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Sample records for host cell nuclear

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

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    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.

  2. Can shale safely host US nuclear waste?

    Science.gov (United States)

    Neuzil, C.E.

    2013-01-01

    "Even as cleanup efforts after Japan’s Fukushima disaster offer a stark reminder of the spent nuclear fuel (SNF) stored at nuclear plants worldwide, the decision in 2009 to scrap Yucca Mountain as a permanent disposal site has dimmed hope for a repository for SNF and other high-level nuclear waste (HLW) in the United States anytime soon. About 70,000 metric tons of SNF are now in pool or dry cask storage at 75 sites across the United States [Government Accountability Office, 2012], and uncertainty about its fate is hobbling future development of nuclear power, increasing costs for utilities, and creating a liability for American taxpayers [Blue Ribbon Commission on America’s Nuclear Future, 2012].However, abandoning Yucca Mountain could also result in broadening geologic options for hosting America’s nuclear waste. Shales and other argillaceous formations (mudrocks, clays, and similar clay-rich media) have been absent from the U.S. repository program. In contrast, France, Switzerland, and Belgium are now planning repositories in argillaceous formations after extensive research in underground laboratories on the safety and feasibility of such an approach [Blue Ribbon Commission on America’s Nuclear Future, 2012; Nationale Genossenschaft für die Lagerung radioaktiver Abfälle (NAGRA), 2010; Organisme national des déchets radioactifs et des matières fissiles enrichies, 2011]. Other nations, notably Japan, Canada, and the United Kingdom, are studying argillaceous formations or may consider them in their siting programs [Japan Atomic Energy Agency, 2012; Nuclear Waste Management Organization (NWMO), (2011a); Powell et al., 2010]."

  3. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  4. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  5. Host cells and cell banking.

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    Stacey, Glyn N; Merten, Otto-Wilhelm

    2011-01-01

    Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus).

  6. Nuclear reprogramming and stem cell creation

    OpenAIRE

    Gurdon, J B; Byrne, J.A.; Simonsson, S.

    2003-01-01

    The transplantation of a somatic cell nucleus to an enucleated egg results in a major reprogramming of gene expression and switch in cell fate. We review the efficiency of nuclear reprogramming by nuclear transfer. The serial transplantation of nuclei from defective first-transfer embryos and the grafting of cells from such embryos to normal host embryos greatly increases the proportion of nuclei that can be seen to have been reprogrammed. We discuss possible reasons for the early failure of ...

  7. Modified host cells with efflux pumps

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    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  8. Feedback between host galaxy and nuclear activity

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    I. Márquez

    2008-01-01

    Full Text Available Uno de los problemas fundamentales en el estudio de la actividad nuclear en galaxias (AGN es comprender cuáles son los mecanismos para el encendido de la emisión no térmica en sus núcleos. Hay que dilucidar tanto el origen del gas que es acretado hacia el agujero negro, como los mecanismos físicos para que se produzca la necesaria pérdida de momento angular y el gas caiga hacia el centro. Dicho de otro modo, el objetivo es entender bajo qué condiciones se enciende la actividad AGN. Aún quedan muchos aspectos al respecto que son objeto de debate. En esta contribución se discuten en concreto el papel que juega la interacción gravitacional y la relevancia del tipo morfológico de la galaxia anfitriona así como del entorno, para entender si la actividad AGN está más relacionada con los efectos de interacción, o si por el contrario es debida a procesos de evolución secular en la galaxia albergadora.

  9. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  10. Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula.

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    Amaury Nars

    Full Text Available N-acetylglucosamine-based saccharides (chitosaccharides are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

  11. The nuclear to host galaxy relation of high redshift quasars

    CERN Document Server

    Kotilainen, J K; Labita, M; Treves, A; Uslenghi, M

    2007-01-01

    We present near-infrared imaging with ESO VLT+ISAAC of the host galaxies of low luminosity quasars in the redshift range 1 < z < 2, aimed at investigating the relationship between the nuclear and host galaxy luminosities at high redshift. This work complements our previous study to trace the cosmological evolution of the host galaxies of high luminosity quasars (Falomo et al. 2004). The new sample includes 15 low luminosity quasars, nine radio-loud (RLQ) and six radio-quiet (RQQ). They have similar distribution of redshift and optical luminosity, and together with the high luminosity quasars they cover a large range (~4 mag) of the quasar luminosity function. The host galaxies of both types of quasars are in the range of massive inactive ellipticals between L* and 10 L*. RLQ hosts are systematically more luminous than RQQ hosts by a factor of ~2. This difference is similar to that found for the high luminosity quasars. This luminosity gap appears to be independent of the rest-frame U-band luminosity but...

  12. Salmonella - at home in the host cell.

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    Preeti eMalik Kale

    2011-06-01

    Full Text Available The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic trigger-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS, although T3SS-independent mechanisms of entry may be important for invasion of certain host cell-types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.

