for the preparation of lipds labelled with stable or radioactive isotopesappears particularly rewarding because the uptake of precursors by the cells and their incorporation into various lipid compounds proceeds rapidly and often quanitatively.This new approach should be useful also for the biosynthesis of lipids whose acyl moieties contain a spn radical, a fluorescent group, or a light-sensitive label. Thus, plant cell cultures constitute valuable new tools for the biosynthetic preparation of a great variety of labelled lipids. (A.G.)

  8. Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

    Science.gov (United States)

    O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

    2014-01-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

  9. Post irradiative stimulation of the lipids synthesis in the cells of Anacystis nidulans

    International Nuclear Information System (INIS)

    Groshev, V.V.; Tiflova, O.A.

    1982-01-01

    Ultraviolet and X-ray irradiations stimulate postradiation synthesis of fatty acids of lipids in cells of Anacystis nidulans. Stimulation degree is proportional to the radiation dose and time of postradiation incubation of cells

  10. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models.

    Science.gov (United States)

    Chrast, Roman; Saher, Gesine; Nave, Klaus-Armin; Verheijen, Mark H G

    2011-03-01

    The integrity of central and peripheral nervous system myelin is affected in numerous lipid metabolism disorders. This vulnerability was so far mostly attributed to the extraordinarily high level of lipid synthesis that is required for the formation of myelin, and to the relative autonomy in lipid synthesis of myelinating glial cells because of blood barriers shielding the nervous system from circulating lipids. Recent insights from analysis of inherited lipid disorders, especially those with prevailing lipid depletion and from mouse models with glia-specific disruption of lipid metabolism, shed new light on this issue. The particular lipid composition of myelin, the transport of lipid-associated myelin proteins, and the necessity for timely assembly of the myelin sheath all contribute to the observed vulnerability of myelin to perturbed lipid metabolism. Furthermore, the uptake of external lipids may also play a role in the formation of myelin membranes. In addition to an improved understanding of basic myelin biology, these data provide a foundation for future therapeutic interventions aiming at preserving glial cell integrity in metabolic disorders.

  12. Identification of Sphingomyelinase on the Surface of Chlamydia pneumoniae: Possible Role in the Entry into Its Host Cells

    Directory of Open Access Journals (Sweden)

    Tuula A. Peñate Medina

    2014-01-01

    Full Text Available We have recently suggested a novel mechanism, autoendocytosis, for the entry of certain microbes into their hosts, with a key role played by the sphingomyelinase-catalyzed topical conversion of sphingomyelin to ceramide, the differences in the biophysical properties of these two lipids providing the driving force. The only requirement for such microbes to utilize this mechanism is that they should have a catalytically active SMase on their outer surface while the target cells should expose sphingomyelin in the external leaflet of their plasma membrane. In pursuit of possible microbial candidates, which could utilize this putative mechanism, we conducted a sequence similarity search for SMase. Because of the intriguing cellular and biochemical characteristics of the poorly understood entry of Chlamydia into its host cells these microbes were of particular interest. SMase activity was measured in vitro from isolated C. pneumoniae elementary bodies (EB and in the lysate from E. coli cells transfected with a plasmid expressing CPn0300 protein having sequence similarity to SMase. Finally, pretreatment of host cells with exogenous SMase resulting in loss plasma membrane sphingomyelin attenuated attachment of EB.

  13. 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.

  14. 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.

  15. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    Energy Technology Data Exchange (ETDEWEB)

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2017-08-22

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  16. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    Science.gov (United States)

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  17. Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition.

    Science.gov (United States)

    Bukholm, G; Tannaes, T; Nedenskov, P; Esbensen, Y; Grav, H J; Hovig, T; Ariansen, S; Guldvog, I

    1997-05-01

    Differences in expression of disease after infection with Helicobacter pylori have so far been connected with host factors and bacterial interstrain variation. In this study, spontaneous and ecology-mediated intrastrain variation was examined. Four clinical isolates of H. pylori were shown to give rise to two colony forms. Bacterial morphology was examined by electron microscopy. Bacterial fractions were examined for proteins using ion exchange chromatography and SDS-PAGE; for lipids using thin-layer chromatography, lipid anion-exchange chromatography, column chromatography on silica gel, 31P-NMR, gas chromatography and mass spectrometry. Bacterial in vitro invasiveness and adhesiveness were examined in two different systems, and urease and VacA toxin were assayed by Western blot analysis. H. pylori was shown to give rise to two colony forms: at normal pH the population was dominated by L colonies. One strain was chosen for further studies. Bacteria from L colonies retained VacA toxin and urease, did not invade or adhere to epithelial cells, and contained normal quantities of phosphatidylethanolamine. In a small frequency, spontaneous S colonies were formed. Bacteria from these colonies released VacA and urease, adhered to and invaded epithelial cells and contained increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine. After addition of HCl to the culture medium (pH6), almost only S colonies were formed. The results demonstrate that environmental factors, such as HCl, can change the bacterial cell wall, and thereby enhance expression of virulence factors of H. pylori in vitro. A similar in vivo variation would have implications for our understanding of the interaction between HCl secretion in the gastric mucosa and H. pylori in the development of peptic ulcer disease.

  18. Exosomal lipids impact notch signaling and induce death of human pancreatic tumoral SOJ-6 cells.

    Directory of Open Access Journals (Sweden)

    Sadia Beloribi

    Full Text Available Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glycoprotein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008 FASEB J. 22; 3358-3369 enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo and depleted in phospholipids (lipids forming liquid-disordered phase, Ld, we designed Synthetic Exosome-Like Nanoparticles (SELN with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.

  19. [Assessment of lipid layer thickness of tear film in the diagnosis of dry-eye syndrome in children after the hematopoietic stem cell transplantation].

    Science.gov (United States)

    Kurpińska, Małgorzata; Gorczyńska, Ewa; Owoc-Lempach, Joanna; Bernacka, Aleksandra; Misiuk-Hojło, Marta; Chybicka, Alicja

    2011-01-01

    Dry eye syndrome (DES), also known as keratoconjunctivitis sicca (KCS) is recognized as the most frequent ocular complication after allogeneic stem cell transplantation (allo-SCT). KCS can appear either due to insufficient tear production or excessive tear evaporation, both resulting in tears hyperosmolarity that leads to ocular damage. The evaporation rate and better film stability is determined primarily by the status of the lipid layer. Observation and classification of tear film lipid layer interference patterns in normal and dry eyes in patients after allogeneic stem cell transplantation with a follow-up time of 6 months-5 years (median 26.54 months). Investigation of the relation between the lipid layer interference patterns in normal and dry eyes and the results of other dry eye examinations and complaints. Relation between DES and conditioning regimes, including total body irradiation and high-dose chemotherapy, immunosuppressive drugs, the time after allogeneic stem cell transplantation and chronic graft-versus-host disease. Precorneal tears lipid layer interference patterns, were examined in 114 eyes in treatment group with the Tearscope-plus. Patient with dry eye were identified on the basis of Schirmer test scores and/or tear breakup time, and positive lissamine and/or fluorescein staining. 42 of 114 eyes (36.8%) developed DES after allo-SCT A significant correlation between thickness of lipid layer and BUT, Schirmer test, lissamine green and fluorescein staining was found in the treatment group. A significant association was found between present chronic GVHD and DES in children. DES was not associated with TBI, corticosteroids, immunosuppressive drugs and the time in the present study. Tears lipid layer interference patterns are highly correlated with the diagnosis of DES. Tears lipid layer interference patterns ( noninvasive method), can be used to diagnose early DES in children after allo-SCT. Chronic GVHD play a major role in development of DES

  20. Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids

    Directory of Open Access Journals (Sweden)

    Ainhoa eARBUES

    2014-12-01

    Full Text Available Mycobacterial pathogens, including Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB, have evolved a remarkable ability to evade the immune system in order to survive and to colonize the host. Among the most important evasion strategies is the capacity of these bacilli to parasitize host macrophages, since these are major effector cells against intracellular pathogens that can be used as long-term cellular reservoirs. Mycobacterial pathogens employ an array of virulence factors that manipulate macrophage function to survive and establish infection. Until recently, however, the role of mycobacterial cell envelope lipids as virulence factors in macrophage subversion has remained elusive. Here, we will address exclusively the proposed role for phthiocerol dimycocerosates (DIM in the modulation of the resident macrophage response and that of phenolic glycolipids (PGL in the regulation of the recruitment and phenotype of incoming macrophage precursors to the site of infection. We will provide a unique perspective of potential additional functions for these lipids, and highlight obstacles and opportunities to further understand their role in the pathogenesis of TB and other mycobacterial diseases.

  1. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    Science.gov (United States)

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  2. Host Cell Restriction Factors that Limit Influenza A Infection

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    Fernando Villalón-Letelier

    2017-12-01

    Full Text Available Viral infection of different cell types induces a unique spectrum of host defence genes, including interferon-stimulated genes (ISGs and genes encoding other proteins with antiviral potential. Although hundreds of ISGs have been described, the vast majority have not been functionally characterised. Cellular proteins with putative antiviral activity (hereafter referred to as “restriction factors” can target various steps in the virus life-cycle. In the context of influenza virus infection, restriction factors have been described that target virus entry, genomic replication, translation and virus release. Genome wide analyses, in combination with ectopic overexpression and/or gene silencing studies, have accelerated the identification of restriction factors that are active against influenza and other viruses, as well as providing important insights regarding mechanisms of antiviral activity. Herein, we review current knowledge regarding restriction factors that mediate anti-influenza virus activity and consider the viral countermeasures that are known to limit their impact. Moreover, we consider the strengths and limitations of experimental approaches to study restriction factors, discrepancies between in vitro and in vivo studies, and the potential to exploit restriction factors to limit disease caused by influenza and other respiratory viruses.

  3. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

    Directory of Open Access Journals (Sweden)

    Katrin Bomans

    Full Text Available Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS. However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day manner.

  4. Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

    Science.gov (United States)

    Liaw, Lucy; Prudovsky, Igor; Koza, Robert A; Anunciado-Koza, Rea V; Siviski, Matthew E; Lindner, Volkhard; Friesel, Robert E; Rosen, Clifford J; Baker, Paul R S; Simons, Brigitte; Vary, Calvin P H

    2016-09-01

    Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

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    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.

  6. Patterns of oligonucleotide sequences in viral and host cell RNA identify mediators of the host innate immune system.

    Directory of Open Access Journals (Sweden)

    Benjamin D Greenbaum

    Full Text Available The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context--which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans--is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.

  7. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

    Science.gov (United States)

    Viswanathan, Vasanthi S; Ryan, Matthew J; Dhruv, Harshil D; Gill, Shubhroz; Eichhoff, Ossia M; Seashore-Ludlow, Brinton; Kaffenberger, Samuel D; Eaton, John K; Shimada, Kenichi; Aguirre, Andrew J; Viswanathan, Srinivas R; Chattopadhyay, Shrikanta; Tamayo, Pablo; Yang, Wan Seok; Rees, Matthew G; Chen, Sixun; Boskovic, Zarko V; Javaid, Sarah; Huang, Cherrie; Wu, Xiaoyun; Tseng, Yuen-Yi; Roider, Elisabeth M; Gao, Dong; Cleary, James M; Wolpin, Brian M; Mesirov, Jill P; Haber, Daniel A; Engelman, Jeffrey A; Boehm, Jesse S; Kotz, Joanne D; Hon, Cindy S; Chen, Yu; Hahn, William C; Levesque, Mitchell P; Doench, John G; Berens, Michael E; Shamji, Alykhan F; Clemons, Paul A; Stockwell, Brent R; Schreiber, Stuart L

    2017-07-27

    Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

  8. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    Science.gov (United States)

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

    Science.gov (United States)

    Niphakis, Micah J; Lum, Kenneth M; Cognetta, Armand B; Correia, Bruno E; Ichu, Taka-Aki; Olucha, Jose; Brown, Steven J; Kundu, Soumajit; Piscitelli, Fabiana; Rosen, Hugh; Cravatt, Benjamin F

    2015-06-18

    Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    Science.gov (United States)

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes. © 2013 Elsevier Ltd. All rights reserved.

  11. 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

  12. Effects of gamma-irradiation on the glycogen and lipid contents of the rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Nahed, R H.A.; Al-Zahaby, Al-Ahmmady, S.; Sanad, S M.K.; Roushdy, H M

    1986-01-01

    Histochemical changes in the glycogen and lipid contents of the rat liver cells were studied at different intervals following whole body gamma-irradiation at the exposure dose level of 600 rads. The glycogen and lipid contents were significantly altered, the changes were time-dependent.

  13. A Molecular Probe for the Detection of Polar Lipids in Live Cells.

    Science.gov (United States)

    Bader, Christie A; Shandala, Tetyana; Carter, Elizabeth A; Ivask, Angela; Guinan, Taryn; Hickey, Shane M; Werrett, Melissa V; Wright, Phillip J; Simpson, Peter V; Stagni, Stefano; Voelcker, Nicolas H; Lay, Peter A; Massi, Massimiliano; Plush, Sally E; Brooks, Douglas A

    2016-01-01

    Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular

  14. Bioactive lipid coating of bone allografts directs engraftment and fate determination of bone marrow-derived cells in rat GFP chimeras

    OpenAIRE

    Das, Anusuya; Segar, Claire E.; Chu, Yihsuan; Wang, Tiffany W.; Lin, Yong; Yang, Chunxi; Du, Xeujun; Ogle, Roy C.; Cui, Quanjun; Botchwey, Edward A.

    2015-01-01

    Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to lo...

  15. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  16. Targeting lipid metabolism of cancer cells: A promising therapeutic strategy for cancer.

    Science.gov (United States)

    Liu, Qiuping; Luo, Qing; Halim, Alexander; Song, Guanbin

    2017-08-10

    One of the most important metabolic hallmarks of cancer cells is deregulation of lipid metabolism. In addition, enhancing de novo fatty acid (FA) synthesis, increasing lipid uptake and lipolysis have also been considered as means of FA acquisition in cancer cells. FAs are involved in various aspects of tumourigenesis and tumour progression. Therefore, targeting lipid metabolism is a promising therapeutic strategy for human cancer. Recent studies have shown that reprogramming lipid metabolism plays important roles in providing energy, macromolecules for membrane synthesis, and lipid signals during cancer progression. Moreover, accumulation of lipid droplets in cancer cells acts as a pivotal adaptive response to harmful conditions. Here, we provide a brief review of the crucial roles of FA metabolism in cancer development, and place emphasis on FA origin, utilization and storage in cancer cells. Understanding the regulation of lipid metabolism in cancer cells has important implications for exploring a new therapeutic strategy for management and treatment of cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts

    Directory of Open Access Journals (Sweden)

    Shizuka eMiura

    2014-08-01

    Full Text Available Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.

  18. Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent.

    Science.gov (United States)

    Opitz, Lars; Zimmermann, Anke; Lehmann, Sylvia; Genzel, Yvonne; Lübben, Holger; Reichl, Udo; Wolff, Michael W

    2008-12-01

    Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.

  19. Recruitment of host's progenitor cells to sites of human amniotic fluid stem cells implantation.

    Science.gov (United States)

    Mirabella, Teodelinda; Poggi, Alessandro; Scaranari, Monica; Mogni, Massimo; Lituania, Mario; Baldo, Chiara; Cancedda, Ranieri; Gentili, Chiara

    2011-06-01

    The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Cell wall lipids from Mycobacterium bovis BCG are inflammatory when inoculated within a gel matrix: characterization of a new model of the granulomatous response to mycobacterial components.

    Science.gov (United States)

    Rhoades, Elizabeth R; Geisel, Rachel E; Butcher, Barbara A; McDonough, Sean; Russell, David G

    2005-05-01

    The chronic inflammatory response to Mycobacterium generates complex granulomatous lesions that balance containment with destruction of infected tissues. To study the contributing factors from host and pathogen, we developed a model wherein defined mycobacterial components and leukocytes are delivered in a gel, eliciting a localized response that can be retrieved and analysed. We validated the model by comparing responses to the cell wall lipids from Mycobacterium bovis bacillus Calmette-Guerin (BCG) to reported activities in other models. BCG lipid-coated beads and bone marrow-derived macrophages (input macrophages) were injected intraperitoneally into BALB/c mice. Input macrophages and recruited peritoneal exudate cells took up fluorescently tagged BCG lipids, and matrix-associated macrophages and neutrophils produced tumor necrosis factor, interleukin-1alpha, and interleukin-6. Leukocyte numbers and cytokine levels were greater in BCG lipid-bearing matrices than matrices containing non-coated or phosphatidylglycerol-coated beads. Leukocytes arrived in successive waves of neutrophils, macrophages and eosinophils, followed by NK and T cells (CD4(+), CD8(+), or gammadelta) at 7 days and B cells within 12 days. BCG lipids also predisposed matrices for adherence and vascularization, enhancing cellular recruitment. We submit that the matrix model presents pertinent features of the murine granulomatous response that will prove to be an adaptable method for study of this complex response.

  1. Donor Satellite Cell Engraftment is Significantly Augmented When the Host Niche is Preserved and Endogenous Satellite Cells are Incapacitated

    Science.gov (United States)

    Boldrin, Luisa; Neal, Alice; Zammit, Peter S; Muntoni, Francesco; Morgan, Jennifer E

    2012-01-01

    Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy. Stem Cells2012;30:1971–1984 PMID:22730231

  2. Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

    Science.gov (United States)

    De Nisco, Nicole J.; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-01-01

    Bacterial effectors are potent manipulators of host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para), delivers effectors into host cells through two type three secretion systems (T3SS). The ubiquitous T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate non-apoptotic cell death. Much is known about how T3SS1 effectors function in isolation, but we wanted to understand how their concerted action globally affects host cell signaling. To assess the host response to T3SS1, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1+) to those in cells infected with V. para lacking T3SS1 (T3SS1−). Overall, the host transcriptional response to both T3SS1+ and T3SS1− V. para was rapid, robust, and temporally dynamic. T3SS1 re-wired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors target host cells at the posttranslational level to cause cytotoxicity, network analysis indicated that V. para T3SS1 also precipitates a host transcriptional response that initially activates cell survival and represses cell death networks. The increased expression of several key pro-survival transcripts mediated by T3SS1 was dependent on a host signaling pathway that is silenced later in infection by the posttranslational action of T3SS1. Taken together, our analysis reveals a complex interplay between roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. PMID:28512145

  3. 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.

  4. 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.

  5. Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes.

    Science.gov (United States)

    Killackey, Samuel A; Sorbara, Matthew T; Girardin, Stephen E

    2016-01-01

    Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general.

  6. Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen.

    Science.gov (United States)

    Abos Gracia, Beatriz; López Relaño, Juan; Revilla, Ana; Castro, Lourdes; Villalba, Mayte; Martín Adrados, Beatriz; Regueiro, Jose Ramon; Fernández-Malavé, Edgar; Martínez Naves, Eduardo; Gómez Del Moral, Manuel

    2017-01-01

    Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses. © 2017 S. Karger AG, Basel.

  7. An Overview of Lipid Droplets in Cancer and Cancer Stem Cells

    KAUST Repository

    Tirinato, Luca; Pagliari, Francesca; Limongi, Tania; Marini, Monica; Falqui, Andrea; Seco, J.; Candeloro, Patrizio; Liberale, Carlo; Di Fabrizio, Enzo M.

    2017-01-01

    For decades, lipid droplets have been considered as the main cellular organelles involved in the fat storage, because of their lipid composition. However, in recent years, some new and totally unexpected roles have been discovered for them: (i) they are active sites for synthesis and storage of inflammatory mediators, and (ii) they are key players in cancer cells and tissues, especially in cancer stem cells. In this review, we summarize the main concepts related to the lipid droplet structure and function and their involvement in inflammatory and cancer processes.

  8. An Overview of Lipid Droplets in Cancer and Cancer Stem Cells

    KAUST Repository

    Tirinato, Luca

    2017-08-13

    For decades, lipid droplets have been considered as the main cellular organelles involved in the fat storage, because of their lipid composition. However, in recent years, some new and totally unexpected roles have been discovered for them: (i) they are active sites for synthesis and storage of inflammatory mediators, and (ii) they are key players in cancer cells and tissues, especially in cancer stem cells. In this review, we summarize the main concepts related to the lipid droplet structure and function and their involvement in inflammatory and cancer processes.

  9. Phage adsorption and lytic propagation in Lactobacillus plantarum: Could host cell starvation affect them?

    OpenAIRE

    Briggiler Marc?, Mari?ngeles; Reinheimer, Jorge; Quiberoni, Andrea

    2015-01-01

    Background Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. Result First, cell growth kinetics ...

  10. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.

    Science.gov (United States)

    Wunsch, Christopher M; Lewis, Janina P

    2015-12-17

    Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal

  11. Epigenetic Modulation of the Biophysical Properties of Drug-Resistant Cell Lipids to Restore Drug Transport and Endocytic Functions

    OpenAIRE

    Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

    2012-01-01

    In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g....

  12. Characterization of membrane lipid fluidity in human embryo cells malignantly transfer med post 238Pu α irradiation

    International Nuclear Information System (INIS)

    Qi Zirong; Sun Ling; Liu Guolian; Shen Zhiyuan

    1992-01-01

    The membrane lipid fluidity of malignantly transformed human embryo cells following 238 Pu α particlce irradiation in vitro has been studied. The results indicate that the ontogenesis depends on irradiation dose (Gy) and the membrane lipid fluidity in malignantly transformed cells is higher than that in normal embryo cells. With the microviscosity (η) of cells plotted against the cell counts, the correlation coefficient (γ) is calculated to be between 0.9936 and 0.9999. Since the malignant transformation of irradiated embryo cells is manifested early on cell membrane lipid, the fluidity of membrane lipid can be used as an oncologic marker

  13. 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.

  14. Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction

    Science.gov (United States)

    Pedrini, Nicolás; Ortiz-Urquiza, Almudena; Huarte-Bonnet, Carla; Zhang, Shizhu; Keyhani, Nemat O.