  13. Spermatozoa-like cell invaders (nuclear vlimata) in human neoplasia.

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    Logothetou-Rella, H

    1993-07-01

    Spermatozoa-like cells (nuclear vlimata) have been identified in malignant cell cultures and embryonic cells, also common in the cytology and histology of all types of human neoplasia even after chemotherapy. A new mechanism of invasion of malignant cells has been described, according to which neoplastic cells behave and function as parasites using host-cells to divide, survive and eventually produce nuclear vlimata (bullets). Nuclear vlimata are the end cell products of incomplete, unequal, assymetrical division of neoplastic cells. The nuclear vlima exhibits similar morphology to spermatozoa and virus (head with, or without, tail) and invades the cytoplasm and/or nucleus of surrounding host-cells by a similar mechanism to sperm-oocyte interaction (fertilization) or viral cell infection, in the events of nuclear vlima-->tumor-->nuclear vlima-->tumor. The nuclear vlima head contains and transfers DNA, and when incorporated into the host-nucleus is indistinguishable from nucleoli and when in the cytoplasm is similar to sperm pronucleus, observed after sperm penetration of the oocyte. Function of nuclear vlimata is directly dependent on the specific extracellular matrix produced by malignant cells, consisting of glycosaminoglycans-protease-membranes. This mechanism of invasion constitutes the link of all scientific information concerning human neoplasia.

  14. Penetration of Bdellovibrio bacteriovorus into host cells.

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    Abram, D; Castro e Melo, J; Chou, D

    1974-05-01

    Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.

  15. Microorganisms in potential host rocks for geological disposal of nuclear waste and their interactions with radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Cherkouk, A.; Liebe, M.; Luetke, L.; Moll, H.; Stumpf, T. [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Inst. of Resource Ecology

    2015-07-01

    The long-term safety of nuclear waste in a deep geological repository is an important issue in our society. Microorganisms indigenous to potential host rocks are able to influence the oxidation state, speciation and therefore the mobility of radionuclides as well as gas generation or canister corrosion. Therefore, for the safety assessment of such a repository it is necessary to know which microorganisms are present in the potential host rocks (e.g. clay, salt) and if these microorganisms can influence the performance of a repository. Microbial diversity in potential host rocks for geological disposal of nuclear waste was analyzed by culture-independent molecular biological methods (e.g. 16S rRNA gene retrieval) as well as enrichment and isolation of indigenous microbes. Among other isolates, a Paenibacillus strain, as a representative of Firmicutes, was recovered in R2A media under anaerobic conditions from Opalinus clay from the Mont Terri in Switzerland. Accumulation experiments and potentiometric titrations showed a strong interaction of Paenibacillus sp. cells with U(VI) within a broad pH range (3-7). Additionally, the interactions of the halophilic archaeal strain Halobacterium noricense DSM 15987, a salt rock representative reference strain, with U(VI) at high ionic strength was investigated. After 48 h the cells were still alive at uranium concentrations up to 60 μM, which demonstrates that Halobacterium noricense can tolerate uranium concentrations up to this level. The formed uranium sorption species were examined with time-resolved laser-induced fluorescence spectroscopy (TRLFS). The results about the microbial communities present in potential host rocks for nuclear waste repositories and their interactions with radionuclides contribute to the safety assessment of a prospective nuclear waste repository.

  16. Bartonella entry mechanisms into mammalian host cells.

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    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.

  17. Producing primate embryonic stem cells by somatic cell nuclear transfer.

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    Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

    2007-11-22

    Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

  18. Bovine somatic cell nuclear transfer.

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    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  19. Exploitation of host cells by Burkholderia pseudomallei.

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    Stevens, Mark P; Galyov, Edouard E

    2004-04-01

    Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways. The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function. Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas. B. pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion. Here we review current understanding of the molecular mechanisms underlying these processes.

  20. Acanthamoeba induces cell-cycle arrest in host cells.

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    Sissons, James; Alsam, Selwa; Jayasekera, Samantha; Kim, Kwang Sik; Stins, Monique; Khan, Naveed Ahmed

    2004-08-01

    Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

  1. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

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    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  2. Spermatozoa-li ke cell invaders, nuclear vlimata, in human neoplasia

    OpenAIRE

    1993-01-01

    Spermatozoa-like cells (nuclear vlimata) have been identified in malignant cell cultures and embryonic cells, also common in the cytology and histology of all types of human neoplasia even after chemotherapy. A new mechanism of invasion of malignant cells has been described, according to which neoplastic cells behave and function as parasites using host-cells to divide, survive and eventually produce nuclear vlimata (bullets). Nuclear vlimata are the end ce...

  3. 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

  4. Nuclear lamina in plant cells

    Institute of Scientific and Technical Information of China (English)

    汪健; 杨澄; 翟中和

    1996-01-01

    By using selective extraction and diethylene glycol distearate (DGD) embedment and embedment-free electron microscopy, the nuclear lamina was demonstrated in carrot and Ginkgo male generative cells. Western blotting revealed that the nuclear lamina was composed of A-type and B-type lamins which contained at least 66-ku and 84-ku or 66-ku and 86-ku polypeptides, respectively. These lamin proteins were localized at the nudear periphery as shown by immunogold-labelling. In situ hybridization for light microscope and electron microscope showed that plant cells have the homologous sequences of animal lamin cDNA. The sorting site of lamin mRNA is mainly distributed in the cytoplasm near the nudear envelope. The data have verified that there indeed exists nudear lamina in plant cells.

  5. Cryptococcal cell morphology affects host cell interactions and pathogenicity.

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    Laura H Okagaki

    Full Text Available Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

  6. Plant cell proliferation inside an inorganic host.

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    Perullini, Mercedes; Rivero, María Mercedes; Jobbágy, Matías; Mentaberry, Alejandro; Bilmes, Sara A

    2007-01-10

    In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

  7. 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...

  8. Influenza virus mRNA trafficking through host nuclear speckles.

    Science.gov (United States)

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-05-27

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

  9. 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.

  10. Nuclear choreography: interpretations from living cells.

    Science.gov (United States)

    Janicki, Susan M; Spector, David L

    2003-04-01

    The advent of green fluorescent protein technology, its use in photobleaching experiments and the development of methods to rapidly acquire images and analyze complex datasets have opened the door to unraveling the mechanisms of nuclear functions in living cells. Studies over the past few years have characterized the movement of chromatin, nuclear proteins and nuclear bodies and, in some cases, correlated their dynamics with energy dependence, cell cycle progression, developmental changes, factor targeting and nuclear position. The mechanisms by which nuclear components move or are restrained have important implications for understanding not only the efficacy of nuclear functions but also the regulation of developmental programs and cellular growth.

  11. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...... (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures...

  12. 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...

  13. 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...

  14. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  15. Nuclear to host galaxy relation of high redshift quasars

    Directory of Open Access Journals (Sweden)

    J. K. Kotilainen

    2008-01-01

    Full Text Available Se presentan imágenes en el cercano infrarrojo de cuásares a 1 < z < 2 con una cobertura amplia (~ 4 mag de la función de luminosidad de cuásares. Las galaxias anfitrionas de cuásares radio-fuertes (RLQ y radio-callados (RQQ tienen luminosidades en el intervalo de las elípticas masivas inactivas entre L* y 10 L*. Las galaxias huésped de los RLQ son más luminosas que las de los RQQ. La brecha en luminosidad es independiente de la luminosidad en la banda-U en reposo pero está correlacionada con la luminosidad en la banda-R en reposo. Esta diferencia en el color de RLQ y RQQ es probablemente debida a una combinación de la diferencia intrínseca en sus SEDs y a un efecto de selección debido a la extinción interna por polvo. Hay una correlación razonable entre las luminosidades nuclear y huésped para RLQ pero no para RQQ. Si la banda-R traza la luminosidad bolométrica y si la luminosidad huésped es proporcional a la masa del agujero negro, los cuásares de alto corrimiento al rojo emiten en un estrecho intervalo de potencias con respecto a su luminosidad de Eddington.

  16. The Nuclear Receptor LXR Limits Bacterial Infection of Host Macrophages through a Mechanism that Impacts Cellular NAD Metabolism

    Directory of Open Access Journals (Sweden)

    Jonathan Matalonga

    2017-01-01

    Full Text Available Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.

  17. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  18. Host cells and methods for production of isobutanol

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-23

    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.

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

    KAUST Repository

    Via, Allegra

    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.

  20. Similarities between the Epstein-Barr Virus (EBV Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a “Bookmarking” Oncoprotein that Alters the Host Cell Epigenotype?

    Directory of Open Access Journals (Sweden)

    Hans Helmut Niller

    2012-09-01

    Full Text Available EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may “bookmark” active genes and ensure their reactivation in postmitotic cells (epigenetic memory. We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis.

  1. 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.

  2. Salmonella – At Home in the Host Cell

    OpenAIRE

    Malik-Kale, Preeti; Jolly, Carrie E.; Lathrop, Stephanie; Winfree, Seth; Luterbach, Courtney; Steele-Mortimer, Olivia

    2011-01-01

    The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic “trigger”-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on c...

  3. 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.

  4. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

  5. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

    Science.gov (United States)

    Alderete, J F; Garza, G E

    1985-01-01

    The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface

  6. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  7. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

    Science.gov (United States)

    Barott, Katie L; Venn, Alexander A; Perez, Sidney O; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-13

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

  8. 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.

  9. Modulation of host-cell MAPkinase signaling during fungal infection

    OpenAIRE

    2015-01-01

    Fungal infections contribute substantially to human suffering and mortality. The interaction between fungal pathogens and their host involves the invasion and penetration of the surface epithelium, activation of cells of the innate immune system and the generation of an effective response to block infection. Numerous host-cell signaling pathways are activated during fungal infection. This review will focus on the main fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus n...

  10. 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.

  11. Reconstitution of nuclear import in permeabilized cells.

    Science.gov (United States)

    Cassany, Aurelia; Gerace, Larry

    2009-01-01

    The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin-cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin alpha, importin beta, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.