    2013-01-01

    Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and four CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of six genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon

  15. Differential modulation by Akkermansia muciniphila and faecalibacterium prausnitzii of host peripheral lipid metabolism and histone acetylation in mouse gut organoids

    NARCIS (Netherlands)

    Lukovac, S.; Belzer, C.; Pellis, L.; Keijser, B.J.; Vos, W.M. de; Montijn, R.C.; Roeselers, G.

    2014-01-01

    The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal

  16. SPOC1-mediated antiviral host cell response is antagonized early in human adenovirus type 5 infection

    DEFF Research Database (Denmark)

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin

    2013-01-01

    , and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its...... viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host...

  17. Physiological and molecular characterization of atypical lipid-dependent Malassezia yeasts from a dog with skin lesions: adaptation to a new host?

    NARCIS (Netherlands)

    Cafarchia, C.; Latrofa, M.S.; Figueredo, L.A.; da Silva Machado, M.L.; Ferreiro, L.; Guillot, J.; Boekhout, T.; Otranto, D.

    2011-01-01

    Three lipid-dependent Malassezia isolates (here named 114A, 114B and 114C) recovered from a dog with skin lesions were phenotypically and genotypically characterized. All presented ovoid cells and buds formed on a narrow base. Most of the results from physiological tests were consistent with those

  18. The lipid content of cisplatin- and doxorubicin-resistant MCF-7 human breast cancer cells.

    Science.gov (United States)

    Todor, I N; Lukyanova, N Yu; Chekhun, V F

    2012-07-01

    To perform the comparative study both of qualitative and quantitative content of lipids in parental and drug resistant breast cancer cells. Parental (MCF-7/S) and resistant to cisplatin (MCF-7/CP) and doxorubicin (MCF-7/Dox) human breast cancer cells were used in the study. Cholesterol, total lipids and phospholipids content were determined by means of thin-layer chromatography. It was found that cholesterol as well as cholesterol ethers content are significantly higher but diacylglycerols, triacyl-glycerols content are significantly lower in resistant cell strains than in parental (sensitive) cells. Moreover the analysis of individual phospholipids showed the increase of sphingomyelin, phosphatidylserine, cardiolipin, phosphatidic acid and the decrease of phosphatidy-lethanolamine, phosphatidylcholine in MCF-7/CP and MCF-7/Dox cells. Obtained results allow to suggest that the lipid profile changes can mediate the modulation of membrane fluidity in drug resistant MCF-7 breast cancer cells.

  19. Characterization of host lymphoid cells in antibody-facilitated bone marrow chimeras

    International Nuclear Information System (INIS)

    McCarthy, S.A.; Griffith, I.J.; Gambel, P.; Francescutti, L.H.; Wegmann, T.G.

    1985-01-01

    The authors have produced stable murine antibody-facilitated (AF) chimeras by the simultaneous injection of P1 bone marrow cells and anti-P2 monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These AF chimeras are healthy, long-lived, and exhibit no overt signs of graft-versus-host disease. They are immunocompetent and tolerant of host, P2-encoded alloantigens. Donor cell engraftment and takeover, monitored by glucosephosphate isomerase isozyme patterns, is usually complete (greater than 95%) in the peripheral blood, bone marrow, and hemopoietic stem cell compartments of long-term (greater than 3 months posttransplantation) AF chimeras. The authors report here, however, that splenic, lymph node, and thymic leukocytes of AF chimeras represent donor/host chimeric populations. Spleen cell populations of AF chimeras exhibit substantial chimera-to-chimera variation in the preponderant residual host cell type(s) present. Interpretations of the implications of these findings are discussed

  20. Molecular model of a type III secretion system needle: Implications for host-cell sensing.

    Science.gov (United States)

    Deane, Janet E; Roversi, Pietro; Cordes, Frank S; Johnson, Steven; Kenjale, Roma; Daniell, Sarah; Booy, Frank; Picking, William D; Picking, Wendy L; Blocker, Ariel J; Lea, Susan M

    2006-08-15

    Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

  1. Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

    Science.gov (United States)

    Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

    2016-03-03

    The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

  2. Positive regulation of prostate cancer cell growth by lipid droplet forming and processing enzymes DGAT1 and ABHD5

    OpenAIRE

    Mitra, Ranjana; Le, Thuc T.; Gorjala, Priyatham; Goodman Jr., Oscar B.

    2017-01-01

    Background Neoplastic cells proliferate rapidly and obtain requisite building blocks by reprogramming metabolic pathways that favor growth. Previously, we observed that prostate cancer cells uptake and store lipids in the form of lipid droplets, providing building blocks for membrane synthesis, to facilitate proliferation and growth. Mechanisms of lipid uptake, lipid droplet dynamics and their contribution to cancer growth have yet to be defined. This work is focused on elucidating the prosta...

  3. Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

    Directory of Open Access Journals (Sweden)

    Christian Kleusch

    2012-01-01

    Full Text Available In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

  4. 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.

  5. Simultaneous treatment (cell disruption and lipid extraction) of wet microalgae using hydrodynamic cavitation for enhancing the lipid yield.

    Science.gov (United States)

    Lee, Ilgyu; Han, Jong-In

    2015-06-01

    Simultaneous treatment (combining with cell disruption and lipid extraction) using hydrodynamic cavitation (HC) was applied to Nannochloropsis salina to demonstrate a simple and integrated way to produce oil from wet microalgae. A high lipid yield from the HC (25.9-99.0%) was observed compared with autoclave (16.2-66.5%) and ultrasonication (5.4-26.9%) in terms of the specific energy input (500-10,000 kJ/kg). The optimal conditions for the simultaneous treatment were established using a statistical approach. The efficiency of the simultaneous method was also demonstrated by comparing each separate treatment. The maximum lipid yield (predicted: 45.9% and experimental: 45.5%) was obtained using 0.89% sulfuric acid with a cavitation number of 1.17 for a reaction time of 25.05 min via response surface methodology. Considering its comparable extractability, energy-efficiency, and potential for scale-up, HC may be a promising method to achieve industrial-scale microalgae operation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

    Science.gov (United States)

    Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

    2017-02-01

    The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Diversity in host clone performance within a Chinese hamster ovary cell line.

    Science.gov (United States)

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers.

  8. 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.

  9. Atherosclerosis: the interplay between lipids and immune cells

    NARCIS (Netherlands)

    Schaftenaar, Frank; Frodermann, Vanessa; Kuiper, Johan; Lutgens, Esther

    2016-01-01

    Cardiovascular disease is the leading cause of mortality worldwide. The underlying cause of the majority of cardiovascular disease is atherosclerosis. In the past, atherosclerosis was considered to be the result of passive lipid accumulation in the vessel wall. However, today's picture of the

  10. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Directory of Open Access Journals (Sweden)

    María Inés Marchesini

    Full Text Available Choloylglycine hydrolase (CGH, E.C. 3.5.1.24 is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  11. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  12. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  13. Oral chronic graft-versus-host disease: analysis of dendritic cells subpopulations*

    Science.gov (United States)

    Botari, Clara Marino Espricigo; Nunes, Adauto José Ferreira; de Souza, Mair Pedro; Orti-Raduan, Érica Sinara Lenharo; Salvio, Ana Gabriela

    2014-01-01

    The graft-versus-host disease is the major cause of morbidity and mortality in patients who have undergone hematopoietic stem cell transplantation. Aiming at contributing to the understanding of the role of myeloid and plasmacytoid dendritic cells, and natural killer cells in chronic graft-versus-host disease, we examined biopsies of jugal mucosa of 26 patients with acute myeloid leukemia who had undergone allogenic hematopoietic stem cell transplantation. Half of these patients developed oral chronic graft-versus-host disease. Microscopic sections were immunohistochemically stained for anti-CD1a, anti-CD123 and anti-CD56. We calculated the number of immunostained cells in the corium per square millimeter and applied the Mann-Whitney test. Results showed a statistically significant increase of myeloid dendritic cells (CD1a+; p=0,02) and natural killer cells (CD56; p=0,04) in patients with oral chronic graft-versus-host disease. CD123 immunostaining showed no statistical difference between groups. It was concluded that myeloid dendritic cells and natural killer cells participate in the development of oral chronic graft-versus-host disease. PMID:25054751

  14. Oral chronic graft-versus-host disease: analysis of dendritic cells subpopulations.

    Science.gov (United States)

    Botari, Clara Marino Espricigo; Nunes, Adauto José Ferreira; Souza, Mair Pedro de; Orti-Raduan, Erica Sinara Lenharo; Salvio, Ana Gabriela

    2014-01-01

    The graft-versus-host disease is the major cause of morbidity and mortality in patients who have undergone hematopoietic stem cell transplantation. Aiming at contributing to the understanding of the role of myeloid and plasmacytoid dendritic cells, and natural killer cells in chronic graft-versus-host disease, we examined biopsies of jugal mucosa of 26 patients with acute myeloid leukemia who had undergone allogenic hematopoietic stem cell transplantation. Half of these patients developed oral chronic graft-versus-host disease. Microscopic sections were immunohistochemically stained for anti-CD1a, anti-CD123 and anti-CD56. We calculated the number of immunostained cells in the corium per square millimeter and applied the Mann-Whitney test. Results showed a statistically significant increase of myeloid dendritic cells (CD1a+; p=0,02) and natural killer cells (CD56; p=0,04) in patients with oral chronic graft-versus-host disease. CD123 immunostaining showed no statistical difference between groups. It was concluded that myeloid dendritic cells and natural killer cells participate in the development of oral chronic graft-versus-host disease.

  15. The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells

    Science.gov (United States)

    Nunn, Abigail D. G.; Scopigno, Tullio; Pediconi, Natalia; Levrero, Massimo; Hagman, Henning; Kiskis, Juris; Enejder, Annika

    2016-06-01

    Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome.

  16. The lipid fraction of human milk initiates adipocyte differentiation in 3T3-L1 cells.

    Science.gov (United States)

    Fujisawa, Yasuko; Yamaguchi, Rie; Nagata, Eiko; Satake, Eiichiro; Sano, Shinichiro; Matsushita, Rie; Kitsuta, Kazunobu; Nakashima, Shinichi; Nakanishi, Toshiki; Nakagawa, Yuichi; Ogata, Tsutomu

    2013-09-01

    The prevalence of childhood obesity has increased worldwide over the past decade. Despite evidence that human milk lowers the risk of childhood obesity, the mechanism is not fully understood. We investigated the direct effect of human milk on differentiation of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with donated human milk only or the combination of the standard hormone mixture; insulin, dexamethasone (DEX), and 3-isobututyl-1-methylxanthine (IBMX). Furthermore, the induction of preadipocyte differentiation by extracted lipids from human milk was tested in comparison to the cells treated with lipid extracts from infant formula. Adipocyte differentiation, specific genes as well as formation of lipid droplets were examined. We clearly show that lipids present in human milk initiate 3T3-L1 preadipocyte differentiation. In contrast, this effect was not observed in response to lipids present in infant formula. The initiation of preadipocyte differentiation by human milk was enhanced by adding the adipogenic hormone, DEX or insulin. The expression of late adipocyte markers in Day 7 adipocytes that have been induced into differentiation with human milk lipid extracts was comparable to those in control cells initiated by a standard adipogenic hormone cocktail. These results demonstrate that human milk contains bioactive lipids that can initiate preadipocyte differentiation in the absence of the standard adipogenic compounds via a unique pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  18. Cell Therapy in Parkinson's Disease: Host Brain Repair Machinery Gets a Boost From Stem Cell Grafts.

    Science.gov (United States)

    Napoli, Eleonora; Borlongan, Cesar V

    2017-06-01

    This commentary highlights the major findings and future research directions arising from the recent publication by Zuo and colleagues in Stem Cells 2017 (in press). Here, we discuss the novel observations that transplanted human neural stem cells can induce endogenous brain repair by specifically stimulating a host of regenerative processes in the neurogenic niche (i.e., subventricular zone [SVZ]) in an animal model of Parkinson's disease. That the identified therapeutic proteomes, neurotrophic factors, and anti-inflammatory cytokines in the SVZ may facilitate brain regeneration and behavioral recovery open a new venue of research for our understanding of the pathology and treatment of Parkinson's disease. Stem Cells 2017;35:1443-1445. © 2017 AlphaMed Press.

  19. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

    Directory of Open Access Journals (Sweden)

    Lidia Daimiel

    2015-09-01

    Full Text Available We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1,2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA. Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data.

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

    Science.gov (United States)

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

    2014-03-01

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

  1. 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...

  2. iNKT Cells and Their potential Lipid Ligands during Viral Infection

    Directory of Open Access Journals (Sweden)

    Anunya eOpasawatchai

    2015-07-01

    Full Text Available Invariant natural killer T (iNKT cells are a unique population of lipid reactive CD1d restricted innate-like T lymphocytes. Despite being a minor population, they serve as an early source of cytokines and promote immunological crosstalk thus bridging innate and adaptive immunity. Diseases ranging from allergy, autoimmunity, and cancer as well as infectious diseases, including viral infection, have been reported to be influenced by iNKT cells. However, it remains unclear how iNKT cells are activated during viral infection, as virus derived lipid antigens have not been reported. Cytokines may activate iNKT cells during infections from influenza and murine cytomegalovirus (MCMV, although CD1d dependent activation is evident in other viral infections. Several viruses, such as dengue virus (DENV, induce CD1d upregulation which correlates with iNKT cell activation. In contrast, Herpes simplex virus type 1 (HSV-1, Human immunodeficiency virus (HIV, Epstein-Barr virus (EBV and Human papiloma virus (HPV promote CD1d downregulation as a strategy to evade iNKT cell recognition. These observations suggest the participation of a CD1d-dependent process in the activation of iNKT cells in response to viral infection. Endogenous lipid ligands, including phospholipids as well as glycosphingolipids, such as glucosylceramide have been proposed to mediate iNKT cell activation. Pro-inflammatory signals produced during viral infection may stimulate iNKT cells through enhanced CD1d dependent endogenous lipid presentation. Furthermore, viral infection may alter lipid composition and inhibit endogenous lipid degradation. Recent advances in this field are reviewed.

  3. Effects of achilline on lipid metabolism gene expression in cell culture

    Directory of Open Access Journals (Sweden)

    A. V. Ratkin

    2016-01-01

    Full Text Available Objective. Evaluation in vitro of the mechanisms of the hypolipidemic effect of sesquiterpene γ-lactone achilline in the hepatoma tissue culture (HTC.Materials and methods.The influence of sesquiterpene γ-lactone achilline and gemfibrozil (comparison drug on the viability, lipid content and expression of key genes of lipid metabolism in the hepatoma tissue culture. The lipid content was assessed by fluorescent method with the vital dye Nile Red, the cell viability was assessed using MTT assay.Results. Cultivation of of cell cultures of rat’s hepatoma cell line HTC for 48 h with achilline in a concentration of from 0.25 to 1.0 mm and gemfibrozil from 0,25 to 0,5 mm did not change cell viability compared to control. In these same concentrations of the test substance reduced the lipid content in the cells, assessed by fluorescent method with the vital dye Nile Red. To study the mechanism of hypolipidemicaction of achillinedetermined the expression of key genes of lipid metabolism in cell culture lines HTC. The possible mechanism of hypolipidemic action of achilline can be attributed to the increased transport and oxidation of long-chain fatty acids in mitochondria, as evidenced by the increase in the gene expression of carnitine-palmitoyltransferase 2 (Cpt2. The decrease in cholesterol level may be due to increased synthesis of bile acids from cholesterol, due to increased gene expression of 7-alphahydroxylase (Cyp7a1. Conclusion. In cell cultures of rat’s hepatoma cell line HTC sesquiterpene γ-lactone achilline reduces the accumulation of lipids in cells, as evidenced by the decrease in the fluorescence of Nile Red, increased gene expression of the carnitine-palmitoyltransferase 2 (Cpt2 gene and 7-alpha-hydroxylase (Cyp7a1.

  4. Liver X receptors balance lipid stores in hepatic stellate cells through Rab18, a retinoid responsive lipid droplet protein.

    Science.gov (United States)

    O'Mahony, Fiona; Wroblewski, Kevin; O'Byrne, Sheila M; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S; Beaven, Simon W

    2015-08-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ(-/-) mice have increased lipid droplet (LD) size, but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ(-/-) and wild-type mice were profiled by gene array during in vitro activation. Lipid content was quantified by high-performance liquid chromatography and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with small interfering RNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ(-/-) HSCs have increased cholesterol and retinyl esters. The retinoid increase drives intrinsic retinoic acid receptor signaling, and activation occurs more rapidly in Lxrαβ(-/-) HSCs. We identify Rab18 as a novel retinoic acid-responsive, LD-associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 guanosine triphosphatase activity and isoprenylation are required for stellate cell LD loss and induction of activation markers. These phenomena are accelerated in Lxrαβ(-/-) HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards LD loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Retinoid and cholesterol metabolism are linked in stellate cells by the LD-associated protein Rab18. Retinoid overload helps explain the profibrotic phenotype of Lxrαβ(-/-) mice, and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. © 2015 by the American Association for the Study of Liver Diseases.

  5. Amelioration of High Cholesterol Diet Caused Lipids Accumulation in Hepatic Cells by Rutin and Ascorbic Acid

    OpenAIRE

    Abdulaziz M. Aleisa

    2013-01-01

    Non Alcoholic Fatty Liver Disease (NAFLD) has become a very common metabolic disorder. It refers to a group of conditions where excess fats are deposited in hepatic cells. Several approaches have been considered for the management of NAFLD including dietary changes, which were reported to suppress hepatic lipids accumulation in previous studies. The present study was designed to investigate the possible synergistic effects of Rutin (RT) and Ascorbic Acid (AA) against lipids accumulation in he...

  6. Effects of host cell sterol composition upon internalization of Yersinia pseudotuberculosis and clustered β1 integrin.

    Science.gov (United States)

    Kim, JiHyun; Fukuto, Hana S; Brown, Deborah A; Bliska, James B; London, Erwin

    2018-01-26

    Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δ inv , Δ yadA , and Δ inv Δ yadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The Δ inv Δ yadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Differentiation of strains of varicella-zoster virus by changes in neutral lipid metabolism in infected cells

    International Nuclear Information System (INIS)

    Jerkofsky, M.; De Siervo, A.J.

    1986-01-01

    Eleven isolates of varicella-zoster virus were tested for their effects on the incorporation of [ 14 C]acetate into lipids in infected human embryonic lung cells. By relative percent, all virus isolates demonstrated a shift from polar lipid synthesis to neutral lipid, especially triglyceride, synthesis. By data expressed as counts per minute per microgram of protein, the VZV strains could be separated into two groups: those strains which depressed lipid synthesis and those strains which did not depress, and may even have stimulated, lipid, especially triglyceride, synthesis. These results may be useful in understanding the development of lipid changes seen in children affected with Reye's syndrome following chickenpox

  8. T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

    Science.gov (United States)

    Wun, Kwok S; Reijneveld, Josephine F; Cheng, Tan-Yun; Ladell, Kristin; Uldrich, Adam P; Le Nours, Jérôme; Miners, Kelly L; McLaren, James E; Grant, Emma J; Haigh, Oscar L; Watkins, Thomas S; Suliman, Sara; Iwany, Sarah; Jimenez, Judith; Calderon, Roger; Tamara, Kattya L; Leon, Segundo R; Murray, Megan B; Mayfield, Jacob A; Altman, John D; Purcell, Anthony W; Miles, John J; Godfrey, Dale I; Gras, Stephanie; Price, David A; Van Rhijn, Ildiko; Moody, D Branch; Rossjohn, Jamie

    2018-04-01

    The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

  9. The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

    Science.gov (United States)

    Mostowy, Serge; Shenoy, Avinash R.

    2016-01-01

    Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

  10. Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication

    International Nuclear Information System (INIS)

    Scaria, A.; Edenberg, H.J.

    1987-01-01

    The authors examined the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV40 DNA synthesis in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m 2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m 2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication. (Auth.)

  11. Raman imaging and lipidomic analysis of lipid droplets in (activated) hepatic stellate cells

    NARCIS (Netherlands)

    Vaandrager, A.B.; Testerink, N.; Ajat, M.; Houweling, M.; Brouwers, J.F.H.M.; Pully, V.V.; van Manen, H.J.; Otto, Cornelis; Helms, J.B.

    2009-01-01

    In eukaryotic cells the excess of hydrophobic molecules is stored in special organelles named lipid droplets (LDs). These droplets contain triacylglycerides, cholesteryl esters and/or retinyl esters, depending on the function of the cell in which they reside. Retinyl esters, the storage form of

  12. Synthetic Lipid (DOPG) Vesicles Accumulate in the Cell Plate Region But Do Not Fuse1

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Vos, J.W.; Lammeren, van A.A.M.; Emons, A.M.C.

    2008-01-01

    Synthetic Lipid (DOPG) Vesicles Accumulate in the Cell Plate Region But Do Not Fuse1,[W],[OA] Agnieszka Esseling-Ozdoba2, Jan W. Vos, André A.M. van Lammeren and Anne Mie C. Emons* Laboratory of Plant Cell Biology, Department of Plant Sciences, Wageningen University, 6703¿BD Wageningen, The

  13. Oxidative stress induced lipid accumulation via SREBP1c activation in HepG2 cells

    International Nuclear Information System (INIS)

    Sekiya, Mika; Hiraishi, Ako; Touyama, Maiko; Sakamoto, Kazuichi

    2008-01-01

    SREBP1c (sterol regulatory element-binding protein 1c) is a metabolic-syndrome-associated transcription factor that controls fatty acid biosynthesis under glucose/insulin stimulation. Oxidative stress increases lipid accumulation, which promotes the generation of reactive oxygen species (ROS). However, we know little about the role of oxidative stress in fatty acid biosynthesis. To clarify the action of oxidative stress in lipid accumulation via SREBP1c, we examined SREBP1c activity in H 2 O 2 -treated mammalian cells. We introduced a luciferase reporter plasmid carrying the SREBP1c-binding site into HepG2 or COS-7 cells. With increasing H 2 O 2 dose, SREBP1c transcriptional activity increased in HepG2 cells but declined in COS-7 cells. RT-PCR analysis revealed that mRNA expression of SREBP1c gene or of SREBP1c-regulated genes rose H 2 O 2 dose-dependently in HepG2 cells but dropped in COS-7 cells. Lipid accumulation and levels of the nuclear form of SREBP1c increased in H 2 O 2 -stimulated HepG2 cells. ROS may stimulate lipid accumulation in HepG2 cells via SREBP1c activation

  14. Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host∶parasite interactions.

    Directory of Open Access Journals (Sweden)

    Olivia Twu

    Full Text Available Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.

  15. Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

    Science.gov (United States)

    Cardoso, Rita; Soares, Helena; Hemphill, Andrew; Leitão, Alexandre

    2016-07-01

    Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

  16. Plant parasitic nematode effectors target host defence and nuclear functions to establish feeding cells

    Directory of Open Access Journals (Sweden)

    Michaël eQuentin

    2013-03-01

    Full Text Available Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells. Effectors synthesised in the oesophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized feeding cells requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defence responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalised within feeding cell nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesised roles in the unique feeding behaviour of these pests.

  17. 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.

  18. Unusually large unit cell of lipid bicontinuous cubic phase: towards nature's length scales

    Science.gov (United States)

    Kim, Hojun; Leal, Cecilia

    Lipid bicontinuous cubic phases are of great interest for drug delivery, protein crystallization, biosensing, and templates for directing hard material assembly. Structural modulations of lipid mesophases regarding phase identity and unit cell size are often necessary to augment loading and gain pore size control. One important example is the need for unit cells large enough to guide the crystallization of bigger proteins without distortion of the templating phase. In nature, bicontinuous cubic constructs achieve unit cell dimensions as high as 300 nm. However, the largest unit cell of lipid mesophases synthesized in the lab is an order of magnitude lower. In fact, it has been predicted theoretically that lipid bicontinuous cubic phases of unit cell dimensions exceeding 30 nm could not exist, as high membrane fluctuations would damp liquid crystalline order. Here we report non-equilibrium assembly methods of synthesizing metastable bicontinuous cubic phases with unit cell dimensions as high as 70 nm. The phases are stable for very long periods and become increasingly ordered as time goes by without changes to unit cell dimensions. We acknowledge the funding source as a NIH.

  19. Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi, a Bloom-Forming Alga in the Ocean[C][W][OPEN

    Science.gov (United States)

    Rosenwasser, Shilo; Mausz, Michaela A.; Schatz, Daniella; Sheyn, Uri; Malitsky, Sergey; Aharoni, Asaph; Weinstock, Eyal; Tzfadia, Oren; Ben-Dor, Shifra; Feldmesser, Ester; Pohnert, Georg; Vardi, Assaf

    2014-01-01

    Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical “arms race” in the ocean. PMID:24920329

  20. Neutron scattering to study membrane systems: from lipid vesicles to living cells.

    Energy Technology Data Exchange (ETDEWEB)

    Nickels, Jonathan D. [ORNL; Chatterjee, Sneha [ORNL; Stanley, Christopher B. [ORNL; Qian, Shuo [ORNL; Cheng, Xiaolin [ORNL; Myles, Dean A A [ORNL; Standaert, Robert F. [ORNL; Elkins, James G. [ORNL; Katsaras, John [ORNL

    2017-03-01

    The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

  1. Intrinsic potential of cell membranes: opposite effects of lipid transmembrane asymmetry and asymmetric salt ion distribution

    DEFF Research Database (Denmark)

    Gurtovenko, Andrey A; Vattulainen, Ilpo

    2009-01-01

    Using atomic-scale molecular dynamics simulations, we consider the intrinsic cell membrane potential that is found to originate from a subtle interplay between lipid transmembrane asymmetry and the asymmetric distribution of monovalent salt ions on the two sides of the cell membrane. It turns out......Cl saline solution and the PE leaflet is exposed to KCl, the outcome is that the effects of asymmetric lipid and salt ion distributions essentially cancel one another almost completely. Overall, our study highlights the complex nature of the intrinsic potential of cell membranes under physiological...... that both the asymmetric distribution of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids across a membrane and the asymmetric distribution of NaCl and KCl induce nonzero drops in the transmembrane potential. However, these potential drops are opposite in sign. As the PC leaflet faces a Na...

  2. 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....

  3. Lipid Accumulation in Peripheral Blood Dendritic Cells and Anticancer Immunity in Patients with Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ryo Arai

    2018-01-01

    Full Text Available We studied the subsets of peripheral blood dendritic cells (DCs and lipid accumulation in DCs to investigate the involvement of DCs in the decreased anticancer immunity of advanced lung cancer patients. We analyzed the population of DC subsets in peripheral blood using flow cytometry. We then determined lipid accumulation in the DCs using BODIPY 650/665, a fluorophore with an affinity for lipids. Compared with healthy controls, the number of DCs in the peripheral blood of treatment-naive cancer patients was significantly reduced. In patients with stage III + IV disease, the numbers of myeloid DCs (mDCs and plasmacytoid DCs were also significantly reduced. Lipid accumulation in DCs evaluated based on the fluorescence intensity of BODIPY 650/665 was significantly higher in stage III + IV lung cancer patients than in the controls. In the subset analysis, the fluorescence was highest for mDCs. The intracellularly accumulated lipids were identified as triglycerides. A decreased mixed leukocyte reaction was observed in the mDCs from lung cancer patients compared with those from controls. Taken together, the results show that lung cancer patients have a notably decreased number of peripheral blood DCs and their function as antigen-presenting cells is decreased due to their high intracellular lipid accumulation. Thereby, anticancer immunity is suppressed.

  4. Traversing the Cell: Agrobacterium T-DNA’s Journey to the Host Genome

    Science.gov (United States)

    Gelvin, Stanton B.

    2012-01-01

    The genus Agrobacterium is unique in its ability to conduct interkingdom genetic exchange. Virulent Agrobacterium strains transfer single-strand forms of T-DNA (T-strands) and several Virulence effector proteins through a bacterial type IV secretion system into plant host cells. T-strands must traverse the plant wall and plasma membrane, traffic through the cytoplasm, enter the nucleus, and ultimately target host chromatin for stable integration. Because any DNA sequence placed between T-DNA “borders” can be transferred to plants and integrated into the plant genome, the transfer and intracellular trafficking processes must be mediated by bacterial and host proteins that form complexes with T-strands. This review summarizes current knowledge of proteins that interact with T-strands in the plant cell, and discusses several models of T-complex (T-strand and associated proteins) trafficking. A detailed understanding of how these macromolecular complexes enter the host cell and traverse the plant cytoplasm will require development of novel technologies to follow molecules from their bacterial site of synthesis into the plant cell, and how these transferred molecules interact with host proteins and sub-cellular structures within the host cytoplasm and nucleus. PMID:22645590

  5. Epigenetic modulation of the biophysical properties of drug-resistant cell lipids to restore drug transport and endocytic functions.

    Science.gov (United States)

    Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

    2012-09-04

    In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

  6. Host cell proteins in biotechnology-derived products: A risk assessment framework.

    Science.gov (United States)

    de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

    2015-11-01

    To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

  7. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

    International Nuclear Information System (INIS)

    Miyamae, Yusaku; Nishito, Yukina; Nakai, Naomi; Nagumo, Yoko; Usui, Takeo; Masuda, Seiji; Kambe, Taiho; Nagao, Masaya

    2016-01-01

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A_1. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

  8. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyamae, Yusaku, E-mail: ymiyamae@lif.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nishito, Yukina; Nakai, Naomi [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagumo, Yoko; Usui, Takeo [Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572 (Japan); Masuda, Seiji; Kambe, Taiho [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagao, Masaya, E-mail: mnagao@kais.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan)

    2016-08-12

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A{sub 1}. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

  9. The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis.

    LENUS (Irish Health Repository)

    Matlawska-Wasowska, Ksenia

    2010-12-01

    Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.

  10. Salmonella modulation of host cell gene expression promotes its intracellular growth.

    Directory of Open Access Journals (Sweden)

    Sebastian Hannemann

    Full Text Available Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS, which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.

  11. Salmonella modulation of host cell gene expression promotes its intracellular growth.

    Science.gov (United States)

    Hannemann, Sebastian; Gao, Beile; Galán, Jorge E

    2013-01-01

    Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.

  12. Microbiota promote secretory cell determination in the intestinal epithelium by modulating host Notch signaling.

    Science.gov (United States)

    Troll, Joshua V; Hamilton, M Kristina; Abel, Melissa L; Ganz, Julia; Bates, Jennifer M; Stephens, W Zac; Melancon, Ellie; van der Vaart, Michiel; Meijer, Annemarie H; Distel, Martin; Eisen, Judith S; Guillemin, Karen

    2018-02-23

    Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer. © 2018. Published by The Company of Biologists Ltd.

  13. Cycle Inhibiting Factors (Cifs: Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

    Directory of Open Access Journals (Sweden)

    Eric Oswald

    2011-03-01

    Full Text Available Cycle inhibiting factors (Cifs are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

  14. The effect of ionizing radiation on lipid metabolism in lymphoid cells

    International Nuclear Information System (INIS)

    Kolomiytseva, I.K.; Novoselova, E.G.; Kulagina, T.P.; Kuzin, A.M.

    1987-01-01

    Lipid metabolism was studied in lymphoid tissues of rats after whole body irradiation with doses producing damage of different degrees to lymphoid cells (4-10 Gy). The content of free cholesterol, cholesterol esters, and total phospholipids was determined in peripheral blood lymphocytes and thymocytes 1-2 h after exposure. Simultaneously, the rate of in vitro incorporation of 2 14 C-acetate into total lipids, phospholipids, and cholesterol of lymphoid cells was estimated. It was shown that exposure of rats to ionizing radiation caused activation of lipogenesis. Cholesterol synthesis was activated after a dose of 4 Gy and decreased with increasing dose. (author)

  15. Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling

    NARCIS (Netherlands)

    Remus, D.M.; Kranenburg, van R.; Swam, van I.I.; Taverne, N.; Bongers, R.S.; Wels, M.; Wells, J.; Bron, P.A.; Kleerebezem, M.

    2012-01-01

    Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well

  16. S1P dependent inter organ trafficking of group 2 innate lymphoid cells suppots host defense

    Science.gov (United States)

    Innate lymphoid cells (ILCs) are considered to be the innate counterparts of adaptive T lymphocytes and play important roles in host defense, tissue repair, metabolic homeostasis, and inflammatory diseases. ILCs are generally thought of as tissue-resident cells, but whether ILCs strictly behave in a...

  17. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

    Directory of Open Access Journals (Sweden)

    Jose C Garcia-Garcia

    2009-06-01

    Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

  18. Atherogenic ω-6 Lipids Modulate PPAR- EGR-1 Crosstalk in Vascular Cells

    Directory of Open Access Journals (Sweden)

    Jia Fei

    2011-01-01

    Full Text Available Atherogenic ω-6 lipids are physiological ligands of peroxisome proliferator-activated receptors (PPARs and elicit pro- and antiatherogenic responses in vascular cells. The objective of this study was to investigate if ω-6 lipids modulated the early growth response-1 (Egr-1/PPAR crosstalk thereby altering vascular function. Rat aortic smooth muscle cells (RASMCs were exposed to ω-6 lipids, linoleic acid (LA, or its oxidized form, 13-HPODE (OxLA in the presence or absence of a PPARα antagonist (MK886 or PPARγ antagonist (GW9662 or PPAR-specific siRNA. Our results demonstrate that ω-6 lipids, induced Egr-1 and monocyte chemotactic protein-1 (MCP-1 mRNA and protein levels at the acute phase (1–4 hrs when PPARα was downregulated and at subacute phase (4–12 hrs by modulating PPARγ, thus resulting in altered monocyte adhesion to RASMCs. We provide novel insights into the mechanism of action of ω-6 lipids on Egr-1/PPAR interactions in vascular cells and their potential in altering vascular function.

  19. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Directory of Open Access Journals (Sweden)

    José B Gama

    2014-08-01

    Full Text Available Buruli ulcer (BU is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1 and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1. In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

  20. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Science.gov (United States)

    Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

    2014-08-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

  1. Unravel lipid accumulation mechanism in oleaginous yeast through single cell systems biology study

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Shiyou; Xiaoliang, Xie

    2017-12-18

    Replacement of petroleum with advanced biofuels is critical for environmental protection needs, sustainable and secure energy demands, and economic development. Bacteria, yeasts, and fungi can naturally synthesize fatty acids, isoprenoids, or polyalkanoates for energy storage, and therefore are currently explored for hydrocarbon fuel production. Oleaginous yeasts can accumulate high levels of lipids in the form of triacylglycerols (TAGs) when encountering stress conditions or imbalanced growth (e.g., growing under excess carbon sources and limited nitrogen conditions). Advantages of using oleaginous yeast as cell factories include short duplication time (< 1 hour), high yield of intracellular droplets, and easy scale-up for industrial production. Currently, various oleaginous yeasts (e.g., Yarrowia, Candida, Rhodotorulla, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces) have been developed as potential advanced biofuel producers. Oleaginous yeast lipid production has two phases: 1) growth phase, where cells utilize the carbon and nitrogen source to build up biomass. And 2) lipid accumulation phase, where they convert carbon source in media into the storage lipid body. (i.e. a high carbon to nitrogen ratio leads to high lipid production). The lipid production varies dramatically when different sugar, e.g. glucose, xylose is used as carbon source. The efficient utilization of all monomeric sugars of hexoses and pentoses from various lignocellulosic biomass processing approaches is the key for economic lignocellulosic biofuel production. In this project, we explored lipid production in oleaginous yeast under different nitrogen and sugar conditions at the single-cell level. To understand the lipid production mechanism and identify genetic features responsive to lipid accumulation in the presence of pentose and nitrogen, we developed an automated chemical imaging and single-cell transcriptomics method to correlate the lipid accumulation with the

  2. Adrenal adenomas: relationship between histologic lipid-rich cells and CT attenuation number

    International Nuclear Information System (INIS)

    Yamada, Takayuki; Ishibashi, Tadashi; Saito, Haruo; Matsuhashi, Toshio; Majima, Kazuhiro; Tsuda, Masashi; Takahashi, Shoki; Moriya, Takuya

    2003-01-01

    Objective: To evaluate the relationship between lipid-rich cells of the adrenal adenoma and precontrast computed tomographic (CT) attenuation numbers in three clinical groups. Materials and Methods: Thirty-five surgically resected adrenal adenomas were used. The clinical diagnoses of the patients included 13 cases of primary aldosteronism, 15 cases of Cushing's syndrome, and 7 non-functioning tumors. The number of lipid-rich clear cells was counted using a microscopic eyepiece grid that contained 100 squares. The results were expressed as the percentages of lipid-rich areas. Results: There was a strong inverse linear relationship between the percentage of lipid-rich cells and the precontrast CT attenuation number (R 2 =0.724, P<0.0001). There were significantly more lipid-rich cells in the primary aldosteronism and non-functioning tumor cases compared to cases of Cushing's syndrome (P=0.007 and 0.015, respectively). The CT attenuation numbers of the primary aldosteronism cases were significantly lower than those of Cushing's syndrome (P=0.0052). Furthermore, the CT attenuation numbers of the non-functioning tumor cases were lower than those of Cushing's syndrome cases. Conclusion: We showed that adrenal adenomas in primary aldosteronism and non-functioning tumors contain significantly more lipid-rich cells than those in Cushing's syndrome. They also showed significantly lower attenuation than that in Cushing's syndrome on CT scans. Our results suggest that precontrast CT attenuation numbers may be helpful in the differentiation of adenomas from non-adenomatous lesions, which include malignancies

  3. Detection of Lipid-Rich Prostate Circulating Tumour Cells with Coherent Anti-Stokes Raman Scattering Microscopy

    International Nuclear Information System (INIS)

    Mitra, Ranjana; Chao, Olivia; Urasaki, Yasuyo; Goodman, Oscar B; Le, Thuc T

    2012-01-01

    Circulating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge. Coherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line. One hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells. Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC

  4. HumanViCe: Host ceRNA network in virus infected cells in human

    Directory of Open Access Journals (Sweden)

    Suman eGhosal

    2014-07-01

    Full Text Available Host-virus interaction via host cellular components has been an important field of research in recent times. RNA interference mediated by short interfering RNAs and microRNAs (miRNA, is a widespread anti-viral defence strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signalling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signalling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g. APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe, a comprehensive database that provides the potential ceRNA networks in virus

  5. 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

  6. 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

  7. Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

    Science.gov (United States)

    Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

    2015-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

  8. Reduction of fatal graft-versus-host disease by 3H--thymidine suicide of donor cells cultured with host cells

    International Nuclear Information System (INIS)

    Cheever, M.A.; Einstein, A.B. Jr.; Kempf, R.A.; Fefer, A.

    1977-01-01

    The effect of the tritiated thymidine ( 3 H-TdR) suicide technique on the ability of donor cells to induce fatal graft-versus-host disease (GVHD) was studied. C57BL/6 (H-2/sup b/) spleen cells were stimulated in vitro with irradiated BALB/c (H-2/sup d/) Moloney lymphoma cells in mixed culture and 3 H-TdR of high-specific activity added to eliminate proliferating cells. The ability of such cells to induce fatal GVHD was assayed by injecting them i.v. into adult BALB/c mice immunosuppressed with cyclophosphamide (180 mg/kg). These cells induced fatal GVHD in fewer mice (52 percent) than did C57BL/6 cells cultured with BALB/c lymphoma cells but without 3 H-TdR (87 percent) and C57BL/6 cells cultured with irradiated C57BL/6 cells with (95 percent) or without 3 H-TdR (86 percent). Thus, the 3 H-TdR suicide technique greatly diminished the ability of cells to induce lethal GVHD

  9. Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

    Science.gov (United States)

    Renault, Andrew D.; Kunwar, Prabhat S.; Lehmann, Ruth

    2010-01-01

    In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells. PMID:20431117

  10. Amalgamation of Chlamydia pneumoniae inclusions with lipid droplets in foam cells in human atherosclerotic plaque.

    Science.gov (United States)

    Bobryshev, Yuri V; Killingsworth, Murray C; Tran, Dihn; Lord, Reginald

    2008-07-01

    Chlamydia pneumoniae (Chlamydophila pneumoniae) infect macrophages and accelerates foam cell formation in in vitro experiments, but whether this might occur in human atherosclerosis is unknown. In the present study, we examined 17 carotid artery segments, obtained by endarterectomy, in which the presence of C. pneumoniae was confirmed by both polymerase chain reaction and immunohistochemistry. Electron microscopy demonstrated the presence of structures with the appearance of elementary, reticulate and aberrant bodies of C. pneumoniae in the cytoplasm of macrophage foam cells. The volume of the cytoplasm that was free from vacuoles and lipid droplets in C. pneumoniae-infected foam cells was dramatically reduced, and a phenomenon of the amalgamation of C. pneumoniae inclusions with lipid droplets was detected. Double immunohistochemistry showed that C. pneumoniae-infected foam cells contained a large number of oxidized low-density lipoproteins. The observations provide support to the hypothesis that C. pneumoniae could affect foam cell formation in human atherosclerosis.

  11. Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

    DEFF Research Database (Denmark)

    Zech, Tobias; Ejsing, Christer S.; Gaus, Katharina

    2009-01-01

    Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin...... and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation...... domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate...

  12. Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target for developing indirect-acting antiviral agents.

    Directory of Open Access Journals (Sweden)

    Andrea D Olmstead

    2012-01-01

    Full Text Available HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV, whose assembly and pathogenesis depend on interaction with lipid droplets (LDs. One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1--or site-1 protease (S1P. SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs, which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow

  13. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

    Science.gov (United States)

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

    2015-10-01

    . More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    Science.gov (United States)

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

    2015-01-01

    immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. PMID:26246569

  15. The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

    Science.gov (United States)

    Krause, Martin R; Regen, Steven L

    2014-12-16

    CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been

  16. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  17. Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall

    International Nuclear Information System (INIS)

    Onimatsu, Hideki; Sugimoto, Ichiro; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2004-01-01

    A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion

  18. Modeling conduction in host-graft interactions between stem cell grafts and cardiomyocytes.

    Science.gov (United States)

    Chen, Michael Q; Yu, Jin; Whittington, R Hollis; Wu, Joseph C; Kovacs, Gregory T A; Giovangrandi, Laurent

    2009-01-01

    Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

  19. Cell surface appearance of unexpected host MHC determinants on thymocytes from radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    Sharrow, S.O.; Mathieson, B.J.; Singer, A.

    1981-01-01

    The phenotypic appearance of cell surface antigens on murine thymocytes from long-term radiation bone marrow chimeras was analyzed using indirect immunofluorescence and flow microfluorometry. Cells maturing in the thymi of these mice were typed for MHC (Kk, I-Ak, H-2b, Kb, and Ib) and non-MHC (Lty 1, Ly 9, and TL) determinants. All cells were of donor origin as determined by non-MHC (Ly) phenotype in P1 leads to P2, P1 x P2 leads to P1, and P1 leads to P2 radiation chimeras. In contrast, the MHC phenotypes of these thymocytes were markedly affected by the host environment. Specifically, H-2 and I-A determinants of both parental phenotypes were detected on thymocytes from P1 leads to P1 x P2 chimeras; I-A determinants of host phenotype were present, whereas I-A determinants of donor phenotype were reduced on thymocytes from P1 x P2 leads to P1 chimeras; and thymocytes from P1 leads to P2 chimeras possessed H-2 and I-A determinants of host phenotype but showed reduction of donor I-A phenotype determinants. The appearance of host cell surface H-2 and I-A determinants on thymocytes from chimeras closely parallels the functional recognition of MHC determinants by T cells from chimeric mice and thus may be significantly related to the development of the self-recognition repertoire by maturing T cells

  20. Invasion of Eukaryotic Cells by Legionella Pneumophila: A Common Strategy for all Hosts?

    Directory of Open Access Journals (Sweden)

    Paul S Hoffman

    1997-01-01

    Full Text Available Legionella pneumophila is an environmental micro-organism capable of producing an acute lobar pneumonia, commonly referred to as Legionnaires’ disease, in susceptible humans. Legionellae are ubiquitous in aquatic environments, where they survive in biofilms or intracellularly in various protozoans. Susceptible humans become infected by breathing aerosols laden with the bacteria. The target cell for human infection is the alveolar macrophage, in which the bacteria abrogate phagolysosomal fusion. The remarkable ability of L pneumophila to infect a wide range of eukaryotic cells suggests a common strategy that exploits very fundamental cellular processes. The bacteria enter host cells via coiling phagocytosis and quickly subvert organelle trafficking events, leading to formation of a replicative phagosome in which the bacteria multiply. Vegetative growth continues for 8 to 10 h, after which the bacteria develop into a short, highly motile form called the ‘mature form’. The mature form exhibits a thickening of the cell wall, stains red with the Gimenez stain, and is between 10 and 100 times more infectious than agar-grown bacteria. Following host cell lysis, the released bacteria infect other host cells, in which the mature form differentiates into a Gimenez-negative vegetative form, and the cycle begins anew. Virulence of L pneumophila is considered to be multifactorial, and there is growing evidence for both stage specific and sequential gene expression. Thus, L pneumophila may be a good model system for dissecting events associated with the host-parasite interactions.