  12. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  13. Modulation of host-cell MAPkinase signaling during fungal infection

    Directory of Open Access Journals (Sweden)

    Nir Osherov

    2015-10-01

    Full Text Available Fungal infections contribute substantially to human suffering and mortality. The interaction between fungal pathogens and their host involves the invasion and penetration of the surface epithelium, activation of cells of the innate immune system and the generation of an effective response to block infection. Numerous host-cell signaling pathways are activated during fungal infection. This review will focus on the main fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans and their ability to activate the host MAP-kinase signaling pathways leading to cytokine secretion, increased cell motility and killing of the pathogen. Both epithelial and innate immune cells specifically recognize fungal antigens and in particular cell surface polysaccharides such as β-glucans and react to them by activating multiple signaling pathways, including those containing MAP-kinase modules. Recent findings suggest that the host response to fungal infection utilizes the MAP-kinase pathway to differentiate between commensal and pathogenic fungi to selectively react only to the pathogenic forms. However, the paucity of relevant publications strongly emphasize that our understanding of host MAP-kinase signaling in response to fungal infection is still at a very early stage. It is clear, based on studies of host MAP-kinase signaling during viral and bacterial infections, that in fungi as well, a wealth of exciting findings await us.

  14. Virus-host co-evolution under a modified nuclear genetic code

    Directory of Open Access Journals (Sweden)

    Derek J. Taylor

    2013-03-01

    Full Text Available Among eukaryotes with modified nuclear genetic codes, viruses are unknown. However, here we provide evidence of an RNA virus that infects a fungal host (Scheffersomyces segobiensis with a derived nuclear genetic code where CUG codes for serine. The genomic architecture and phylogeny are consistent with infection by a double-stranded RNA virus of the genus Totivirus. We provide evidence of past or present infection with totiviruses in five species of yeasts with modified genetic codes. All but one of the CUG codons in the viral genome have been eliminated, suggesting that avoidance of the modified codon was important to viral adaptation. Our mass spectroscopy analysis indicates that a congener of the host species has co-opted and expresses a capsid gene from totiviruses as a cellular protein. Viral avoidance of the host’s modified codon and host co-option of a protein from totiviruses suggest that RNA viruses co-evolved with yeasts that underwent a major evolutionary transition from the standard genetic code.

  15. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    Directory of Open Access Journals (Sweden)

    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.

  16. Herpesvirus Genome Integration into Telomeric Repeats of Host Cell Chromosomes.

    Science.gov (United States)

    Osterrieder, Nikolaus; Wallaschek, Nina; Kaufer, Benedikt B

    2014-11-01

    It is well known that numerous viruses integrate their genetic material into host cell chromosomes. Human herpesvirus 6 (HHV-6) and oncogenic Marek's disease virus (MDV) have been shown to integrate their genomes into host telomeres of latently infected cells. This is unusual for herpesviruses as most maintain their genomes as circular episomes during the quiescent stage of infection. The genomic DNA of HHV-6, MDV, and several other herpesviruses harbors telomeric repeats (TMRs) that are identical to host telomere sequences (TTAGGG). At least in the case of MDV, viral TMRs facilitate integration into host telomeres. Integration of HHV-6 occurs not only in lymphocytes but also in the germline of some individuals, allowing vertical virus transmission. Although the molecular mechanism of telomere integration is poorly understood, the presence of TMRs in a number of herpesviruses suggests it is their default program for genome maintenance during latency and also allows efficient reactivation.

  17. Host epithelial geometry regulates breast cancer cell invasiveness

    Science.gov (United States)

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  18. 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.

  19. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    Science.gov (United States)

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  20. Aquatic viruses induce host cell death pathways and its application.

    Science.gov (United States)

    Reshi, Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-01-04

    Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell's death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Do Nuclear Star Clusters and Supermassive Black Holes Follow the Same Host-Galaxy Correlations?

    Directory of Open Access Journals (Sweden)

    Peter Erwin

    2012-01-01

    Full Text Available Studies have suggested that there is a strong correlation between the masses of nuclear star clusters (NSCs and their host galaxies, a correlation which is said to be an extension of the well-known correlations between supermassive black holes (SMBHs and their host galaxies. But careful analysis of disk galaxies—including 2D bulge/disk/bar decompositions—shows that while SMBHs correlate with the stellar mass of the bulge component of galaxies, the masses of NSCs correlate much better with the total galaxy stellar mass. In addition, the mass ratio MNSC/M⋆, tot for NSCs in spirals (at least those with Hubble types Sc and later is typically an order of magnitude smaller than the mass ratio MBH/M⋆, bul of SMBHs. The absence of a universal “central massive object” correlation argues against common formation and growth mechanisms for both SMBHs and NSCs. We also discuss evidence for a break in the NSC-host galaxy correlation, galaxies with Hubble types earlier than Sbc appear to host systematically more massive NSCs than do types Sc and later.