  1. Molecular mechanism of intracellular lipid accumulation: Suppressive effect of PycnogenolR in liver cells

    Directory of Open Access Journals (Sweden)

    Shoichiro Ikuyama

    2013-09-01

    Full Text Available ABSTRACTCells are physiologically ready to accumulate lipids such as triacylglycerides in the cytoplasm.Five classes of perilipin (PLIN family proteins are known to be involved in the process of intracellular lipid accumulation. PLIN2 is expressed ubiquitously including adipocytes, hepatocytes and macrophages. Over-expression of PLIN2 is demonstrated in the lesions of fatty liver diseases and atherosclerosis. Suppression of PLIN2 expression prevents from developing these pathological conditions in animal models, suggesting that PLIN2 could be a therapeutic target molecule for excessive intracellular lipid accumulation which leads to various metabolic derangements. The PLIN2 gene promoter has two important cis-acting elements in close proximity:AP-1 element which mediates inflammatory signals and PPRE which mediates free fatty acid effect. In NMuLi mouse liver cells, FFA such as oleic acid requires both functional AP-1 and PPRE simultaneously to stimulate the promoter activity, indicating the presence of intimate interaction of inflammatory and metabolic signals on this gene. PycnogenolR, French maritime pine bark extracts, suppressed the oleic acid-induced PLIN2 expression and lipid accumulation in NMuLi cells. We found that PycnogenolR did not suppress the PLIN2 promoter activity or AP-1 binding to DNA. Instead, PycnogenolRfacilitates the PLIN2 mRNA degradation, leading to suppression of lipid accumulation. This effect seems to be independent of antioxidant effect of PycnogenolR.We raise the idea that PLIN2 is a putative target molecule for prevention of pathological condition induced by excessive lipid accumulation, and this class of natural compounds could be putative therapeutic modalities.Key words: PycnogenolR, lipid droplet, perilipin, fatty liver disease

  2. Albumin-associated lipids regulate human embryonic stem cell self-renewal.

    Directory of Open Access Journals (Sweden)

    Francesc R Garcia-Gonzalo

    Full Text Available BACKGROUND: Although human embryonic stem cells (hESCs hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect. CONCLUSIONS/SIGNIFICANCE: Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.

  3. Lipid Cell and Micropapillary Variants of Urothelial Carcinoma of the Ureter

    Directory of Open Access Journals (Sweden)

    Yu Miyama

    2015-11-01

    Full Text Available We report on a case of urothelial carcinoma (UC with lipid cell and micropapillary variants in the ureter. A 64-year-old man presented with gross hematuria. Urinary cytology revealed the presence of atypical urothelial cells. Computed tomography and drip infusion/retrograde pyelography identified a mass-occupying lesion in the left mid-ureter, as well as left hydronephrosis. A clinical diagnosis of left ureteral cancer was given and the patient underwent left nephroureterectomy. Microscopically, the major component of the tumor was a conventional high-grade UC. In the invasive region, however, lipid cell and micropapillary variants of UC were also observed. Upon immunohistochemical analysis, all of the components were diffusely positive for cytokeratin 7 and p53. Intense membranous expression of human epidermal growth factor receptor 2 (HER2 was also observed in both the lipid cell and micropapillary variants of UC, whereas weak and incomplete staining was observed in most regions of the conventional UC. The pathological stage was pT3 N2. Multiple times, the patient experienced recurrence of the UC in the urinary bladder and urethra. Although the patient underwent total cystectomy and urethrectomy, 52 months following the initial surgery, signs of local recurrence developed, as well as multiple lymph node and bone metastases. The patient died 75 months following the initial surgery. To the best of our knowledge, this is the first reported case of a lipid cell variant of ureteral UC. The overexpression of HER2 may be associated with both the lipid cell and micropapillary variants of UC.

  4. Exercise performance, red blood cell deformability, and lipid peroxidation: effects of fish oil and vitamin E

    NARCIS (Netherlands)

    Oostenbrug, G. S.; Mensink, R. P.; Hardeman, M. R.; de Vries, T.; Brouns, F.; Hornstra, G.

    1997-01-01

    Previous studies have indicated that fish oil supplementation increases red blood cell (RBC) deformability, which may improve exercise performance. Exercise alone, or in combination with an increase in fatty acid unsaturation, however, may enhance lipid peroxidation. Effects of a bicycle time trial

  5. Identification of proteins associated with lipid of Jurlat T-cell line by mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Pompach, Petr; Man, Petr; Novák, Petr; Fišerová, Anna; Havlíček, Vladimír; Bezouška, Karel

    2003-01-01

    Roč. 87, - (2003), s. 319 ISSN 0165-2478 Institutional research plan: CEZ:AV0Z5020903; CEZ:MSM 113100001 Keywords : lipid * rafts * t- cell Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003

  6. Native and enzymatically modified wheat (Triticum aestivum L.) endogenous lipids in bread making: a focus on gas cell stabilization mechanisms.

    Science.gov (United States)

    Gerits, Lien R; Pareyt, Bram; Masure, Hanne G; Delcour, Jan A

    2015-04-01

    Lipopan F and Lecitase Ultra lipases were used in straight dough bread making to study how wheat lipids affect bread loaf volume (LV) and crumb structure setting. Lipase effects on LV were dose and dough piece weight dependent. The bread quality improving mechanisms exerted by endogenous lipids were studied in terms of gluten network strengthening, which indirectly stabilizes gas cells, and in terms of direct interfacial gas cell stabilization. Unlike diacetyl tartaric esters of mono- and diacylglycerols (DATEM, used as control), lipase use did not impact dough extensibility. The effect on dough extensibility was therefore related to its lipid composition at the start of mixing. Both lipases and DATEM strongly increase the levels of polar lipids in dough liquor and their availability for and potential accumulation at gas cell interfaces. Lipases form lysolipids that emulsify other lipids. We speculate that DATEM competes with (endogenous) polar lipids for interacting with gluten proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

    Science.gov (United States)

    Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

    2016-04-01

    Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Lipid droplet organelle distribution in populations of dividing cells studied by simulation

    International Nuclear Information System (INIS)

    Dalhaimer, Paul

    2013-01-01

    One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number—and to a lesser extent volume—in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies. (paper)

  9. Lipid droplet organelle distribution in populations of dividing cells studied by simulation

    Science.gov (United States)

    Dalhaimer, Paul

    2013-06-01

    One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number—and to a lesser extent volume—in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

  10. Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells.

    Science.gov (United States)

    Byk, T; Haddada, H; Vainchenker, W; Louache, F

    1998-11-20

    Adenoviral vectors have the potential to infect a large number of cell types including quiescent cells. Their use in hematopoietic cells is limited by the episomal form of their DNA, leading to transgene loss in the progeny cells. However, the use of this vector may be interesting for short-term in vitro modifications of primitive human hematopoietic cells. Therefore, we have investigated the ability of adenovirus to transduce cord blood CD34+ cells. Several promoters were tested using the lacZ reporter gene. The PGK and CMV promoters induced transgene expression in 18-25% of the cells, whereas the HTLV-I and especially the RSV promoter were almost inactive. To improve infection efficiency, adenovirus was complexed with cationic lipids. Lipofectamine, Cellfectin, and RPR120535b, but not Lipofectin, Lipofectace, or DOTAP, markedly improved transgene expression in CD34+ cells (from 19 to 35%). Lipofectamine strongly enhanced infection efficiency of the poorly infectable primitive CD34+CD38low cells (from 11 to 28%) whereas the more mature CD34+CD38+ cells were only slightly affected (from 24 to 31%). Lipofectamine tripled the infection of CFU-GMs and LTC-ICs derived from the CD34+CD38low cell fraction (from 4 to 12% and from 5 to 16%, respectively) and doubled that of BFU-Es (from 13 to 26%). We conclude that cationic lipids can markedly increase the efficiency of adenovirus-mediated gene transfer into primitive hematopoietic cells.

  11. 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.

  12. Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

    Directory of Open Access Journals (Sweden)

    Quadri Luis EN

    2009-04-01

    Full Text Available Abstract Background Cystic fibrosis (CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown. Methods This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection. Results CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1, a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP, a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7 and stearoyl-CoA desaturase 2 (Scd2, two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC. Conclusion These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

  13. Lipid-load in peripheral blood mononuclear cells: Impact of food-consumption, dietary-macronutrients, extracellular lipid availability and demographic factors.

    Science.gov (United States)

    Ameer, Fatima; Munir, Rimsha; Usman, Hina; Rashid, Rida; Shahjahan, Muhammad; Hasnain, Shahida; Zaidi, Nousheen

    2017-04-01

    Lipid-load in peripheral blood mononuclear cells (PBMCs) has recently gained attention of the researchers working on nutritional regulation of metabolic health. Previous works have indicated that the metabolic circuitries in the circulating PBMCs are influenced by dietary-intake and macronutrient composition of diet. In the present work, we analyzed the impact of diet and dietary macronutrients on PBMCs' lipid-load. The overall analyses revealed that dietary carbohydrates and fats combinatorially induce triglyceride accumulation in PBMCs. On the other hand, dietary fats were shown to induce significant decrease in PBMCs' cholesterol-load. The effects of various demographic factors -including age, gender and body-weight- on PBMCs' lipid-load were also examined. Body-weight and age were both shown to affect PBMC's lipid-load. Our study fails to provide any direct association between extracellular lipid availability and cholesterol-load in both, freshly isolated and cultured PBMCs. The presented work significantly contributes to the current understanding of the impact of food-consumption, dietary macronutrients, extracellular lipid availability and demographic factors on lipid-load in PBMCs. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

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    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.

  15. Plasma lipid pattern and red cell membrane structure in β-thalassemia patients in Jakarta

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    Seruni K.U. Freisleben

    2011-08-01

    Full Text Available Background: Over the last 10 years, we have investigated thalassemia patients in Jakarta to obtain a comprehensive picture of iron overload, oxidative stress, and cell damage.Methods: In blood samples from 15 transfusion-dependent patients (group T, 5 non-transfused patients (group N and 10 controls (group C, plasma lipids and lipoproteins, lipid-soluble vitamin E, malondialdehyde (MDA and thiol status were measured. Isolated eryhtrocyte membranes were investigated with electron paramagnetic resonance (EPR spectroscopy using doxyl-stearic acid and maleimido-proxyl spin lables. Data were analyzed statistically with ANOVA.Results: Plasma triglycerides were higher and cholesterol levels were lower in thalassemic patients compared to controls. Vitamin E, group C: 21.8 vs T: 6.2 μmol/L and reactive thiols (C: 144 vs. T: 61 μmol/L were considerably lower in transfused patients, who exert clear signs of oxidative stress (MDA, C: 1.96 vs T: 9.2 μmol/L and of tissue cell damage, i.e., high transaminases plasma levels. Non-transfused thalassemia patients have slight signs of oxidative stress, but no significant indication of cell damage. Erythrocyte membrane parameters from EPR spectroscopy differ considerably between all groups. In transfusion-dependent patients the structure of the erythrocyte membrane and the gradients of polarity and fluidity are destroyed in lipid domains; binding capacity of protein thiols in the membrane is lower and immobilized.Conclusion: In tranfusion-dependent thalassemic patients, plasma lipid pattern and oxidative stress are associated with structural damage of isolated erythrocyte membranes as measured by EPR spectroscopy with lipid and proteinthiol spin labels. (Med J Indones 2011; 20:178-84Keywords: electron paramagnetic resonance spectroscopy, erythrocyte membrane, lipoproteins, oxidative stress, thalassemia, plasma lipids.

  16. High dietary level of synthetic vitamin E on lipid peroxidation, membrane fatty acid composition and cytotoxicity in breast cancer xenograft and in mouse host tissue

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    Barnes Christopher J

    2003-03-01

    Full Text Available Abstract Background d-α-tocopherol is a naturally occurring form of vitamin E not previously known to have antitumor activity. Synthetic vitamin E (sE is a commonly used dietary supplement consisting of a mixture of d-α-tocopherol and 7 equimolar stereoisomers. To test for antilipid peroxidation and for antitumor activity of sE supplementation, two groups of nude mice bearing a MDA-MB 231 human breast cancer tumor were fed an AIN-76 diet, one with and one without an additional 2000 IU/kg dry food (equivalent to 900 mg of all-rac-α-tocopherol or sE. This provided an intake of about 200 mg/kg body weight per day. The mice were killed at either 2 or 6 weeks after the start of dietary intervention. During necropsy, tumor and host tissues were excised for histology and for biochemical analyses. Results Tumor growth was significantly reduced by 6 weeks of sE supplementation. Thiobarbituric acid reactive substances, an indicator of lipid peroxidation, were suppressed in tumor and in host tissues in sE supplemented mice. In the sE treated mice, the fatty acid composition of microsomal and mitochondrial membranes of tumor and host tissues had proportionately less linoleic acid (n-6 C 18-2, similar levels of arachidonic acid (n-6 C 20-4, but more docosahexanoic acid (n-3 C 22-6. The sE supplementation had no significant effect on blood counts or on intestinal histology but gave some evidence of cardiac toxicity as judged by myocyte vacuoles and by an indicator of oxidative stress (increased ratio of Mn SOD mRNA over GPX1 mRNA. Conclusions At least one of the stereoisomers in sE has antitumor activity. Synthetic vitamin E appears to preferentially stabilize membrane fatty acids with more double bonds in the acyl chain. Although sE suppressed tumor growth and lipid peroxidation, it may have side-effects in the heart.

  17. Knowledge to Predict Pathogens: Legionella pneumophila Lifecycle Critical Review Part I Uptake into Host Cells

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    Alexis L. Mraz

    2018-01-01

    Full Text Available Legionella pneumophila (L. pneumophila is an infectious disease agent of increasing concern due to its ability to cause Legionnaires’ Disease, a severe community pneumonia, and the difficulty in controlling it within water systems. L. pneumophila thrives within the biofilm of premise plumbing systems, utilizing protozoan hosts for protection from disinfectants and other environmental stressors. While there is a great deal of information regarding how L. pneumophila interacts with protozoa and human macrophages (host for human infection, the ability to use this data in a model to attempt to predict a concentration of L. pneumophila in a water system is not known. The lifecycle of L. pneumophila within host cells involves three processes: uptake, growth, and egression from the host cell. The complexity of these three processes would risk conflation of the concepts; therefore, this review details the available information regarding how L. pneumophila invades host cells (uptake within the context of data needed to model this process, while a second review will focus on growth and egression. The overall intent of both reviews is to detail how the steps in L. pneumophila’s lifecycle in drinking water systems affect human infectivity, as opposed to detailing just its growth and persistence in drinking water systems.

  18. Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

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    Lin, Yi-Han; Machner, Matthias P

    2017-06-15

    Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

  19. Dynamic Quantification of Host Schwann Cell Migration into Peripheral Nerve Allografts

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    Whitlock, Elizabeth L.; Myckatyn, Terence M.; Tong, Alice Y.; Yee, Andrew; Yan, Ying; Magill, Christina K.; Johnson, Philip J.; Mackinnon, Susan E.

    2010-01-01

    Host Schwann cell (SC) migration into nerve allografts is the limiting factor in the duration of immunosuppression following peripheral nerve allotransplantation, and may be affected by different immunosuppressive regimens. Our objective was to compare SC migration patterns between clinical and experimental immunosuppression regimens both over time and at the harvest endpoint. Eighty mice that express GFP under the control of the Schwann cell specific S100 promoter were engrafted with allogeneic, nonfluorescent sciatic nerve grafts. Mice received immunosuppression with either tacrolimus (FK506), or experimental T-cell triple costimulation blockade (CSB), consisting of CTLA4-immunoglobulin fusion protein, anti-CD40 monoclonal antibody, and anti-inducible costimulator monoclonal antibody. Migration of GFP-expressing host SCs into wild-type allografts was assessed in vivo every 3 weeks until 15 weeks postoperatively, and explanted allografts were evaluated for immunohistochemical staining patterns to differentiate graft from host SCs. Immunosuppression with tacrolimus exhibited a plateau of SC migration, characterized by significant early migration (< 3 weeks) followed by a constant level of host SCs in the graft (15 weeks). At the endpoint, graft fluorescence was decreased relative to surrounding host nerve, and donor SCs persisted within the graft. CSB-treated mice displayed gradually increasing migration of host SCs into the graft, without the plateau noted in tacrolimus-treated mice, and also maintained a population of donor SCs at the 15-week endpoint. SC migration patterns are affected by immunosuppressant choice, particularly in the immediate postoperative period, and the use of a single treatment of CSB may allow for gradual population of nerve allografts with host SCs. PMID:20633557

  20. Multiple factors and processes involved in host cell killing by bacteriophage Mu: characterization and mapping.

    Science.gov (United States)

    Waggoner, B T; Marrs, C F; Howe, M M; Pato, M L

    1984-07-15

    The regions of bacteriophage Mu involved in host cell killing were determined by infection of a lambda-immune host with 12 lambda pMu-transducing phages carrying different amounts of Mu DNA beginning at the left end. Infecting lambda pMu phages containing 5.0 (+/- 0.2) kb or less of the left end of Mu DNA did not kill the lambda-immune host, whereas lambda pMu containing 5.1 kb did kill, thus locating the right end of the kil gene between approximately 5.0 and 5.1 kb. For the Kil+ phages the extent of killing increased as the multiplicity of infection (m.o.i.) increased. In addition, killing was also affected by the presence of at least two other regions of Mu DNA: one, located between 5.1 and 5.8 kb, decreased the extent of killing; the other, located between 6.3 and 7.9 kb, greatly increased host cell killing. Killing was also assayed after lambda pMu infection of a lambda-immune host carrying a mini-Mu deleted for most of the B gene and the middle region of Mu DNA. Complementation of mini-Mu replication by infecting B+ lambda pMu phages resulted in killing of the lambda-immune, mini-Mu-containing host, regardless of the presence or absence of the Mu kil gene. The extent of host cell killing increased as the m.o.i. of the infecting lambda pMu increased, and was further enhanced by both the presence of the kil gene and the region located between 6.3 and 7.9 kb. These distinct processes of kil-mediated killing in the absence of replication and non-kil-mediated killing in the presence of replication were also observed after induction of replication-deficient and kil mutant prophages, respectively.

  1. Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

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    Ricardo Chaves Vilela

    2012-09-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

  2. Mesenchymal stromal cells in the antimicrobial host response of hematopoietic stem cell recipients with graft-versus-host disease--friends or foes?

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    Balan, A; Lucchini, G; Schmidt, S; Schneider, A; Tramsen, L; Kuçi, S; Meisel, R; Bader, P; Lehrnbecher, T

    2014-10-01

    Mesenchymal stromal cells (MSCs) are multipotent cells, which exhibit broad immunosuppressive activities. Moreover, they may be administered irrespectively of human leukocyte antigen (HLA) compatibility, without inducing life-threatening immunological reactions, as they express no HLA class II and limited HLA class I antigens under resting conditions. These characteristics have made MSC an appealing candidate for cell therapy after hematopoietic stem cell transplantation (HSCT), for example, for treatment of graft-versus-host disease (GvHD) or for graft rejection prevention/treatment in allogeneic HSCT recipients. Unfortunately, information regarding the effect of MSC infusion on the host response to infectious agents is scarce, and study results on infectious complications in patients receiving MSC are conflicting. The present review focuses on the available data from in vitro studies and animal models regarding the interaction of MSC with bacterial, viral and fungal pathogens. In a clinical part, we present the current information on infectious complications in allogeneic HSCT recipients who had received MSCs as prophylaxis or treatment of GvHD disease.

  3. Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

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    Peyret, Ariane; Ibarboure, Emmanuel; Le Meins, Jean-François; Lecommandoux, Sebastien

    2018-01-01

    Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)- b -poly(ethylene oxide) (PBut- b -PEO) and outer monolayer of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm 2 s -1 at 25 °C and D = 2.3 ± 0.7 μm 2 s -1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

  4. Lipid Metabolism in Vascular Smooth Muscle Cells Infuenced by HCMV Infection

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    Lingfang Li

    2016-10-01

    Full Text Available Background: The present study was designed to observe the infection of human cytomegalovirus (HCMV to human vascular smooth muscle cells (VSMCs, and the effect of viral infection on lipid metabolism in VSMCs. Methods: The cytopathic effects were observed by inverted microscopy and viral infection were examined by electron microscopy and RT-PCR. The lipid metabolism related gene profiling of VSMCs after HCMV infection was assayed by cDNA assay and the abnormal expression of genes were validated by quantitative RT-PCR. The content of cholesterol in VSMCs after HCMV infection was assayed by cholesterol detection kit. Results: VSMCs showed obvious cytopathic effects after HCMV infection. Intact viral particles could be detected in VSMCs using electron microscope. By use of RT-PCR technology, IE gene of HCMV could be amplified from VSMCs. The expression of cell lipid metabolism related gene profiling showed obvious disorders. The expression levels of HMG-CoA synthase and HMG-CoA reductase after infection increased significantly. The cellular cholesterol content (µmol/106 cells was significantly higher than that of mock infected group at 72h post infection. Conclusion: HCMV can infect VSMCs and the infection can affect cellular lipid metabolism related gene expression, which get involved in the occurrence and development of atherosclerosis (AS.

  5. Predicting the subcellular localization of viral proteins within a mammalian host cell

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    Thomas DY

    2006-04-01

    Full Text Available Abstract Background The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. Results Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. Conclusion Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.

  6. Antimicrobial role of human meibomian lipids at the ocular surface.

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    Mudgil, Poonam

    2014-10-14

    Human meibomian lipids form the outermost lipid layer of the tear film and serve many important functions to maintain its integrity. Although not investigated earlier, these lipids may have antimicrobial properties that help in strengthening the innate host defense of tears at the ocular surface. The aim of this study was to investigate the antimicrobial role of human meibomian lipids. Ocular pathogenic bacteria, Staphylococcus aureus 31, Pseudomonas aeruginosa 19, Pseudomonas aeruginosa 20, and Serratia marcescens 35, were grown in the presence and absence of human meibomian lipids in an artificial tear solution at the physiological temperature. Viable counts were obtained to note the number of bacteria surviving the treatment with meibomian lipids. Bacterial cells were imaged using scanning electron microscopy to observe the damages caused by meibomian lipids. Viable count results showed that in the presence of meibomian lipids, growth of all bacteria was considerably lower. Scanning electron microscopy showed that meibomian lipids caused extensive cellular damage to bacteria as manifested in smaller size, loss of aggregation, abnormal phenotype, cellular distortion, damaged cell wall, and cell lysis. This is the first-ever report of the antimicrobial role of human meibomian lipids. These lipids possess antimicrobial properties against both Gram-positive and Gram-negative bacteria and are involved in the innate host defense of tears in protecting the ocular surface against microbial pathogens. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  7. Industrial production of clotting factors: Challenges of expression, and choice of host cells.

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    Kumar, Sampath R

    2015-07-01

    The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Anaplasma phagocytophilum Manipulates Host Cell Apoptosis by Different Mechanisms to Establish Infection

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    Pilar Alberdi

    2016-07-01

    Full Text Available Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human and animal granulocytic anaplasmosis and tick-borne fever of ruminants. This obligate intracellular bacterium evolved to use common strategies to establish infection in both vertebrate hosts and tick vectors. Herein, we discuss the different strategies used by the pathogen to modulate cell apoptosis and establish infection in host cells. In vertebrate neutrophils and human promyelocytic cells HL-60, both pro-apoptotic and anti-apoptotic factors have been reported. Tissue-specific differences in tick response to infection and differential regulation of apoptosis pathways have been observed in adult female midguts and salivary glands in response to infection with A. phagocytophilum. In tick midguts, pathogen inhibits apoptosis through the Janus kinase/signal transducers and activators of transcription (JAK/STAT pathway, while in salivary glands, the intrinsic apoptosis pathways is inhibited but tick cells respond with the activation of the extrinsic apoptosis pathway. In Ixodes scapularis ISE6 cells, bacterial infection down-regulates mitochondrial porin and manipulates protein processing in the endoplasmic reticulum and cell glucose metabolism to inhibit apoptosis and facilitate infection, whereas in IRE/CTVM20 tick cells, inhibition of apoptosis appears to be regulated by lower caspase levels. These results suggest that A. phagocytophilum uses different mechanisms to inhibit apoptosis for infection of both vertebrate and invertebrate hosts.

  9. Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ.

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    van der Linde, Karina; Timofejeva, Ljudmilla; Egger, Rachel L; Ilau, Birger; Hammond, Reza; Teng, Chong; Meyers, Blake C; Doehlemann, Gunther; Walbot, Virginia

    2018-03-01

    Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize ( Zea mays ), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus Ustilago maydis , to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zm mac1-1 anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses. © 2018 American Society of Plant Biologists. All rights reserved.

  10. Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

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    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

  11. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

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    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

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

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    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.

  13. Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing.

    Science.gov (United States)

    Duguay, Brett A; Huang, Kate Wei-Chen; Kulka, Marianna

    2018-04-18

    Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes. ©2018 Society for Leukocyte Biology.

  14. Host Cell Responses to Persistent Mycoplasmas - Different Stages in Infection of HeLa Cells with Mycoplasma hominis

    Science.gov (United States)

    Hopfe, Miriam; Deenen, René; Degrandi, Daniel; Köhrer, Karl; Henrich, Birgit

    2013-01-01

    Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of

  15. Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

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    Ladislav Nedbal

    2010-09-01

    Full Text Available Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C=C stretching mode and 1,445 cm−1 (CH2 scissoring mode as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

  16. Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

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    Brennan S. Dirk

    2016-10-01

    Full Text Available Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1 is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED and photoactivation and localization microscopy (PALM have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET and bimolecular fluorescence complementation (BiFC have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

  17. Treg cell-IgA axis in maintenance of host immune homeostasis with microbiota

    OpenAIRE

    Feng, Ting; Elson, Charles O.; Cong, Yingzi

    2010-01-01

    The intestine is the home to a vast diversity of microbiota and a complex of mucosal immune system. Multiple regulatory mechanisms control host immune responses to microbiota and maintain intestinal immune homeostasis. This mini review will provide evidence indicating a Treg cell-IgA axis and such axis playing a major role in maintenance of intestinal homeostasis.

  18. Bright fluorescent Streptococcus pneumoniae for live cell imaging of host-pathogen interactions

    NARCIS (Netherlands)

    Kjos, M.; Aprianto, R.; Fernandes, V.E.; Andrew, P.W.; Strijp, van J.A.G.; Nijland, R.; Veening, J.W.

    2015-01-01

    Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people, but at the same time one of the major causes of infectious diseases such as pneumonia, meningitis and sepsis. The shift from commensal to pathogen and its interaction with host cells is poorly understood. One of the

  19. Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

    NARCIS (Netherlands)

    Kjos, Morten; Aprianto, Rieza; Fernandes, Vitor E.; Andrew, Peter W.; van Strijp, Jos A. G.; Nijland, Reindert; Veening, Jan-Willem

    Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the

  20. Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense.

    NARCIS (Netherlands)

    Vos, J.B.; Sterkenburg, M.A. van; Rabe, K.F.; Schalkwijk, J.; Hiemstra, P.S.; Datson, N.A.

    2005-01-01

    The airway epithelium responds to microbial exposure by altering expression of a variety of genes to increase innate host defense. We aimed to delineate the early transcriptional response in human primary bronchial epithelial cells exposed for 6 h to a mixture of IL-1beta and TNF-alpha or

  1. Complexities in human herpesvirus-6A and -6B binding to host cells

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Höllsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...

  2. The Paracoccidioides cell wall: past and present layers towards understanding interaction with the host

    Directory of Open Access Journals (Sweden)

    Rosana ePuccia

    2011-12-01

    Full Text Available The cell wall of pathogenic fungi plays import roles in interaction with the host, so that its composition and structure may determine the course of infection. Here we present an overview of the current and past knowledge on the cell wall constituents of Paracoccidioides brasiliensis and P. lutzii. These are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis, a systemic granulomatous and debilitating disease. Focus is given on cell wall carbohydrate and protein contents, their immune-stimulatory features, adhesion properties, drug target characteristics, and morphological phase specificity. We offer a journey towards the future understanding of the dynamic life that takes place in the cell wall and of the changes that it may suffer when living in the human host.

  3. Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

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    Yaakov Maman

    2011-10-01

    Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

  4. Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

    Science.gov (United States)

    Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

    2016-04-01

    The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. Synchronous induction of detachment and reattachment of symbiotic Chlorella spp. from the cell cortex of the host Paramecium bursaria.

    Science.gov (United States)

    Kodama, Yuuki; Fujishima, Masahiro

    2013-09-01

    Paramecium bursaria harbor several hundred symbiotic Chlorella spp. Each alga is enclosed in a perialgal vacuole membrane, which can attach to the host cell cortex. How the perialgal vacuole attaches beneath the host cell cortex remains unknown. High-speed centrifugation (> 1000×g) for 1min induces rapid detachment of the algae from the host cell cortex and concentrates the algae to the posterior half of the host cell. Simultaneously, most of the host acidosomes and lysosomes accumulate in the anterior half of the host cell. Both the detached algae and the dislocated acidic vesicles recover their original positions by host cyclosis within 10min after centrifugation. These recoveries were inhibited if the host cytoplasmic streaming was arrested by nocodazole. Endosymbiotic algae during the early reinfection process also show the capability of desorption after centrifugation. These results demonstrate that adhesion of the perialgal vacuole beneath the host cell cortex is repeatedly inducible, and that host cytoplasmic streaming facilitates recovery of the algal attachment. This study is the first report to illuminate the mechanism of the induction to desorb for symbiotic algae and acidic vesicles, and will contribute to the understanding of the mechanism of algal and organelle arrangements in Paramecium. Copyright © 2013 Elsevier GmbH. All rights reserved.

  6. The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol

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    Flores Rhonda

    2011-04-01

    Full Text Available Abstract Background The periplasmic High Temperature Requirement protein A (HtrA plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA in chlamydial pathogenesis is not clear. Results The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor. Conclusion Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

  7. Ferromagnetic filled carbon nanotubes and nanoparticles: synthesis and lipid-mediated delivery into human tumor cells

    International Nuclear Information System (INIS)

    Moench, I.; Meye, A.; Leonhardt, A.; Kraemer, K.; Kozhuharova, R.; Gemming, T.; Wirth, M.P.; Buechner, B.

    2005-01-01

    We describe the synthesis and the properties of Fe-filled multi-walled carbon nanotubes (MWNTs) and nanoparticles (NP) produced by chemical vapor deposition (CVD). We have employed ferrocene as a starting substance and oxidized Si-wafers as substrates. The magnetic properties and the interaction of the material with bladder cancer cells were determined. After the addition of NP suspensions to cultured cells, no adhesion of the nanoparticles/nanotubes (NT/NP) to the cell membrane and also no cellular uptake were observed. However, the preincubation of the (NT/NP) suspension with cationic lipid caused an efficient delivery of the lipid-nanostructure complexes into the cytoplasm within 2 h after adding to the culture medium

  8. Effects of Krill Oil on serum lipids of hyperlipidemic rats and human SW480 cells

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    Qian Wen-Bin

    2008-08-01

    Full Text Available Abstract Background Cardiovascular disease (CVD and colon cancer incidence are known to be closely related to dietary factors. This article evaluated effects of krill oil (KO on serum lipids of hyperlipidemia rats and human colon cancer cells (SW480. Serum lipids of rats fed with high fat diet (HFD and different doses of KO were measured by automatic analyzer. Effect of KO on viability of cells was determined by methyl thiazolyl tetrazolium (MTT assay. Results Except for higher dose group, body weights decreased significantly. Total cholesterol (TC, LDL-cholesterol (LDL-C of all dose groups, Triglycerides (TG of low and mid dose groups descended significantly, while there were no significant differences of HDL-cholesterol (HDL-C, compared with control group. Treatment of colon cancer cells with KO also resulted in time-dependent inhibition of cell growth. Conclusion Our findings indicated that the consumption of KO may provide benefits to control serum lipid levels in certain diseases and inhibit growth of colon cancer cells. Therefore, KO may be a good candidate for development as a functional food and nutraceutical.

  9. Mesenchymal Stem Cells Enhance Liver Regeneration via Improving Lipid Accumulation and Hippo Signaling

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    Yang Liu

    2018-01-01

    Full Text Available The liver has the potential to regenerate after injury. It is a challenge to improve liver regeneration (LR after liver resection in clinical practice. Bone morrow-derived mesenchymal stem cells (MSCs have shown to have a role in various liver diseases. To explore the effects of MSCs on LR, we established a model of 70% partial hepatectomy (PHx. Results revealed that infusion of MSCs could improve LR through enhancing cell proliferation and cell growth during the first 2 days after PHx, and MSCs could also restore liver synthesis function. Infusion of MSCs also improved liver lipid accumulation partly via mechanistic target of rapamycin (mTOR signaling and enhanced lipid β-oxidation support energy for LR. Rapamycin-induced inhibition of mTOR decreased liver lipid accumulation at 24 h after PHx, leading to impaired LR. And after infusion of MSCs, a proinflammatory environment formed in the liver, evidenced by increased expression of IL-6 and IL-1β, and thus the STAT3 and Hippo-YAP pathways were activated to improve cell proliferation. Our results demonstrated the function of MSCs on LR after PHx and provided new evidence for stem cell therapy of liver diseases.

  10. The ins and outs of phosphosignalling in Plasmodium: Parasite regulation and host cell manipulation.

    Science.gov (United States)

    Carvalho, Teresa Gil; Morahan, Belinda; John von Freyend, Simona; Boeuf, Philippe; Grau, Georges; Garcia-Bustos, Jose; Doerig, Christian

    2016-07-01

    Signal transduction and kinomics have been rapidly expanding areas of investigation within the malaria research field. Here, we provide an overview of phosphosignalling pathways that operate in all stages of the Plasmodium life cycle. We review signalling pathways in the parasite itself, in the cells it invades, and in other cells of the vertebrate host with which it interacts. We also discuss the potential of these pathways as novel targets for antimalarial intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Coliphage 186 replication is delayed when the host cell is UV irradiated before infection

    International Nuclear Information System (INIS)

    Hooper, I.; Woods, W.H.; Egan, B.

    1981-01-01

    In contrast to results with injections by lambda and P2, the latent period for infection by coliphage 186 is extended when the host cell is UV irradiated before infection. We find that 186 replication is significantly delayed in such a cell, even though the phage itself has not been irradiated. In contrast, replication of the closely related phage P2 under the same conditions is not affected

  12. A role for host cell exocytosis in InlB-mediated internalisation of Listeria monocytogenes.

    Science.gov (United States)

    Van Ngo, Hoan; Bhalla, Manmeet; Chen, Da-Yuan; Ireton, Keith

    2017-11-01

    The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB-mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB-dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB-mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB-mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection. © 2017 John Wiley & Sons Ltd.

  13. Encapsulation in lipid-core nanocapsules overcomes lung cancer cell resistance to tretinoin.

    Science.gov (United States)

    Schultze, Eduarda; Ourique, Aline; Yurgel, Virginia Campello; Begnini, Karine Rech; Thurow, Helena; de Leon, Priscila Marques Moura; Campos, Vinicius Farias; Dellagostin, Odir Antônio; Guterres, Silvia R; Pohlmann, Adriana R; Seixas, Fabiana Kömmling; Beck, Ruy Carlos Ruver; Collares, Tiago

    2014-05-01

    Tretinoin is a retinoid derivative that has an antiproliferative effect on several kinds of tumours. Human lung adenocarcinoma epithelial cell lines (A549) exhibit a profound resistance to the effects of tretinoin. Nanocarriers seem to be a good alternative to overcomecellular resistance to drugs. The aim of this study was to test whether tretinoin-loaded lipid-core nanocapsules exert anantitumor effect on A549 cells. A549 cells were incubated with free tretinoin (TTN), blank nanocapsules (LNC) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC). Data from evaluation of DNA content and Annexin V binding assay by flow cytometry showed that TTN-LNC induced apoptosis and cell cycle arrest at the G1-phase while TTN did not. TTN-LNC showed higher cytotoxic effects than TTN on A549 cells evaluated by MTT and LIVE/DEAD cell viability assay. Gene expression profiling identified up-regulated expression of gene p21 by TTN-LNC, supporting the cell cycle arrest effect. These results showed for the first time that TTN-LNC are able to overcome the resistance of adenocarcinoma cell line A549 to treatment with TTN by inducing apoptosis and cell cycle arrest, providing support for their use in applications in lung cancer therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Molecular mechanisms of Porphyromonas gingivalis-host cell interaction on periodontal diseases

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    Masaaki Nakayama

    2017-11-01

    Full Text Available Porphyromonas gingivalis (P. gingivalis is a major oral pathogen and associated with periodontal diseases including periodontitis and alveolar bone loss. In this review, we indicate that two virulence factors, which are hemoglobin receptor protein (HbR and cysteine proteases “gingipains”, expressed by P. gingivalis have novel functions on the pathogenicity of P. gingivalis. P. gingivalis produces three types of gingipains and concomitantly several adhesin domains. Among the adhesin domains, hemoglobin receptor protein (HbR, also called HGP15, has the function of induction of interleukin-8 (IL-8 expression in human gingival epithelial cells, indicating the possibility that HbR is associated with P. gingivalis-induced periodontal inflammation. On bacteria-host cells contact, P. gingivalis induces cellular signaling alteration in host cells. Phosphatidylinositol 3-kinase (PI3K and Akt are well known to play a pivotal role in various cellular physiological functions including cell survival and glucose metabolism in mammalian cells. Recently, we demonstrated that gingipains attenuate the activity of PI3K and Akt, which might have a causal influence on periodontal diseases by chronic infection to the host cells from the speculation of molecular analysis. In this review, we discuss new molecular and biological characterization of the virulence factors from P. gingivalis.

  15. Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

    Science.gov (United States)

    Yang, Eun Sun; Yang, Joon-Hyuck; Park, Ji Eun; Park, Jeen-Woo

    2005-01-01

    Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

  16. Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

    Science.gov (United States)

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-04-01

    Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/Escherichia coli (E. coli) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Membrane-lipid therapy: A historical perspective of membrane-targeted therapies - From lipid bilayer structure to the pathophysiological regulation of cells.

    Science.gov (United States)

    Escribá, Pablo V

    2017-09-01

    Our current understanding of membrane lipid composition, structure and functions has led to the investigation of their role in cell signaling, both in healthy and pathological cells. As a consequence, therapies based on the regulation of membrane lipid composition and structure have been recently developed. This novel field, known as Membrane Lipid Therapy, is growing and evolving rapidly, providing treatments that are now in use or that are being studied for their application to oncological disorders, Alzheimer's disease, spinal cord injury, stroke, diabetes, obesity, and neuropathic pain. This field has arisen from relevant discoveries on the behavior of membranes in recent decades, and it paves the way to adopt new approaches in modern pharmacology and nutrition. This innovative area will promote further investigation into membranes and the development of new therapies with molecules that target the cell membrane. Due to the prominent roles of membranes in the cells' physiology and the paucity of therapeutic approaches based on the regulation of the lipids they contain, it is expected that membrane lipid therapy will provide new treatments for numerous pathologies. The first on-purpose rationally designed molecule in this field, minerval, is currently being tested in clinical trials and it is expected to enter the market around 2020. However, it seems feasible that during the next few decades other membrane regulators will also be marketed for the treatment of human pathologies. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017. Published by Elsevier B.V.

  18. Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

    Directory of Open Access Journals (Sweden)

    Elena Ufimtseva

    2015-01-01

    Full Text Available Tuberculosis (TB is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

  19. Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

    Science.gov (United States)

    2015-01-01

    Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals. PMID:26064970

  20. High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation.

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    Kim Cheng

    Full Text Available Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS. HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.

  1. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

    Directory of Open Access Journals (Sweden)

    Mathias J Gerl

    Full Text Available Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1 HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

  2. The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

    Directory of Open Access Journals (Sweden)

    Yvette A. Hayman

    2017-03-01

    Full Text Available Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR and aspiration events. The observation of lipid-laden macrophages (LLMs within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

  3. The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

    Science.gov (United States)

    Hayman, Yvette A; Sadofsky, Laura R; Williamson, James D; Hart, Simon P; Morice, Alyn H

    2017-01-01

    Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR) and aspiration events. The observation of lipid-laden macrophages (LLMs) within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD) pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

  4. Lipid-Based Nanovectors for Targeting of CD44-Overexpressing Tumor Cells

    Directory of Open Access Journals (Sweden)

    Silvia Arpicco

    2013-01-01

    Full Text Available Hyaluronic acid (HA is a naturally occurring glycosaminoglycan that exists in living systems, and it is a major component of the extracellular matrix. The hyaluronic acid receptor CD44 is found at low levels on the surface of epithelial, haematopoietic, and neuronal cells and is overexpressed in many cancer cells particularly in tumour initiating cells. HA has been therefore used as ligand attached to HA-lipid-based nanovectors for the active targeting of small or large active molecules for the treatment of cancer. This paper describes the different approaches employed for the preparation, characterization, and evaluation of these potent delivery systems.

  5. Models of dynamic extraction of lipid tethers from cell membranes

    International Nuclear Information System (INIS)

    Nowak, Sarah A; Chou, Tom

    2010-01-01

    When a ligand that is bound to an integral membrane receptor is pulled, the membrane and the underlying cytoskeleton can deform before either the membrane delaminates from the cytoskeleton or the ligand detaches from the receptor. If the membrane delaminates from the cytoskeleton, it may be further extruded and form a membrane tether. We develop a phenomenological model for this process by assuming that deformations obey Hooke's law up to a critical force at which the cell membrane locally detaches from the cytoskeleton and a membrane tether forms. We compute the probability of tether formation and show that tethers can be extruded only within an intermediate range of force loading rates and pulling velocities. The mean tether length that arises at the moment of ligand detachment is computed as are the force loading rates and pulling velocities that yield the longest tethers

  6. Ultrastructural characteristics of nurse cell-larva complex of four species of Trichinella in several hosts

    Directory of Open Access Journals (Sweden)

    Sacchi L.

    2001-06-01

    Full Text Available The nurse cell-larva complex of nematodes of the genus Trichinella plays an Important role in the survival of the larva in decaying muscles, frequently favouring the transmission of the parasite in extreme environmental conditions. The ultrastructure of the nurse cell-larva complex in muscles from different hosts infected with T. nativa (a walrus and a polar bear, T. spiralis (horses and humans, T. pseudospiralis (a laboratory mouse and T. papuae (a laboratory mouse were examined. Analysis with transmission electron microscope showed that the typical nurse cell structure was present in all examined samples, irrespective of the species of larva, of the presence of a collagen capsule, of the age of infection and of the host species, suggesting that there exists a molecular mechanism that in the first stage of larva invasion is similar for encapsulated and non-encapsulated species.