  3. Do Nuclear Star Clusters and Black Holes Follow the Same Host-Galaxy Correlations?

    CERN Document Server

    Erwin, Peter

    2011-01-01

    Studies have suggested that there is a strong correlation between the masses of nuclear star clusters (NSCs) and their host galaxies, a correlation which said to be an extension of the well-known correlations between supermassive black holes (SMBHs) and their host galaxies. But careful analysis of disk galaxies -- including 2D bulge/disk/bar decompositions -- shows that while SMBHs correlate with the stellar mass of the bulge component of galaxies, the masses of NSCs correlate much better with the total galaxy stellar mass. In addition, the mass ratio M_nsc/M_star,tot for NSCs in spirals (at least those with Hubble types Sc and later) is typically an order of magnitude smaller than the mass ratio M_bh/M_star, bulge of SMBHs. The absence of a universal "central massive object" correlation argues against common formation and growth mechanisms for both SMBHs and NSCs. We also discuss evidence for a break in the NSC-host galaxy correlation: galaxies with Hubble types earlier than Sbc appear to host systematically...

  4. Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei

    Directory of Open Access Journals (Sweden)

    Sara H. G. Sinclair

    2015-02-01

    Full Text Available Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA, via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other obligate intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.

  5. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

    Indian Academy of Sciences (India)

    Qingpo Liu; Qingzhong Xue

    2005-04-01

    A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm species Oryza sativa, Zea mays, Triticum aestivum and Arabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes in O. sativa, Z. mays, and T. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.

  7. Relationships between host and symbiont cell cycles in sea anemones and their symbiotic dinoflagellates.

    Science.gov (United States)

    Dimond, James L; Pineda, Rea R; Ramos-Ascherl, Zullaylee; Bingham, Brian L

    2013-10-01

    The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

  8. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    OpenAIRE

    1980-01-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphologica...

  9. Lipid exchange between Borrelia burgdorferi and host cells.

    Directory of Open Access Journals (Sweden)

    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.

  10. Mechanisms of outer membrane vesicle entry into host cells.

    Science.gov (United States)

    O'Donoghue, Eloise J; Krachler, Anne Marie

    2016-11-01

    Bacterial outer membrane vesicles (OMVs) are nano-sized compartments consisting of a lipid bilayer that encapsulates periplasm-derived, luminal content. OMVs, which pinch off of Gram-negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV-host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

  11. Plasmodium species: master renovators of their host cells.

    Science.gov (United States)

    de Koning-Ward, Tania F; Dixon, Matthew W A; Tilley, Leann; Gilson, Paul R

    2016-08-01

    Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host.

  12. Host cell modulation by human, animal and plant pathogens.

    Science.gov (United States)

    Andersson, Siv G E; Kempf, Volkhard A J

    2004-04-01

    Members of the alpha-proteobacteria display a broad range of interactions with higher eukaryotes. Some are pathogens of humans, such as Rickettsia and Bartonella that are associated with diseases like epidemic typhus, trench fever, cat scratch disease and bacillary angiomatosis. Others like the Brucella cause abortions in pregnant animals. Yet other species have evolved elaborate interactions with plants; in this group we find both plant symbionts and parasites. Despite radically different host preferences, extreme genome size variations and the absence of toxin genes, similarities in survival strategies and host cell interactions can be recognized among members of the alpha-proteobacteria. Here, we review some of these similarities, with a focus on strategies for modulation of the host target cell.

  13. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

    Science.gov (United States)

    Snustad, D P; Bursch, C J; Parson, K A; Hefeneider, S H

    1976-04-01

    The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and

  14. Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

    Science.gov (United States)

    Licona-Limón, Paula; Henao-Mejia, Jorge; Temann, Angela U; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De'broski R; Flavell, Richard A

    2013-10-17

    Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis.

    Science.gov (United States)

    Santos, António J M; Durkin, Charlotte H; Helaine, Sophie; Boucrot, Emmanuel; Holden, David W

    2016-07-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S Typhimurium delays epithelial cell turnover in the intestine.

  16. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  17. Host Cell Factors as Antiviral Targets in Arenavirus Infection

    Directory of Open Access Journals (Sweden)

    Elsa B. Damonte

    2012-09-01

    Full Text Available Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.

  18. Host cell factors as antiviral targets in arenavirus infection.

    Science.gov (United States)

    Linero, Florencia N; Sepúlveda, Claudia S; Giovannoni, Federico; Castilla, Viviana; García, Cybele C; Scolaro, Luis A; Damonte, Elsa B

    2012-09-01

    Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.

  19. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    Science.gov (United States)

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  20. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    Directory of Open Access Journals (Sweden)

    Burleigh Barbara A

    2009-05-01

    Full Text Available Abstract Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

  1. Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

    Science.gov (United States)

    Sukarieh, R; Sonenberg, N; Pelletier, J

    2010-05-01

    Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

  2. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  3. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    Science.gov (United States)

    Markwell, M A; Paulson, J C

    1980-10-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells.