  7. Lymphotoxin organizes contributions to host defense and metabolic illness from innate lymphoid cells.

    Science.gov (United States)

    Upadhyay, Vaibhav; Fu, Yang-Xin

    2014-04-01

    The lymphotoxin (LT)-pathway is a unique constituent branch of the Tumor Necrosis Superfamily (TNFSF). Use of LT is a critical mechanism by which fetal innate lymphoid cells regulate lymphoid organogenesis. Within recent years, adult innate lymphoid cells have been discovered to utilize this same pathway to regulate IL-22 and IL-23 production for host defense. Notably, genetic studies have linked polymorphisms in the genes encoding LTα to several phenotypes contributing to metabolic syndrome. The role of the LT-pathway may lay the foundation for a bridge between host immune response, microbiota, and metabolic syndrome. The contribution of the LT-pathway to innate lymphoid cell function and metabolic syndrome will be visited in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while β-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research.

  9. Lipid somersaults

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Menon, Anant K.

    2016-01-01

    Membrane lipids diffuse rapidly in the plane of the membrane but their ability to flip spontaneously across a membrane bilayer is hampered by a significant energy barrier. Thus spontaneous flip-flop of polar lipids across membranes is very slow, even though it must occur rapidly to support diverse...... aspects of cellular life. Here we discuss the mechanisms by which rapid flip-flop occurs, and what role lipid flipping plays in membrane homeostasis and cell growth. We focus on conceptual aspects, highlighting mechanistic insights from biochemical and in silico experiments, and the recent, ground......-breaking identification of a number of lipid scramblases....

  10. Beet yellow stunt virus in cells of Sonchus oleraceus L. and its relation to host mitochondria.

    Science.gov (United States)

    Esau, K

    1979-10-15

    In Sonchus oleraceus L. (Asteraceae) infected with the beet yellow stunt virus (BYSV) the virions are found in phloem cells, including the sieve elements. In parenchymatous phloem cells, the virus is present mainly in the cytoplasm. In some parenchymatous cells, containing massive accumulations of virus, the flexuous rodlike virus particles are found partly inserted into mitochondrial cristae. The mitochondrial envelope is absent where virus is present in the cristae. A similar relation between virus and host mitochondria apparently has not been recorded for any other plant virus.

  11. Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells.

    Science.gov (United States)

    Shi, H B; Yu, K; Luo, J; Li, J; Tian, H B; Zhu, J J; Sun, Y T; Yao, D W; Xu, H F; Shi, H P; Loor, J J

    2015-10-01

    Milk fat originates from the secretion of cytosolic lipid droplets (CLD) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; gene symbol PLIN2) is a CLD-binding protein that is crucial for synthesis of mature CLD. Our hypothesis was that ADRP regulates CLD production and metabolism in goat mammary epithelial cells (GMEC) and thus plays a role in determining milk fat content. To understand the role of ADRP in ruminant milk fat metabolism, ADRP (PLIN2) was overexpressed or knocked down in GMEC using an adenovirus system. Immunocytochemical staining revealed that ADRP localized to the surface of CLD. Supplementation with oleic acid (OA) enhanced its colocalization with CLD surface and enhanced lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knocked down. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggests that some other compensatory factors may also aid in the process of CLD formation. The ADRP reversed the decrease of CLD accumulation induced by adipose triglyceride lipase. This is highly suggestive of ADRP promoting triacylglycerol stability within CLD by preventing access to adipose triglyceride lipase. Collectively, these data provide direct in vitro evidence that ADRP plays a key role in CLD formation and stability in GMEC. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Global impact of Salmonella type III secretion effector SteA on host cells

    International Nuclear Information System (INIS)

    Cardenal-Muñoz, Elena; Gutiérrez, Gabriel; Ramos-Morales, Francisco

    2014-01-01

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration

  13. Global impact of Salmonella type III secretion effector SteA on host cells

    Energy Technology Data Exchange (ETDEWEB)

    Cardenal-Muñoz, Elena; Gutiérrez, Gabriel; Ramos-Morales, Francisco

    2014-07-11

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

  14. CD1b-mycolic acid tetramers demonstrate T-cell fine specificity for mycobacterial lipid tails

    NARCIS (Netherlands)

    Van Rhijn, Ildiko; Iwany, Sarah K; Fodran, Peter; Cheng, Tan-Yun; Gapin, Laurent; Minnaard, Adriaan J; Moody, D Branch

    Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1-presented lipid antigens contain

  15. Incorporation of [14C]-palmitate into lipids of Brassica cells during the induction of freezing tolerance

    International Nuclear Information System (INIS)

    Lynch, D.V.; Joseph, R.A.

    1989-01-01

    Changes in plasma membrane lipid composition have been causally related to increased freezing tolerance. Studies of lipid metabolism during ABA induction of freezing tolerance in Brassica napus suspension cultures were undertaken. Cells were labeled with [ 14 C]-palmitate four days after transfer to fresh medium (control) or medium containing ABA (which increases freezing tolerance). At times between one and 20 hrs after labeling, ABA-treated cells incorporated almost twice the amount of label as controls cells. Approximately 80% of the radioactivity was associated with neutral lipids in ABA-treated cells and controls. Incorporation of label into total cellular polar lipids was 4.9 x 10 5 dpm/mg protein for control cells and 1 x 10 6 dpm/mg protein for cells transferred to medium containing ABA. Analysis of lipids following alkaline hydrolysis indicated that incorporation of [ 14 C]-palmitate into glucosylceramide of ABA-treated cells was less than 60% of control values when expressed relative to that of the total polar lipids. Incorporation into ceramides was also depressed in ABA-treated cells

  16. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    International Nuclear Information System (INIS)

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-01-01

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway

  17. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  18. LipidPedia: a comprehensive lipid knowledgebase.

    Science.gov (United States)

    Kuo, Tien-Chueh; Tseng, Yufeng Jane

    2018-04-10

    Lipids are divided into fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, prenol lipids and polyketides. Fatty acyls and glycerolipids are commonly used as energy storage, whereas glycerophospholipids, sphingolipids, sterols and saccharolipids are common used as components of cell membranes. Lipids in fatty acyls, glycerophospholipids, sphingolipids and sterols classes play important roles in signaling. Although more than 36 million lipids can be identified or computationally generated, no single lipid database provides comprehensive information on lipids. Furthermore, the complex systematic or common names of lipids make the discovery of related information challenging. Here, we present LipidPedia, a comprehensive lipid knowledgebase. The content of this database is derived from integrating annotation data with full-text mining of 3,923 lipids and more than 400,000 annotations of associated diseases, pathways, functions, and locations that are essential for interpreting lipid functions and mechanisms from over 1,400,000 scientific publications. Each lipid in LipidPedia also has its own entry containing a text summary curated from the most frequently cited diseases, pathways, genes, locations, functions, lipids and experimental models in the biomedical literature. LipidPedia aims to provide an overall synopsis of lipids to summarize lipid annotations and provide a detailed listing of references for understanding complex lipid functions and mechanisms. LipidPedia is available at http://lipidpedia.cmdm.tw. yjtseng@csie.ntu.edu.tw. Supplementary data are available at Bioinformatics online.

  19. Demonstration of clonable alloreactive host T cells in a primate model for bone marrow transplantation

    International Nuclear Information System (INIS)

    Reisner, Y.; Ben-Bassat, I.; Douer, D.; Kaploon, A.; Schwartz, E.; Ramot, B.

    1986-01-01

    The phenomenon of marrow rejection following supralethal radiochemotherapy was explained in the past mainly by non-T-cell mechanisms known to be resistant to high-dose irradiation. In the present study a low but significant number of radiochemoresistant-clonable T cells was found in the peripheral blood and spleen of Rhesus monkeys following the cytoreductive protocol used for treatment of leukemia patients prior to bone marrow transplantation. More than 95% of the clonable cells are concentrated in the spleen 5 days after transplant. The cells possess immune memory as demonstrated by the generation of alloreactive-specific cytotoxicity. The present findings suggest that host-versus-graft activity may be mediated by alloreactive T cells. It is hoped that elimination of such cells prior to bone marrow transplantation will increase the engraftment rate of HLA-nonidentical marrow in leukemia patients

  20. Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membrances

    Energy Technology Data Exchange (ETDEWEB)

    James, R.; Branton, D.

    1973-01-01

    The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35/sup 0/C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching. Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity. The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.

  1. Preoperative serum lipids as prognostic predictors in esophageal squamous cell carcinoma patients with esophagectomy.

    Science.gov (United States)

    Chen, Pengxiang; Han, Lihui; Wang, Cong; Jia, Yibin; Song, Qingxu; Wang, Jianbo; Guan, Shanghui; Tan, Bingxu; Liu, Bowen; Jia, Wenqiao; Cui, Jianfeng; Zhou, Wei; Cheng, Yufeng

    2017-06-20

    This study was to evaluate the prognostic significance of serum lipids in esophageal squamous cell carcinoma patients who underwent esophagectomy. Preoperative serum lipids were collected from 214 patients who were diagnosed with esophageal squamous cell carcinoma. All of the patients received esophagectomy in Qilu Hospital of Shandong University from January 2007 to December 2008. The records and data were analyzed retrospectively. We found that low total cholesterol (for T stage, p = 0.006; for TNM stage, p = 0.039) and low-density lipoprotein cholesterol (for T stage, p = 0.031; for TNM stage, p = 0.035) were associated with advanced T stage and TNM stage. Kaplan-Meier survival analysis indicated that low total cholesterol and low-density lipoprotein cholesterol were associated with shorter disease-free survival(for total cholesterol, p = 0.045; for low-density lipoprotein cholesterol, p squamous cell carcinoma patients who underwent esophagectomy. LHR can serve as a promising serum lipids-based prognostic indicator.

  2. Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells

    Directory of Open Access Journals (Sweden)

    Jonathan Laiño

    2016-08-01

    Full Text Available Researchers have demonstrated that lactic acid bacteria (LAB with immunomodulatory capabilities (immunobiotics exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS, that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals.

  3. Nanomimics of host cell membranes block invasion and expose invasive malaria parasites.

    Science.gov (United States)

    Najer, Adrian; Wu, Dalin; Bieri, Andrej; Brand, Françoise; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

    2014-12-23

    The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.

  4. Characterisation of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    Directory of Open Access Journals (Sweden)

    Remco eStam

    2013-10-01

    Full Text Available Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centres on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signalling. Here, we characterised three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localisation of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organisation, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

  5. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    Energy Technology Data Exchange (ETDEWEB)

    Boschi, Federico, E-mail: federico.boschi@univr.it [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy); Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona (Italy); Rizzatti, Vanni; Zamboni, Mauro [Department of Medicine, Geriatric Section, University of Verona, Piazzale Stefani 1, 37126 Verona (Italy); Sbarbati, Andrea [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy)

    2014-02-15

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

  6. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    International Nuclear Information System (INIS)

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-01-01

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

  7. The Pla Protease of Yersinia pestis Degrades Fas Ligand to Manipulate Host Cell Death and Inflammation

    Science.gov (United States)

    Caulfield, Adam J.; Walker, Margaret E.; Gielda, Lindsay M.; Lathem, Wyndham W.

    2014-01-01

    SUMMARY Pneumonic plague is a deadly respiratory disease caused by Yersinia pestis. The bacterial protease Pla contributes to disease progression and manipulation of host immunity, but the mechanisms by which this occurs are largely unknown. Here we show that Pla degrades the apoptotic signaling molecule Fas ligand (FasL) to prevent host cell apoptosis and inflammation. Wild-type Y. pestis, but not a Pla mutant (Δpla), degrades FasL, which results in decreased downstream caspase-3/7 activation and reduced apoptosis. Similarly, lungs of mice challenged with wild-type Y. pestis show reduced levels of FasL and activated caspase-3/7 compared to Δpla infection. Consistent with a role for FasL in regulating immune responses, Δpla infection results in aberrant pro-inflammatory cytokine levels. The loss of FasL or inhibition of caspase activity alters host inflammatory responses and enables enhanced Y. pestis outgrowth in the lungs. Thus, by degrading FasL, Y. pestis manipulates host cell death pathways to facilitate infection. PMID:24721571

  8. Lipidomic data on lipid droplet triglyceride remodelling associated with protection of breast cancer cells from lipotoxic stress

    Directory of Open Access Journals (Sweden)

    Eva Jarc

    2018-06-01

    Full Text Available The data presented here is related to the research article entitled “Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress” by E. Jarc et al., Biochim. Biophys. Acta 1863 (2018 247–265. Elevated uptake of unsaturated fatty acids and lipid droplet accumulation are characteristic of aggressive cancer cells and have been associated with the cellular stress response. The present study provides lipidomic data on the triacylglycerol (TAG and phosphatidylcholine (PC composition of MDA-MB-231 breast cancer cells exposed to docosahexaenoic acid (DHA; 22:6, ω-3. Datasets provide information on the changes in lipid composition induced by depletion of adipose triglyceride lipase (ATGL and by exogenous addition of secreted phospholipase A2 (sPLA2 in DHA-treated cells. The presented alterations in lipid composition, mediated by targeting lipid droplet biogenesis and lipolysis, are associated with protection from lipotoxicity and allow further investigation into the role of lipid droplets in the resistance of cancer cells to lipotoxic stress. Keywords: Lipid droplets, Lipidomics, Adipose triglyceride lipase, Polyunsaturated fatty acid, Cancer, Phospholipase A2

  9. The Structural Basis for Lipid and Endotoxin Binding in RP105-MD-1, and Consequences for Regulation of Host Lipopolysaccharide Sensitivity.

    Science.gov (United States)

    Ortiz-Suarez, Maite L; Bond, Peter J

    2016-01-05

    MD-1 is a member of the MD-2-related lipid-recognition (ML) family, and associates with RP105, a cell-surface protein that resembles Toll-like receptor 4 (TLR4). The RP105⋅MD-1 complex has been proposed to play a role in fine-tuning the innate immune response to endotoxin such as bacterial lipopolysaccharide (LPS) via TLR4⋅MD-2, but controversy surrounds its mechanism. We have used atomically detailed simulations to reveal the structural basis for ligand binding and consequent functional dynamics of MD-1 and the RP105 complex. We rationalize reports of endogenous phospholipid binding, by showing that they prevent collapse of the malleable MD-1 fold, before refining crystallographic models and uncovering likely binding modes for LPS analogs. Subsequent binding affinity calculations reveal that endotoxin specificity arises from the entropic cost of expanding the MD-1 cavity to accommodate bulky lipid tails, and support the role of MD-1 as a "sink" that sequesters endotoxin from TLR4 and stabilizes RP105/TLR4 interactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite–host interaction

    Directory of Open Access Journals (Sweden)

    An

    2015-07-01

    Full Text Available Namrata Anand,1 Rupinder K Kanwar,2 Mohan Lal Dubey,1 R K Vahishta,3 Rakesh Sehgal,1,* Anita K Verma,4 Jagat R Kanwar2,*1Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 2Nanomedicine Laboratory of Immunology and Molecular Biomedical Research, School of Medicine, Molecular and Medical Research Strategic Research Centre, Faculty of Health, Deakin University, Geelong, VIC, Australia; 3Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, 4Nanobiotech Laboratory, Department of Zoology, Kirorimal College, University of Delhi, Delhi, India*These authors contributed equally to this workBackground: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs and macrophages (human monocytic cell line-derived macrophages THP1 cells.Methods: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections.Results: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene

  11. The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane.

    Science.gov (United States)

    Liu, Hengjun; Maruyama, Hisataka; Masuda, Taisuke; Honda, Ayae; Arai, Fumihito

    2016-01-01

    Influenza virus infection can result in changes in the cellular ion levels at 2-3 h post-infection. More H(+) is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H(+) during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H(+) from the intracellular compartment. Increased H(+) export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5-0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.

  12. Differential Regulation of Mas-Related G Protein-Coupled Receptor X2-Mediated Mast Cell Degranulation by Antimicrobial Host Defense Peptides and Porphyromonas gingivalis Lipopolysaccharide.

    Science.gov (United States)

    Gupta, Kshitij; Idahosa, Chizobam; Roy, Saptarshi; Lee, Donguk; Subramanian, Hariharan; Dhingra, Anuradha; Boesze-Battaglia, Kathleen; Korostoff, Jonathan; Ali, Hydar

    2017-10-01

    Porphyromonas gingivalis is a keystone pathogen that contributes to periodontal pathogenesis by disrupting host-microbe homeostasis and promoting dysbiosis. The virulence of P. gingivalis likely reflects an alteration in the lipid A composition of its lipopolysaccharide (LPS) from the penta-acylated ( Pg LPS 1690 ) to the tetra-acylated ( Pg LPS 1435/1449 ) form. Mast cells play an important role in periodontitis, but the mechanisms of their activation and regulation remain unknown. The expression of epithelium- and neutrophil-derived host defense peptides (HDPs) (LL-37 and human β-defensin-3), which activate mast cells via Mas-related G protein-coupled receptor X2 (MRGPRX2), is increased in periodontitis. We found that MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are elevated in patients with chronic periodontitis. Furthermore, HDPs stimulated degranulation in a human mast cell line (LAD2) and in RBL-2H3 cells stably expressing MRGPRX2 (RBL-MRGPRX2). Pg LPS 1690 caused substantial inhibition of HDP-induced mast cell degranulation, but Pg LPS 1435/1449 had no effect. A fluorescently labeled HDP (FAM-LL-37) bound to RBL-MRGPRX2 cells, and Pg LPS 1690 inhibited this binding, but Pg LPS 1435/1449 had no effect. These findings suggest that low-level inflammation induced by HDP/MRGPRX2-mediated mast cell degranulation contributes to gingival homeostasis but that sustained inflammation due to elevated levels of both HDPs and MRGPRX2-expressing mast cells promotes periodontal disease. Furthermore, differential regulation of HDP-induced mast cell degranulation by Pg LPS 1690 and Pg LPS 1435/1449 may contribute to the modulation of disease progression. Copyright © 2017 American Society for Microbiology.

  13. Rodent Plasmodium-infected red blood cells: imaging their fates and interactions within their hosts.

    Science.gov (United States)

    Claser, Carla; Malleret, Benoit; Peng, Kaitian; Bakocevic, Nadja; Gun, Sin Yee; Russell, Bruce; Ng, Lai Guan; Rénia, Laurent

    2014-02-01

    Malaria, a disease caused by the Plasmodium parasite, remains one of the most deadly infectious diseases known to mankind. The parasite has a complex life cycle, of which only the erythrocytic stage is responsible for the diverse pathologies induced during infection. To date, the disease mechanisms that underlie these pathologies are still poorly understood. In the case of infections caused by Plasmodium falciparum, the species responsible for most malaria related deaths, pathogenesis is thought to be due to the sequestration of infected red blood cells (IRBCs) in deep tissues. Other human and rodent malaria parasite species are also known to exhibit sequestration. Here, we review the different techniques that allow researchers to study how rodent malaria parasites modify their host cells, the distribution of IRBCs in vivo as well as the interactions between IRBCs and host tissues. © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

  14. Host epithelial cell invasion by Campylobacter jejuni: trigger or zipper mechanism?

    Directory of Open Access Journals (Sweden)

    Tadhg eÓ Cróinín

    2012-03-01

    Full Text Available Campylobacter jejuni, a spiral-shaped Gram-negative pathogen, is a highly frequent cause of gastrointestinal foodborne illness in humans worldwide. Clinical outcome of C. jejuni infections ranges from mild to severe diarrheal disease, and some other complications including reactive arthritis and Guillain–Barré syndrome. This review article highlights various C. jejuni pathogenicity factors, host cell determinants and proposed signaling mechanisms involved in human host cell invasion and their potential role in the development of C. jejuni-mediated disease. A model is presented which outlines the various important interactions of C. jejuni with the intestinal epithelium, and we discuss the pro’s and con’s for the zipper over the trigger mechanism of invasion. Future work should clarify the contradictory role of some previously identified factors, and should identify and characterize novel virulence determinants, which are crucial to provide fresh insights into the diversity of strategies employed by this pathogen to cause disease.

  15. Recognizing the SINEs of Infection: Regulation of Retrotransposon Expression and Modulation of Host Cell Processes

    Directory of Open Access Journals (Sweden)

    William Dunker

    2017-12-01

    Full Text Available Short interspersed elements (SINEs are a family of retrotransposons evolutionarily derived from cellular RNA polymerase III transcripts. Over evolutionary time, SINEs have expanded throughout the human genome and today comprise ~11% of total chromosomal DNA. While generally transcriptionally silent in healthy somatic cells, SINE expression increases during a variety of types of stresses, including DNA virus infection. The relevance of SINE expression to viral infection was largely unexplored, however, recent years have seen great progress towards defining the impact of SINE expression on viral replication and host gene expression. Here we review the origin and diversity of SINE elements and their transcriptional control, with an emphasis on how their expression impacts host cell biology during viral infection.

  16. Staphylococcus aureus ?-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

    OpenAIRE

    Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

    2013-01-01

    Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol...

  17. The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B.

    Directory of Open Access Journals (Sweden)

    Jillian C Carmichael

    2018-05-01

    Full Text Available All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1, direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor, we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B, and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4 blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease.

  18. The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B.

    Science.gov (United States)

    Carmichael, Jillian C; Yokota, Hiroki; Craven, Rebecca C; Schmitt, Anthony; Wills, John W

    2018-05-01

    All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease.

  19. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  20. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

    Directory of Open Access Journals (Sweden)

    Bernard Thay

    Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

  1. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    International Nuclear Information System (INIS)

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

    2014-01-01

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

  2. The Role of B Cell Targeting in Chronic Graft-Versus-Host Disease

    Directory of Open Access Journals (Sweden)

    Ruben Rhoades

    2017-10-01

    Full Text Available Chronic graft-versus-host disease (cGVHD is a leading cause of late morbidity and mortality following allogeneic stem cell transplantation. Current therapies, including corticosteroids and calcineurin inhibitors, are only effective in roughly 50% of cases; therefore, new treatment strategies are under investigation. What was previously felt to be a T cell disease has more recently been shown to involve activation of both T and B cells, as well as a number of cytokines. With a better understanding of its pathophysiology have come more expansive preclinical and clinical trials, many focused on B cell signaling. This report briefly reviews our current understanding of cGVHD pathophysiology and reviews clinical and preclinical trials with B cell-targeted agents.

  3. γδ T cells in homeostasis and host defence of epithelial barrier tissues.

    Science.gov (United States)

    Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

    2017-12-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

  4. Aspirin suppresses the abnormal lipid metabolism in liver cancer cells via disrupting an NFκB-ACSL1 signaling.

    Science.gov (United States)

    Yang, Guang; Wang, Yuan; Feng, Jinyan; Liu, Yunxia; Wang, Tianjiao; Zhao, Man; Ye, Lihong; Zhang, Xiaodong

    2017-05-06

    Abnormal lipid metabolism is a hallmark of tumorigenesis. Hence, the alterations of metabolism enhance the development of hepatocellular carcinoma (HCC). Aspirin is able to inhibit the growth of cancers through targeting nuclear factor κB (NF-κB). However, the role of aspirin in disrupting abnormal lipid metabolism in HCC remains poorly understood. In this study, we report that aspirin can suppress the abnormal lipid metabolism of HCC cells through inhibiting acyl-CoA synthetase long-chain family member 1 (ACSL1), a lipid metabolism-related enzyme. Interestingly, oil red O staining showed that aspirin suppressed lipogenesis in HepG2 cells and Huh7 cells in a dose-dependent manner. In addition, aspirin attenuated the levels of triglyceride and cholesterol in the cells, respectively. Strikingly, we identified that aspirin was able to down-regulate ACSL1 at the levels of mRNA and protein. Moreover, we validated that aspirin decreased the nuclear levels of NF-κB in HepG2 cells. Mechanically, PDTC, an inhibitor of NF-κB, could down-regulate ACSL1 at the levels of mRNA and protein in the cells. Functionally, PDTC reduced the levels of lipid droplets, triglyceride and cholesterol in HepG2 cells. Thus, we conclude that aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling. Our finding provides new insights into the mechanism by which aspirin inhibits abnormal lipid metabolism of HCC. Therapeutically, aspirin is potentially available for HCC through controlling abnormal lipid metabolism. Copyright © 2017. Published by Elsevier Inc.

  5. Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress

    Directory of Open Access Journals (Sweden)

    Paul-Christian Burda

    2017-04-01

    Full Text Available A crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2 reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

  6. Phylogeographic origin of Helicobacter pylori determines host-adaptive responses upon coculture with gastric epithelial cells.

    Science.gov (United States)

    Sheh, Alexander; Chaturvedi, Rupesh; Merrell, D Scott; Correa, Pelayo; Wilson, Keith T; Fox, James G

    2013-07-01

    While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA, vacA, and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.

  7. Complexities in human herpesvirus-6A and -6B binding to host cells

    International Nuclear Information System (INIS)

    Pedersen, Simon Metz; Hoellsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher affinity than others for the virus. Within recent years, elucidation of the viral complex has identified additional HHV-6A and -6B specific glycoproteins. Thus, gH-gL associates with a gQ1-gQ2 dimer to form a heterotetrameric complex. In addition, a novel complex consisting of gH-gL-gO has been described that does not bind CD46. Accumulating evidence suggests that an additional HHV-6A and -6B receptor exists. The previous simple picture of HHV-6A/B-host cell contact therefore includes more layers of complexities on both the viral and the host cell side of the interaction

  8. Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells

    International Nuclear Information System (INIS)

    Tujulin, E.; Macellaro, A.; Norlander, L.; Liliehoeoek, B.

    1998-01-01

    The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intra-vacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as weal as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell. Key words: Coxiella burnetii; internalisation; endocytosis (authors)

  9. Genome-wide host responses against infectious laryngotracheitis virus vaccine infection in chicken embryo lung cells

    Directory of Open Access Journals (Sweden)

    Lee Jeongyoon

    2012-04-01

    Full Text Available Abstract Background Infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1 infection causes high mortality and huge economic losses in the poultry industry. To protect chickens against ILTV infection, chicken-embryo origin (CEO and tissue-culture origin (TCO vaccines have been used. However, the transmission of vaccine ILTV from vaccinated- to unvaccinated chickens can cause severe respiratory disease. Previously, host cell responses against virulent ILTV infections were determined by microarray analysis. In this study, a microarray analysis was performed to understand host-vaccine ILTV interactions at the host gene transcription level. Results The 44 K chicken oligo microarrays were used, and the results were compared to those found in virulent ILTV infection. Total RNAs extracted from vaccine ILTV infected chicken embryo lung cells at 1, 2, 3 and 4 days post infection (dpi, compared to 0 dpi, were subjected to microarray assay using the two color hybridization method. Data analysis using JMP Genomics 5.0 and the Ingenuity Pathway Analysis (IPA program showed that 213 differentially expressed genes could be grouped into a number of functional categories including tissue development, cellular growth and proliferation, cellular movement, and inflammatory responses. Moreover, 10 possible gene networks were created by the IPA program to show intermolecular connections. Interestingly, of 213 differentially expressed genes, BMP2, C8orf79, F10, and NPY were expressed distinctly in vaccine ILTV infection when compared to virulent ILTV infection. Conclusions Comprehensive knowledge of gene expression and biological functionalities of host factors during vaccine ILTV infection can provide insight into host cellular defense mechanisms compared to those of virulent ILTV.

  10. Combined Enzymatic and Mechanical Cell Disruption and Lipid Extraction of Green Alga Neochloris oleoabundans

    Science.gov (United States)

    Wang, Dongqin; Li, Yanqun; Hu, Xueqiong; Su, Weimin; Zhong, Min

    2015-01-01

    Microalgal biodiesel is one of the most promising renewable fuels. The wet technique for lipids extraction has advantages over the dry method, such as energy-saving and shorter procedure. The cell disruption is a key factor in wet oil extraction to facilitate the intracellular oil release. Ultrasonication, high-pressure homogenization, enzymatic hydrolysis and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication were employed in this study to disrupt the cells of the microalga Neochloris oleoabundans. The cell disruption degree was investigated. The cell morphology before and after disruption was assessed with scanning and transmission electron microscopy. The energy requirements and the operation cost for wet cell disruption were also estimated. The highest disruption degree, up to 95.41%, assessed by accounting method was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization. A lipid recovery of 92.6% was also obtained by the combined process. The combined process was found to be more efficient and economical compared with the individual process. PMID:25853267

  11. Combined Enzymatic and Mechanical Cell Disruption and Lipid Extraction of Green Alga Neochloris oleoabundans

    Directory of Open Access Journals (Sweden)

    Dongqin Wang

    2015-04-01

    Full Text Available Microalgal biodiesel is one of the most promising renewable fuels. The wet technique for lipids extraction has advantages over the dry method, such as energy-saving and shorter procedure. The cell disruption is a key factor in wet oil extraction to facilitate the intracellular oil release. Ultrasonication, high-pressure homogenization, enzymatic hydrolysis and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication were employed in this study to disrupt the cells of the microalga Neochloris oleoabundans. The cell disruption degree was investigated. The cell morphology before and after disruption was assessed with scanning and transmission electron microscopy. The energy requirements and the operation cost for wet cell disruption were also estimated. The highest disruption degree, up to 95.41%, assessed by accounting method was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization. A lipid recovery of 92.6% was also obtained by the combined process. The combined process was found to be more efficient and economical compared with the individual process.

  12. Critical composition fluctuations in artificial and cell-derived lipid membranes

    Science.gov (United States)

    Honerkamp-Smith, Aurelia

    2014-03-01

    Cell plasma membranes contain a mixture of lipid types which can segregate into coexisting liquids, a thermodynamic phenomenon which may contribute to biological functions. Simplified, artificial three-component lipid vesicles can be prepared which display a critical miscibility transition near room temperature. We found that such vesicles exhibit concentration fluctuations whose size, composition, and timescales vary consistently with critical exponents for two-dimensional conserved order parameter systems. However, the critical miscibility transition is also observed in vesicles formed directly from the membranes of living cells, despite their more complex composition and the presence of membrane proteins. I will describe our critical fluctuation measurements and also review a variety of more recent work by other researchers. Proximity to a critical point alters the spatial distribution and aggregation tendencies of proteins, and makes lipid mixtures more susceptible to domain formation by protein-mediated interactions, such as adhesion zones. Recent work suggests that critical temperature depression may also be relevant to the mechanism of anaesthetic action.

  13. Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells.

    Science.gov (United States)

    Falke, Joseph J; Ziemba, Brian P

    2014-09-01

    The chemotactic migration of eukaryotic ameboid cells up concentration gradients is among the most advanced forms of cellular behavior. Chemotaxis is controlled by a complex network of signaling proteins bound to specific lipids on the cytoplasmic surface of the plasma membrane at the front of the cell, or the leading edge. The central lipid players in this leading edge signaling pathway include the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), both of which play multiple roles. The products of PI(4,5)P2 hydrolysis, diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3), are also implicated as important players. Together, these leading edge phosphoinositides and their degradation products, in concert with a local Ca(2+) signal, control the recruitment and activities of many peripheral membrane proteins that are crucial to the leading edge signaling network. The present critical review summarizes the current molecular understanding of chemotactic signaling at the leading edge, including newly discovered roles of phosphoinositide lipids and Ca(2+), while highlighting key questions for future research. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

    Science.gov (United States)

    Riestra, Angelica M.; Gandhi, Shiv; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J.

    2015-01-01

    Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis. PMID:26684303

  15. 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.

  16. Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

    Science.gov (United States)

    Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2018-06-27

    The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

  17. An in silico pipeline to filter the Toxoplasma gondii proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation.

    Science.gov (United States)

    Syn, Genevieve; Blackwell, Jenefer M; Jamieson, Sarra E; Francis, Richard W

    2018-01-01

    Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.

  18. Single Cell Synchrotron FT-IR Microspectroscopy Reveals a Link between Neutral Lipid and Storage Carbohydrate Fluxes in S. cerevisiae

    Science.gov (United States)

    Jamme, Frédéric; Vindigni, Jean-David; Méchin, Valérie; Cherifi, Tamazight; Chardot, Thierry; Froissard, Marine

    2013-01-01

    In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated. PMID:24040242

  19. Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Frédéric Jamme

    Full Text Available In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins. We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated.

  20. Antioxidant Potential and Modulatory Effects of Restructured Lipids from the Amazonian Palms on Liver Cells

    Directory of Open Access Journals (Sweden)

    Andrea de Oliveira Falcão

    2017-01-01

    Full Text Available Enzymatic interesterification is used to manipulate oil and fat in order to obtain improved restructured lipids with desired technological properties. However, with raw materials containing significant amounts of bioactive compounds, the influence of this enzymatic process on the bioactivity of the final product is still not clear. Thus, the aim of this study is to evaluate the antioxidant potential and modulatory effects of two raw materials from the Amazonian area, buriti oil and murumuru fat, before and after lipase interesterification, on human hepatoma cells (HepG2. The results indicate that minor bioactive compounds naturally found in the raw materials and their antioxidant capacity are preserved after enzymatic interesterification, and that the restructured lipids modulate HepG2 endogenous antioxidant enzyme.

  1. Lipid Droplets: A New Player in Colorectal Cancer Stem Cells Unveiled by Spectroscopic Imaging

    KAUST Repository

    Tirinato, Luca; Liberale, Carlo; Di Franco, Simone; Candeloro, Patrizio; Benfante, Antonina; La Rocca, Rosanna; Potze, Lisette; Marotta, Roberto; Ruffilli, Roberta; Rajamanickam, Vijayakumar P.; Malerba, Mario; De Angelis, Francesco; Falqui, Andrea; Carbone, Ennio; Todaro, Matilde; Medema, Jan Paul; Stassi, Giorgio; Di Fabrizio, Enzo M.

    2015-01-01

    The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

  2. Sialoglycoconjugates in Trypanosoma cruzi-host cell interaction: possible biological model - a review

    Directory of Open Access Journals (Sweden)

    Alane Beatriz Vermelho

    1994-03-01

    Full Text Available A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.

  3. Mesenchymal Stromal Cells: What Is the Mechanism in Acute Graft-Versus-Host Disease?

    Directory of Open Access Journals (Sweden)

    Neil Dunavin

    2017-07-01

    Full Text Available After more than a decade of preclinical and clinical development, therapeutic infusion of mesenchymal stromal cells is now a leading investigational strategy for the treatment of acute graft-versus-host disease (GVHD. While their clinical use continues to expand, it is still unknown which of their immunomodulatory properties contributes most to their therapeutic activity. Herein we describe the proposed mechanisms, focusing on the inhibitory activity of mesenchymal stromal cells (MSCs at immunologic checkpoints. A deeper understanding of the mechanism of action will allow us to design more effective treatment strategies.

  4. Enhanced lipid production with undetoxified corncob hydrolysate by Rhodotorula glutinis using a high cell density culture strategy.

    Science.gov (United States)

    Liu, Yating; Wang, Yanping; Liu, Hongjuan; Zhang, Jian'an

    2015-03-01

    In recent years, energy crisis and environmental issues such as greenhouse effect, global warming, etc. has roused peoples' concern. Biodiesel, as renewable energy, has attracted much attention to deal with such problems. This work studied the lipid production by Rhodotorula glutinis with undetoxified corncob hydrolysate. The results indicated that R. glutinis had high tolerance to the inhibitors in corncob hydrolysate and it could utilize undetoxified corncob hydrolysate directly for lipid production. The cell grew well with undetoxified hydrolysate in the batch culture of 5L fermentor with the optimized C/N ratio of 75, lipid titer and lipid content reached 5.5g/L and 36.4%, respectively. High cell density culture with two-stage nitrogen feeding strategy was studied to enhance the lipid production, biomass, lipid concentration and lipid content of 70.8, 33.5g/L and 47.2% were obtained. The results indicated the potential application for lipid production by R. glutinis with corncob hydrolysate directly. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Positive regulation of prostate cancer cell growth by lipid droplet forming and processing enzymes DGAT1 and ABHD5.

    Science.gov (United States)

    Mitra, Ranjana; Le, Thuc T; Gorjala, Priyatham; Goodman, Oscar B

    2017-09-06

    Neoplastic cells proliferate rapidly and obtain requisite building blocks by reprogramming metabolic pathways that favor growth. Previously, we observed that prostate cancer cells uptake and store lipids in the form of lipid droplets, providing building blocks for membrane synthesis, to facilitate proliferation and growth. Mechanisms of lipid uptake, lipid droplet dynamics and their contribution to cancer growth have yet to be defined. This work is focused on elucidating the prostate cancer-specific modifications in lipid storage pathways so that these modified gene products can be identified and therapeutically targeted. To identify genes that promote lipid droplet formation and storage, the expression profiles of candidate genes were assessed and compared between peripheral blood mononuclear cells and prostate cancer cells. Subsequently, differentially expressed genes were inhibited and growth assays performed to elucidate their role in the growth of the cancer cells. Cell cycle, apoptosis and autophagy assays were performed to ascertain the mechanism of growth inhibition. Our results indicate that DGAT1, ABHD5, ACAT1 and ATGL are overexpressed in prostate cancer cells compared to PBMCs and of these overexpressed genes, DGAT1 and ABHD5 aid in the growth of the prostate cancer cells. Blocking the expression of both DGAT1 and ABHD5 results in inhibition of growth, cell cycle block and cell death. DGAT1 siRNA treatment inhibits lipid droplet formation and leads to autophagy where as ABHD5 siRNA treatment promotes accumulation of lipid droplets and leads to apoptosis. Both the siRNA treatments reduce AMPK phosphorylation, a key regulator of lipid metabolism. While DGAT1 siRNA reduces phosphorylation of ACC, the rate limiting enzyme in de novo fat synthesis and triggers phosphorylation of raptor and ULK-1 inducing autophagy and cell death, ABHD5 siRNA decreases P70S6 phosphorylation, leading to PARP cleavage, apoptosis and cell death. Interestingly, DGAT-1 is involved

  6. Impact of host cell variation on the neutralization of HIV-1 in vitro.

    Science.gov (United States)

    Polonis, Victoria R; Schuitemaker, Hanneke; Bunnik, Evelien M; Brown, Bruce K; Scarlatti, Gabriella

    2009-09-01

    In this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. The data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. HIV-vaccine-induced antibody performance in the current neutralization assays may function as a 'gatekeeper' for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

  7. Lipid production in association of filamentous fungi with genetically modified cyanobacterial cells.

    Science.gov (United States)

    Miranda, Ana F; Taha, Mohamed; Wrede, Digby; Morrison, Paul; Ball, Andrew S; Stevenson, Trevor; Mouradov, Aidyn

    2015-01-01

    Numerous strategies have evolved recently for the generation of genetically modified or synthetic microalgae and cyanobacteria designed for production of ethanol, biodiesel and other fuels. In spite of their obvious attractiveness there are still a number of challenges that can affect their economic viability: the high costs associated with (1) harvesting, which can account for up to 50 % of the total biofuel's cost, (2) nutrients supply and (3) oil extraction. Fungal-assisted bio-flocculation of microalgae is gaining increasing attention due to its high efficiency, no need for added chemicals and low energy inputs. The implementation of renewable alternative carbon, nitrogen and phosphorus sources from agricultural wastes and wastewaters for growing algae and fungi makes this strategy economically attractive. This work demonstrates that the filamentous fungi, Aspergillus fumigatus can efficiently flocculate the unicellular cyanobacteria Synechocystis PCC 6803 and its genetically modified derivatives that have been altered to enable secretion of free fatty acids into growth media. Secreted free fatty acids are potentially used by fungal cells as a carbon source for growth and ex-novo production of lipids. For most of genetically modified strains the total lipid yields extracted from the fungal-cyanobacterial pellets were found to be higher than additive yields of lipids and total free fatty acids produced by fungal and Synechocystis components when grown in mono-cultures. The synergistic effect observed in fungal-Synechocystis associations was also found in bioremediation rates when animal husbandry wastewater was used an alternative source of nitrogen and phosphorus. Fungal assisted flocculation can complement and assist in large scale biofuel production from wild-type and genetically modified Synechocystis PCC 6803 strains by (1) efficient harvesting of cyanobacterial cells and (2) producing of high yields of lipids accumulated in fungal-cyanobacterial pellets.

  8. Lipid droplets, perilipins and cytokeratins--unravelled liaisons in epithelium-derived cells.

    Directory of Open Access Journals (Sweden)

    Hans Heid

    Full Text Available Lipid droplets (LDs are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs. These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5. Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron- in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF cytokeratins 8 and 18 (also designated as keratins K8 and K18. Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.

  9. PEG-detachable lipid-polymer hybrid nanoparticle for delivery of chemotherapy drugs to cancer cells.

    Science.gov (United States)

    Du, Jiang-bo; Song, Yan-feng; Ye, Wei-liang; Cheng, Ying; Cui, Han; Liu, Dao-zhou; Liu, Miao; Zhang, Bang-le; Zhou, Si-yuan

    2014-08-01

    The experiment aimed to increase the drug-delivery efficiency of poly-lactic-co-glycolic acid (PLGA) nanoparticles. Lipid-polymer hybrid nanoparticles (LPNs-1) were prepared using PLGA as a hydrophobic core and FA-PEG-hyd-DSPE as an amphiphilic shell. Uniform and spherical nanoparticles with an average size of 185 nm were obtained using the emulsification solvent evaporation method. The results indicated that LPNs-1 showed higher drug loading compared with naked PLGA nanoparticles (NNPs). Drug release from LPNs-1 was faster in an acidic environment than in a neutral environment. LPNs-1 showed higher cytotoxicity on KB cells, A549 cells, MDA-MB-231 cells, and MDA-MB-231/ADR cells compared with free doxorubicin (DOX) and NNPs. The results also showed that, compared with free DOX and NNPs, LPNs-1 delivered more DOX to the nuclear of KB cells and MDA-MB-231/ADR cells. LPNs-1 induced apoptosis in KB cells and MDA-MB-231/ADR cells in a dose-dependent manner. The above data indicated that DOX-loaded LPNs-1 could kill not only normal tumor cells but also drug-resistant tumor cells. These results indicated that modification of PLGA nanoparticles with FA-PEG-hyd-DSPE could considerably increase the drug-delivery efficiency and LPNs-1 had potential in the delivery of chemotherapeutic agents in the treatment of cancer.

  10. Influence of in vitro supplementation with lipids from conventional and Alpine milk on fatty acid distribution and cell growth of HT-29 cells

    Directory of Open Access Journals (Sweden)

    Dänicke Sven

    2011-08-01

    Full Text Available Abstract Background To date, the influence of milk and dairy products on carcinogenesis remains controversial. However, lipids of ruminant origin such as conjugated linoleic acids (CLA are known to exhibit beneficial effects in vitro and in vivo. The aim of the present study was to determine the influence of milk lipids of different origin and varying quality presenting as free fatty acid (FFA solutions on cellular fatty acid distribution, cellular viability, and growth of human colon adenocarcinoma cells (HT-29. Methods FAME of conventional and Alpine milk lipids (MLcon, MLalp and cells treated with FFA derivatives of milk lipids were analyzed by means of GC-FID and Ag+-HPLC. Cellular viability and growth of the cells were determined by means of CellTiter-Blue®-assay and DAPI-assay (4',6-diamidino-2-phenylindole dihydrochloride, respectively. Results Supplementation with milk lipids significantly decreased viability and growth of HT-29 cells in a dose- and time-dependent manner. MLalp showed a lower SFA/MUFA ratio, a 8 fold increased CLA content, and different CLA profile compared to MLcon but did not demonstrate additional growth-inhibitory effects. In addition, total concentration and fatty acid distribution of cellular lipids were altered. In particular, treatment of the cells yielded highest amounts of two types of milk specific major fatty acids (μg FA/mg cellular protein after 8 h of incubation compared to 24 h; 200 μM of MLcon (C16:0, 206 ± 43, 200 μM of MLalp (C18:1 c9, (223 ± 19. Vaccenic acid (C18:1 t11 contained in milk lipids was converted to c9,t11-CLA in HT-29 cells. Notably, the ratio of t11,c13-CLA/t7,c9-CLA, a criterion for pasture feeding of the cows, was significantly changed after incubation for 8 h with lipids from MLalp (3.6 - 4.8, compared to lipids from MLcon (0.3 - 0.6. Conclusions Natural lipids from conventional and Alpine milk showed similar growth inhibitory effects. However, different changes in cellular

  11. Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice

    International Nuclear Information System (INIS)

    Nueesch, Juerg P.F.; Lachmann, Sylvie; Rommelaere, Jean

    2005-01-01

    During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of α/β tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott-Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production

  12. Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale.