  4. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  5. Host cell kinases and the hepatitis C virus life cycle.

    Science.gov (United States)

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Recombinant host cells and media for ethanol production

    Science.gov (United States)

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  7. Co-evolution of nuclear star clusters, massive black holes and their host galaxies

    CERN Document Server

    Antonini, Fabio; Silk, Joseph

    2015-01-01

    Studying how nuclear star clusters (NSCs) form and how they are related to the growth of the central massive black holes (MBHs) and their host galaxies is fundamental for our understanding of the evolution of galaxies and the processes that have shaped their central structures. We present the results of a semi-analytical galaxy formation model that follows the evolution of dark matter halos along merger trees, as well as that of the baryonic components. This model allows us to study the evolution of NSCs in a cosmological context, by taking into account the growth of NSCs due to both dynamical friction-driven migration of stellar clusters and star formation triggered by infalling gas, while also accounting for dynamical heating from (binary) MBHs. We find that in-situ star formation contributes a significant fraction (up to ~40%) of the total mass of NSCs in our model. Both NSC growth through in-situ star formation and through star cluster migration are found to generate NSC -- host galaxy scaling correlation...

  8. Nuclear crisis relocation: Issues for a host community-the case of Greenfield, Massachusetts, USA

    Science.gov (United States)

    Platt, Rutherford H.

    1986-03-01

    US civil defense planning for nuclear attack since 1974 has emphasized the doctrine of “crisis relocation.” Under this doctrine, some 150 million people would evacuate from urban areas and other probable targets to rural “host communities.” The population of the latter would “stay put” to assist the relocatees. Local communities would be responsible for the welfare of up to ten times their normal population for an indefinite period of time. This study examined certain implications of crisis relocation for the town of Greenfield, Massachusetts, USA, a typical host community. Various assumptions were articulated regarding the timing of events, the season of year, weather, and social behavior. Assumptions were favorable to the success of crisis relocation. Nevertheless, Greenfield would face impossible burdens in attempting to provide fallout protection, water, food, medical care, and civil order. Additional pressures would arise from adjoining communities which are functionally dependent upon Greenfield for normal goods and services, but which would receive their own allotment of relocatees. Crisis relocation is not taken seriously in Greenfield and virtually no preparations have been made to implement it.

  9. THE COMPLEX ORGANIZATION OF EUKARYOTIC CELL NUCLEUS (III: THE NUCLEAR MATRIX AND THE NUCLEAR LAMINA

    Directory of Open Access Journals (Sweden)

    Cristian S. Cimpeanu

    2013-07-01

    Full Text Available A large variety of nuclear fibrous proteins (such as actin, myosin, lamin B, transcription factors, topoisomerases, etc represent constitutive elements of complex structures present in the eukaryotic nuclei: the nuclear matrix and the nuclear lamina, repectively. These nuclear compartments, with fibrous network-like structure, play crucialroles in structural organization of nuclei, chromatin remodeling, DNA transcription, signals transduction, cell cycle regulation, embryonic development and other nuclear basic processes.

  10. Inhibition of the host translation shutoff response by herpes simplex virus 1 triggers nuclear envelope-derived autophagy.

    Science.gov (United States)

    Radtke, Kerstin; English, Luc; Rondeau, Christiane; Leib, David; Lippé, Roger; Desjardins, Michel

    2013-04-01

    Macroautophagy is a cellular pathway that degrades intracellular pathogens and contributes to antigen presentation. Herpes simplex virus 1 (HSV-1) infection triggers both macroautophagy and an additional form of autophagy that uses the nuclear envelope as a source of membrane. The present study constitutes the first in-depth analysis of nuclear envelope-derived autophagy (NEDA). We established LC3a as a marker that allowed us to distinguish between NEDA and macroautophagy in both immunofluorescence and flow cytometry. NEDA was observed in many different cell types, indicating that it is a general response to HSV-1 infection. This autophagic pathway is known to depend on the viral protein γ34.5, which can inhibit macroautophagy via binding to beclin-1. Using mutant viruses, we were able to show that binding of beclin-1 by γ34.5 had no effect on NEDA, demonstrating that NEDA is regulated differently than macroautophagy. Instead, NEDA was triggered in response to γ34.5 binding to protein phosphatase 1α, an interaction used by the virus to prevent host cells from shutting off protein translation. NEDA was not triggered when late viral protein production was inhibited with acyclovir or hippuristanol, indicating that the accumulation of these proteins might stress infected cells. Interestingly, expression of the late viral protein gH was sufficient to rescue NEDA in the context of infection with a virus that otherwise does not support strong late viral protein expression. We argue that NEDA is a cellular stress response triggered late during HSV-1 infection and might compensate for the viral alteration of the macroautophagic response.

  11. Brucella T4SS: the VIP pass inside host cells.

    Science.gov (United States)

    Lacerda, Thais Lourdes Santos; Salcedo, Suzana Pinto; Gorvel, Jean-Pierre

    2013-02-01

    For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

  12. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    Science.gov (United States)

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis.