    Science.gov (United States)

    Striberny, Bernd; Krause, Kirsten

    2015-01-01

    The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas.

  13. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

    Directory of Open Access Journals (Sweden)

    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  14. How the growth rate of host cells affects cancer risk in a deterministic way

    Science.gov (United States)

    Draghi, Clément; Viger, Louise; Denis, Fabrice; Letellier, Christophe

    2017-09-01

    It is well known that cancers are significantly more often encountered in some tissues than in other ones. In this paper, by using a deterministic model describing the interactions between host, effector immune and tumor cells at the tissue level, we show that this can be explained by the dependency of tumor growth on parameter values characterizing the type as well as the state of the tissue considered due to the "way of life" (environmental factors, food consumption, drinking or smoking habits, etc.). Our approach is purely deterministic and, consequently, the strong correlation (r = 0.99) between the number of detectable growing tumors and the growth rate of cells from the nesting tissue can be explained without evoking random mutation arising during DNA replications in nonmalignant cells or "bad luck". Strategies to limit the mortality induced by cancer could therefore be well based on improving the way of life, that is, by better preserving the tissue where mutant cells randomly arise.

  15. Study on the relationship between red blood cell immunity and lipid peroxidation in patients with endometriosis

    International Nuclear Information System (INIS)

    Yang Jingxiu; Shi Shaohong; Wang Yuping; Xie Xueqin; Qin Jibao

    2005-01-01

    Objective: To assess the relationship between red blood cell immunity and lipid peroxidation (LPO) in patients with endometriosis. Methods: The percentage of positive red blood cell c3b receptor rosette (RBC c3b -RR) and red blood cell immune complex rosette (RBC-ICR) were examined in 54 patients with endometriosis and 30 controls. Serum levels of malondialdehyde (MDA), superoxidase (SOD) and glutathione peroxidase (GSH-PX) were measured by chemocolorimetry in these subjects. Results: Percentage of positive RBC-ICR and MDA levels were significantly higher in patients with endometriosis than those in controls (P c3b RR, SOD, GSH-PX, SOD/MDA ratio were significantly lower in patients with endometriosis than those in controls (P c3b -RR was negatively correlated with MDA levels (r= -0. 4428, P < 0.05) and RBC-ICRR was positively correlated with MDA(r=0.5488, P0.05). Conclusion: The lower red cell immune adhesion function was closely associated with the disturbance of metabolism of lipid peroxidation in patients with endometriosis. (authors)

  16. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

    Science.gov (United States)

    Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-10-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  17. Lipidomic data on lipid droplet triglyceride remodelling associated with protection of breast cancer cells from lipotoxic stress.

    Science.gov (United States)

    Jarc, Eva; Eichmann, Thomas O; Zimmermann, Robert; Petan, Toni

    2018-06-01

    The data presented here is related to the research article entitled "Lipid droplets induced by secreted phospholipase A 2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress" by E. Jarc et al., Biochim. Biophys. Acta 1863 (2018) 247-265. Elevated uptake of unsaturated fatty acids and lipid droplet accumulation are characteristic of aggressive cancer cells and have been associated with the cellular stress response. The present study provides lipidomic data on the triacylglycerol (TAG) and phosphatidylcholine (PC) composition of MDA-MB-231 breast cancer cells exposed to docosahexaenoic acid (DHA; 22:6, ω-3). Datasets provide information on the changes in lipid composition induced by depletion of adipose triglyceride lipase (ATGL) and by exogenous addition of secreted phospholipase A 2 (sPLA 2 ) in DHA-treated cells. The presented alterations in lipid composition, mediated by targeting lipid droplet biogenesis and lipolysis, are associated with protection from lipotoxicity and allow further investigation into the role of lipid droplets in the resistance of cancer cells to lipotoxic stress.

  18. TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    De Miguel, Diego; Gallego-Lleyda, Ana; Erviti-Ardanaz, Sandra; Anel, Alberto; Martinez-Lostao, Luis; Ayuso, José María; Fernández, Luis José; Ochoa, Ignacio; Pazo-Cid, Roberto; Del Agua, Celia

    2016-01-01

    Purpose. Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. Methods/patients. LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. Results. LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. Conclusion. The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment. (paper)

  19. Oridonin Loaded Solid Lipid Nanoparticles Enhanced Antitumor Activity in MCF-7 Cells

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-01-01

    Full Text Available Oridonin (ORI, a famous diterpenoid from Chinese herbal medicine, has drawn rising attention for its remarkable apoptosis and autophagy-inducing activity in human cancer therapy, while clinical application of ORI is limited by its strong hydrophobicity and rapid plasma clearance. The purpose of this study was to evaluate whether the antitumor activity of ORI could be enhanced by loading into solid lipid nanoparticles (SLNs. ORI-loaded SLNs were prepared by hot high pressure homogenization with narrow size distribution and good entrapment efficacy. MTT assay indicated that ORI-loaded SLNs enhanced the inhibition of proliferation against several human cancer cell lines including breast cancer MCF-7 cells, hepatocellular carcinoma HepG 2 cells, and lung carcinoma A549 cells compared with free ORI, while no significant enhancement of toxicity to human mammary epithelial MCF-10A cells was shown. Meanwhile, flow cytometric analysis demonstrated that ORI-SLNs induced more significant cell cycle arrest at S and decreased cell cycle arrest at G1/G0 phase in MCF-7 cells than bulk ORI solution. Hoechst 33342 staining and Annexin V/PI assay indicated that apoptotic rates of cells treated with ORI-loaded SLNs were higher compared with free ORI. In summary, our data indicated that SLNs may be a potential carrier for enhancing the antitumor effect of hydrophobic drug ORI.

  20. Early host cell targets of Yersinia pestis during primary pneumonic plague.

    Directory of Open Access Journals (Sweden)

    Roger D Pechous

    Full Text Available Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase.

  1. Towards identifying host cell-type specific response patterns to bacterial endosymbiosis

    DEFF Research Database (Denmark)

    Gavrilovic, Srdjan

    The establishment of Symbiotic Nitrogen Fixation (SNF) is a complex process. It requires highly sophisticated signal exchanges between host plant and bacteria in order to fine-tune the molecular mechanisms necessary for optimal performance of the symbiosis, which ultimately determines the evoluti......The establishment of Symbiotic Nitrogen Fixation (SNF) is a complex process. It requires highly sophisticated signal exchanges between host plant and bacteria in order to fine-tune the molecular mechanisms necessary for optimal performance of the symbiosis, which ultimately determines......, and whole plant transformants were regenerated. These will form a basis for isolating transcriptionally active mRNA fractions associated with ribosomes and 21 nt long small RNAs from targeted cell populations....

  2. Lipid accumulation in human breast cancer cells injured by iron depletors.

    Science.gov (United States)

    De Bortoli, Maida; Taverna, Elena; Maffioli, Elisa; Casalini, Patrizia; Crisafi, Francesco; Kumar, Vikas; Caccia, Claudio; Polli, Dario; Tedeschi, Gabriella; Bongarzone, Italia

    2018-04-03

    Current insights into the effects of iron deficiency in tumour cells are not commensurate with the importance of iron in cell metabolism. Studies have predominantly focused on the effects of oxygen or glucose scarcity in tumour cells, while attributing insufficient emphasis to the inadequate supply of iron in hypoxic regions. Cellular responses to iron deficiency and hypoxia are interlinked and may strongly affect tumour metabolism. We examined the morphological, proteomic, and metabolic effects induced by two iron chelators-deferoxamine (DFO) and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)-on MDA-MB-231 and MDA-MB-157 breast cancer cells. These chelators induced a cytoplasmic massive vacuolation and accumulation of lipid droplets (LDs), eventually followed by implosive, non-autophagic, and non-apoptotic death similar to methuosis. Vacuoles and LDs are generated by expansion of the endoplasmic reticulum (ER) based on extracellular fluid import, which includes unsaturated fatty acids that accumulate in LDs. Typical physiological phenomena associated with hypoxia are observed, such as inhibition of translation, mitochondrial dysfunction, and metabolic remodelling. These survival-oriented changes are associated with a greater expression of epithelial/mesenchymal transcription markers. Iron starvation induces a hypoxia-like program able to scavenge nutrients from the extracellular environment, and cells assume a hypertrophic phenotype. Such survival strategy is accompanied by the ER-dependent massive cytoplasmic vacuolization, mitochondrial dysfunctions, and LD accumulation and then evolves into cell death. LDs containing a greater proportion of unsaturated lipids are released as a consequence of cell death. The consequence of the disruption of iron metabolism in tumour tissue and the effects of LDs on intercellular communication, cancer-inflammation axis, and immunity remain to be explored. Considering the potential benefits, these are crucial

  3. Aspirin suppresses the abnormal lipid metabolism in liver cancer cells via disrupting an NFκB-ACSL1 signaling

    International Nuclear Information System (INIS)

    Yang, Guang; Wang, Yuan; Feng, Jinyan; Liu, Yunxia; Wang, Tianjiao; Zhao, Man; Ye, Lihong; Zhang, Xiaodong

    2017-01-01

    Abnormal lipid metabolism is a hallmark of tumorigenesis. Hence, the alterations of metabolism enhance the development of hepatocellular carcinoma (HCC). Aspirin is able to inhibit the growth of cancers through targeting nuclear factor κB (NF-κB). However, the role of aspirin in disrupting abnormal lipid metabolism in HCC remains poorly understood. In this study, we report that aspirin can suppress the abnormal lipid metabolism of HCC cells through inhibiting acyl-CoA synthetase long-chain family member 1 (ACSL1), a lipid metabolism-related enzyme. Interestingly, oil red O staining showed that aspirin suppressed lipogenesis in HepG2 cells and Huh7 cells in a dose-dependent manner. In addition, aspirin attenuated the levels of triglyceride and cholesterol in the cells, respectively. Strikingly, we identified that aspirin was able to down-regulate ACSL1 at the levels of mRNA and protein. Moreover, we validated that aspirin decreased the nuclear levels of NF-κB in HepG2 cells. Mechanically, PDTC, an inhibitor of NF-κB, could down-regulate ACSL1 at the levels of mRNA and protein in the cells. Functionally, PDTC reduced the levels of lipid droplets, triglyceride and cholesterol in HepG2 cells. Thus, we conclude that aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling. Our finding provides new insights into the mechanism by which aspirin inhibits abnormal lipid metabolism of HCC. Therapeutically, aspirin is potentially available for HCC through controlling abnormal lipid metabolism. - Highlights: • Aspirin inhibits the levels of liquid droplets, triglyceride and cholesterol in HCC cells. • Aspirin is able to down-regulate ACSL1 in HCC cells. • NF-κB inhibitor PDTC can down-regulate ACSL1 and reduces lipogenesis in HCC cells. • Aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling.

  4. On the lack of host-cell reactivation of UV-irradiated phage T5

    International Nuclear Information System (INIS)

    Chiang, T.; Harm, W.

    1976-01-01

    Previously reported experiments have shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5. The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host-cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the 'second-step-transfer' region of T5 DNA (involving the remainder of the genome). Superinfection with T4v 1 phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner

  5. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  6. Dendritic cell chimerism in oral mucosa of transplanted patients affected by graft-versus-host disease.

    Science.gov (United States)

    Pérez, Claudio A; Rabanales, Ramón; Rojas-Alcayaga, Gonzalo; Larrondo, Milton; Escobar, Alejandro F; López, Mercedes N; Salazar-Onfray, Flavio; Alfaro, Jorge I; González, Fermín E

    2016-02-01

    Graft-versus-host disease (GVHD) is one of the main complications after haematopoietic stem cell transplantation. Clinical features of GVHD include either an acute (aGVHD) or a chronic (cGVHD) condition that affects locations such as the oral mucosa. While the involvement of the host's dendritic cells (DCs) has been demonstrated in aGVHD, the origin (donor/host) and mechanisms underlying oral cGVHD have not been completely elucidated. In this study, we intend to determine the origin of DCs present in mucosal tissue biopsies from the oral cavity of transplanted patients affected by cGVHD. We purified DCs, from oral biopsies of three patients with cGVHD, through immunobeads and subsequently performed DNA extraction. The origin of the obtained DCs was determined by PCR amplification of 13 informative short tandem repeat (STR) alleles. We also characterised the DCs phenotype and the inflammatory infiltrate from biopsies of two patients by immunohistochemistry. Clinical and histological features of the biopsies were concordant with oral cGVHD. We identified CD11c-, CD207- and CD1a-positive cells in the epithelium and beneath the basal layer. Purification of DCs from the mucosa of patients affected by post-transplantation cGVHD was >95%. PCR-STR data analysis of DCs DNA showed that 100% of analysed cells were of donor origin in all of the evaluated patients. Our results demonstrate that resident DCs isolated from the oral tissue of allotransplanted patients affected by cGVHD are originated from the donor. Further research will clarify the role of DCs in the development and/or severity of oral cGVHD. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Moringa oleifera Lam. improves lipid metabolism during adipogenic differentiation of human stem cells.

    Science.gov (United States)

    Barbagallo, I; Vanella, L; Distefano, A; Nicolosi, D; Maravigna, A; Lazzarino, G; Di Rosa, M; Tibullo, D; Acquaviva, R; Li Volti, G

    2016-12-01

    Moringa oleifera Lam., a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of conditions, including inflammation, cancer and metabolic disorders. We investigated the effect of Moringa oleifera Lam. on adipogenic differentiation of human adipose-derived mesenchymal stem cells and its impact on lipid metabolism and cellular antioxidant systems. We showed that Moringa oleifera Lam. treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor alpha (PPARα), and coactivator 1 alpha (PGC1α). In addition, Moringa oleifera Lam. induces heme oxygenase-1 (HO-1), a well established protective and antioxidant enzyme. Finally Moringa oleifera Lam. significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. Our results suggest that Moringa oleifera Lam. may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis.

  8. Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

    Science.gov (United States)

    Ho, James C S; Storm, Petter; Rydström, Anna; Bowen, Ben; Alsin, Fredrik; Sullivan, Louise; Ambite, Inès; Mok, K H; Northen, Trent; Svanborg, Catharina

    2013-06-14

    Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

  9. Immunohistochemical localization of host and donor-derived cells in the regenerating thymus of radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    Ceredig, R.; Schreyer, M.

    1984-01-01

    The anatomical distribution of CBA (Thy-1.2) host and AKR (Thy-1.1) donor-derived cells in the regenerating thymus of AKR → CBA radiation bone marrow chimeras was investigated. Cryostat sections of chimeric thymuses were incubated with biotin-conjugated monoclonal anti-Thy-1 antibodies specific for host and donor-derived cells and the distribution of the corresponding Thy-1 antigen revealed by the immunoperoxidase staining technique. The thymus was initially repopulated by Thy-1.2 + host-derived cells, but by 28 days following bone marrow reconstitution the few remaining host cells were found mostly in the thymus medulla. However, occasional Thy-1.2 + cells were still present in extramedullary, primarily cortical, sites. Donor-derived (Thy-1.1 + ) cells were first seen in the 11-day chimeric thymus as single cells frequently closely associated with blood vessels in medullary areas. By 17 days, the cortex contained many Thy-1.1 + cells, although occasional single positive cells were still present in the medulla. Changes in the anatomical distribution of host and donor-derived cells in the regenerating chimeric thymus appeared to correlate with changes in their Thy-1 fluorescence profile as determined by flow microfluorometry. (Auth.)

  10. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  11. CD8 T-cells and E-cadherin in host responses against oropharyngeal candidiasis

    Science.gov (United States)

    Quimby, K.; Lilly, E.A.; Zacharek, M.; McNulty, K.; Leigh, J.E.; Vazquez, J.E.; Fidel, P.L.

    2011-01-01

    Oropharyngeal candidiasis (OPC) is the most common oral infection in HIV+ persons. Previous studies suggest a role for CD8+ T-cells against OPC when CD4+ T-cells are lost, but enhanced susceptibility to infection occurs when CD8+ T-cell migration is inhibited by reduced tissue E-cadherin. Objective Conduct a longitudinal study of tissue CD8+ T-cells and E-cadherin expression before, during, and after episodes of OPC. Methods Oral fungal burden was monitored and tissue was evaluated for CD8+ T-cells and E-cadherin over a one-year period in HIV+ persons with a history of, or an acute episode of OPC. Results While longitudinal analyses precluded formal interpretations, point prevalence analyses of the dataset revealed that when patients experiencing OPC were successfully treated, tissue E-cadherin expression was similar to patients who had not experienced OPC, and higher numbers of CD8+ T-cells were distributed throughout OPC− tissue under normal expression of E-cadherin. Conclusion These results suggest that 1) reduction in tissue E-cadherin expression in OPC+ patients is not permanent, and 2) high numbers of CD8+ T-cells can be distributed throughout OPC− tissue under normal E-cadherin expression. Together these results extend our previous studies and continue to support a role for CD8+ T-cells in host defense against OPC. PMID:21958417

  12. Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

    Science.gov (United States)

    Jung, Sook-In; Rodriguez, Natalie; Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G; Park, Hyunsook

    2017-01-01

    The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.

  13. Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis

    International Nuclear Information System (INIS)

    Jones, Steven; Boisvert, Annie; Naghi, Andrada; Hullin-Matsuda, Françoise; Greimel, Peter; Kobayashi, Toshihide; Papadopoulos, Vassilios; Culty, Martine

    2016-01-01

    Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10 μM GEN + 10 μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN + MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10 μM combined GEN + MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN + MEHP. We propose a working model for GEN + MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally

  14. The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways.

    Science.gov (United States)

    De Nisco, Nicole J; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-05-16

    Bacterial effectors potently manipulate host signaling pathways. The marine bacterium Vibrio parahaemolyticus ( V. para ) delivers effectors into host cells through two type 3 secretion systems (T3SSs). T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate nonapoptotic cell death. To understand how the concerted action of T3SS1 effectors globally affects host cell signaling, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1 + ) to those in cells infected with V. para lacking T3SS1 (T3SS1 - ). Overall, the host transcriptional response to both T3SS1 + and T3SS1 - V. para was rapid, robust, and temporally dynamic. T3SS1 rewired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors targeted host cells at the posttranslational level to cause cytotoxicity, V. para T3SS1 also precipitated a host transcriptional response that initially activated cell survival and repressed cell death networks. The increased expression of several key prosurvival transcripts mediated by T3SS1 depended on a host signaling pathway that is silenced posttranslationally later in infection. Together, our analysis reveals a complex interplay between the roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. Copyright © 2017, American Association for the Advancement of Science.

  15. Study on the effect of paclitaxel nanostructure lipid carrier cooperated with radiation on the KB Cells

    International Nuclear Information System (INIS)

    Liu Min; Li Zhihui; Xu Yujie

    2011-01-01

    Objective: To investigate the cytotoxicity effect of paclitaxel nanostructure lipid carrier (TAX-NLC) cooperated with radiation treatment on the KB cells. Methods: The cytotoxicity effect of TAX-NLC compared with free paclitaxel (TAX) on the KB cells was measured by MTT assay, and the cell cycle distribution was analyzed by flow cytometry. Results: The cytotoxicity effect of TAX-NLC was stronger than that of the free TAX. And the cooperative effect between the TAX-NLC and ionized radiation were observed, the cooperative effect of TAX-NLC was stronger than that of the free TAX. Flow cytometric analysis indicated that the rearrangement of cell cycle in KB cells were induced by TAX-NLC. The more G2/M phase cells were observed in KB cells treated by TAX-NLC compared with free TAX. The effect of TAX-NLC in the rearrangement of cell cycle was stronger than that of the free TAX. Conclusion: The cytotoxicity effect of TAX-NLC is stronger than that of the free TAX. And the cooperative effect of TAX-NLC is stronger than that of the free TAX. (authors)

  16. Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

    2015-08-27

    Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

  17. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

    Science.gov (United States)

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

    2013-04-01

    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

  18. Cell wall composition profiling of parasitic giant dodder (Cuscuta reflexa) and its hosts: a priori differences and induced changes.

    Science.gov (United States)

    Johnsen, Hanne R; Striberny, Bernd; Olsen, Stian; Vidal-Melgosa, Silvia; Fangel, Jonatan U; Willats, William G T; Rose, Jocelyn K C; Krause, Kirsten

    2015-08-01

    Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  19. Modulation of Host Osseointegration during Bone Regeneration by Controlling Exogenous Stem Cells Differentiation Using a Material Approach.

    Science.gov (United States)

    Yu, Xiaohua; Wang, Liping; Xia, Zengmin; Chen, Li; Jiang, Xi; Rowe, David; Wei, Mei

    2014-02-01

    Stem cell-based tissue engineering for large bone defect healing has attracted enormous attention in regenerative medicine. However, sufficient osseointegration of the grafts combined with exogenous stem cells still remains a major challenge. Here we developed a material approach to modulate the integration of the grafts to the host tissue when exogenous bone marrow stromal cells (BMSCs) were used as donor cells. Distinctive osseointegration of bone grafts was observed as we varied the content of hydroxyapatite (HA) in the tissue scaffolds implanted in a mouse femur model. More than 80% of new bone was formed in the first two weeks of implantation in high HA content scaffold but lack of host integration while only less than 5% of the new bone was formed during this time period in the no HA group but with much stronger host integration. Cell origin analysis leveraging GFP reporter indicates new bone in HA containing groups was mainly derived from donor BMSCs. In comparison, both host and donor cells were found on new bone surface in the no HA groups which led to seamless bridging between host tissue and the scaffold. Most importantly, host integration during bone formation is closely dictated to the content of HA present in the scaffolds. Taken together, we demonstrate a material approach to modulate the osseointegration of bone grafts in the context of exogenous stem cell-based bone healing strategy which might lead to fully functional bone tissue regeneration.

  20. Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

    Science.gov (United States)

    Plénat, Thomas; Boichot, Sylvie; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2005-12-01

    Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.