  13. Infection strategies of intestinal parasite pathogens and host cell responses

    Directory of Open Access Journals (Sweden)

    Bruno Martorell Di Genova

    2016-03-01

    Full Text Available Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading cause worldwide of diarrheal illness in humans. Diseases caused by these organisms, Giardiasis, Cryptosporidiosis and Amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these tree pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite-host interaction and in the mechanisms implicated in the diseases pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways and cell death.

  14. Host Cell Autophagy in Immune Response to Zoonotic Infections

    Directory of Open Access Journals (Sweden)

    Panagiotis Skendros

    2012-01-01

    Full Text Available Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered within double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. Accumulating evidence supports the pivotal role of autophagy in host defense against intracellular pathogens implicating both innate and adaptive immunity. Many of these pathogens cause common zoonotic infections worldwide. The induction of the autophagic machinery by innate immune receptors signaling, such as TLRs, NOD1/2, and p62/SQSTM1 in antigen-presenting cells results in inhibition of survival and elimination of invading pathogens. Furthermore, Th1 cytokines induce the autophagic process, whereas autophagy also contributes to antigen processing and MHC class II presentation, linking innate to adaptive immunity. However, several pathogens have developed strategies to avoid autophagy or exploit autophagic machinery to their advantage. This paper focuses on the role of host cell autophagy in the regulation of immune response against intracellular pathogens, emphasizing on selected bacterial and protozoan zoonoses.

  15. Centrality of host cell death in plant-microbe interactions.

    Science.gov (United States)

    Dickman, Martin B; Fluhr, Robert

    2013-01-01

    Programmed cell death (PCD) is essential for proper growth, development, and cellular homeostasis in all eukaryotes. The regulation of PCD is of central importance in plant-microbe interactions; notably, PCD and features associated with PCD are observed in many host resistance responses. Conversely, pathogen induction of inappropriate cell death in the host results in a susceptible phenotype and disease. Thus, the party in control of PCD has a distinct advantage in these battles. PCD processes appear to be of ancient origin, as indicated by the fact that many features of cell death strategy are conserved between animals and plants; however, some of the details of death execution differ. Mammalian core PCD genes, such as caspases, are not present in plant genomes. Similarly, pro- and antiapoptotic mammalian regulatory elements are absent in plants, but, remarkably, when expressed in plants, successfully impact plant PCD. Thus, subtle structural similarities independent of sequence homology appear to sustain operational equivalence. The vacuole is emerging as a key organelle in the modulation of plant PCD. Under different signals for cell death, the vacuole either fuses with the plasmalemma membrane or disintegrates. Moreover, the vacuole appears to play a key role in autophagy; evidence suggests a prosurvival function for autophagy, but other studies propose a prodeath phenotype. Here, we describe and discuss what we know and what we do not know about various PCD pathways and how the host integrates signals to activate salicylic acid and reactive oxygen pathways that orchestrate cell death. We suggest that it is not cell death as such but rather the processes leading to cell death that contribute to the outcome of a given plant-pathogen interaction.

  16. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  17. Nuclear microscopy of rat colon epithelial cells

    Science.gov (United States)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  18. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell.

    Science.gov (United States)

    Rolando, Monica; Buchrieser, Carmen

    2014-12-01

    Intracellular bacterial pathogens modulate the host response to persist and replicate inside a eukaryotic cell and cause disease. Legionella pneumophila, the causative agent of Legionnaires' disease, is present in freshwater environments and represents one of these pathogens. During coevolution with protozoan cells, L. pneumophila has acquired highly sophisticated and diverse strategies to hijack host cell processes. It secretes hundreds of effectors into the host cell, and these manipulate host signaling pathways and key cellular processes. Recently it has been shown that L. pneumophila is also able to alter the transcription and translation machinery of the host and to exploit epigenetic mechanisms in the cells it resides in to counteract host responses.

  19. The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells.

    Science.gov (United States)

    van Damme, Mireille; Bozkurt, Tolga O; Cakir, Cahid; Schornack, Sebastian; Sklenar, Jan; Jones, Alexandra M E; Kamoun, Sophien

    2012-01-01

    Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8(R469A;D470A) resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.

  20. The Irish Potato Famine Pathogen Phytophthora infestans Translocates the CRN8 Kinase into Host Plant Cells

    Science.gov (United States)

    van Damme, Mireille; Bozkurt, Tolga O.; Cakir, Cahid; Schornack, Sebastian; Sklenar, Jan; Jones, Alexandra M. E.; Kamoun, Sophien

    2012-01-01

    Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8R469A;D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells. PMID:22927814

  1. The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells.

    Directory of Open Access Journals (Sweden)

    Mireille van Damme

    Full Text Available Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS. The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8(R469A;D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.

  2. 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.

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

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    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.

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

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    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

  5. Stepwise adaptation of murine cytomegalovirus to cells of a foreign host for identification of host range determinants.

    Science.gov (United States)

    Ostermann, Eleonore; Pawletko, Kerstin; Indenbirken, Daniela; Schumacher, Uwe; Brune, Wolfram

    2015-06-01

    Ever since their first isolation 60 years ago, cytomegaloviruses have been recognized as being highly species specific. They replicate only in cells of their own or a closely related host species, while cells of phylogenetically more distant hosts are usually not permissive for viral replication. For instance, human cytomegalovirus replicates in human and chimpanzee fibroblasts but not in rodent cells, and murine cytomegalovirus (MCMV) replicates in cells of mice and rats but not in primate cells. However, the viral and cellular factors determining the narrow host range of cytomegaloviruses have remained largely unknown. We show that MCMV can be adapted stepwise to replicate in cultured human retinal pigment epithelial (RPE-1) cells and human fibroblasts. The human RPE-1 cells used for the initial adaptation step showed a pronounced contact inhibition and produced very low level of interferon-β transcripts upon cytomegalovirus infection, suggesting that these cells provide a particularly favorable environment for adaptation. By whole genome sequencing of the 230 kbp viral genomes of several adapted mutants, a limited number of mutations were detected. Comparison of several human cell-adapted MCMV clones and introduction of specific mutations into the wild-type MCMV genome by site-directed mutagenesis allows for the identification of viral host range determinants and provides the basis for elucidating the molecular basis of the cytomegalovirus host species specificity.

  6. The nuclear-cytoplasmic shuttling of virion host shutoff RNase is enabled by pUL47 and an embedded nuclear export signal and defines the sites of degradation of AU-rich and stable cellular mRNAs.

    Science.gov (United States)

    Shu, Minfeng; Taddeo, Brunella; Roizman, Bernard

    2013-12-01

    The herpes simplex virus host shutoff RNase (VHS-RNase) is the major early block of host responses to infection. VHS-RNase is introduced into cells during infection and selectively degrades stable mRNAs made before infection and the normally short-lived AU-rich stress response mRNAs induced by sensors of innate immunity. Through its interactions with pUL47, another tegument protein, it spares from degradation viral mRNAs. Analyses of embedded motifs revealed that VHS-RNase contains a nuclear export signal (NES) but not a nuclear localization signal. To reconcile the potential nuclear localization with earlier studies showing that VHS-RNase degrades mRNAs in polyribosomes, we constructed a mutant in which NES was ablated. Comparison of the mutant and wild-type VHS-RNases revealed the following. (i) On infection, VHS-RNase is transported to the nucleus, but only the wild-type protein shuttles between the nucleus and cytoplasm. (ii) Both VHS-RNases localized in the cytoplasm following transfection. On cotransfection with pUL47, a fraction of VHS-RNase was translocated to the nucleus, suggesting that pUL47 may enable nuclear localization of VHS-RNase. (iii) In infected cells, VHS-RNase lacking NES degraded the short-lived AU-rich mRNAs but not the stable mRNAs. In transfected cells, both wild-type and NES mutant VHS-RNases effectively degraded cellular mRNAs. Our results suggest that the stable mRNAs are degraded in the cytoplasm, whereas the AU-rich mRNAs may be degraded in both cellular compartments. The selective sparing of viral mRNAs may take place during the nuclear phase in the course of interaction of pUL47, VHS-RNase, and nascent viral mRNAs.

  7. Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Tian, J; Song, J; Li, H; Yang, D; Li, X; Ouyang, H; Lai, L

    2012-08-01

    Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.

  8. Inkjet printing of silk nest arrays for cell hosting.

    Science.gov (United States)

    Suntivich, Rattanon; Drachuk, Irina; Calabrese, Rossella; Kaplan, David L; Tsukruk, Vladimir V

    2014-04-14

    An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 μm and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure. These "locked-in" silk nests remained anchored to the substrate during incubation in cell growth media to provide a biotemplated platform for printing-in, immobilization, encapsulation and growth of cells. The process of inkjet-assisted printing is versatile and can be applied on any type of substrate, including rigid and flexible, with scalability and facile formation.

  9. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

    Science.gov (United States)

    Fallon, Ann M; Baldridge, Gerald D; Carroll, Elissa M; Kurtz, Cassandra M

    2014-09-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

  10. Damage-plasticity model of the host rock in a nuclear waste repository

    Science.gov (United States)

    Koudelka, Tomáš; Kruis, Jaroslav

    2016-06-01

    The paper describes damage-plasticity model for the modelling of the host rock environment of a nuclear waste repository. Radioactive Waste Repository Authority in Czech Republic assumes the repository to be in a granite rock mass which exhibit anisotropic behaviour where the strength in tension is lower than in compression. In order to describe this phenomenon, the damage-plasticity model is formulated with the help of the Drucker-Prager yield criterion which can be set to capture the compression behaviour while the tensile stress states is described with the help of scalar isotropic damage model. The concept of damage-plasticity model was implemented in the SIFEL finite element code and consequently, the code was used for the simulation of the Äspö Pillar Stability Experiment (APSE) which was performed in order to determine yielding strength under various conditions in similar granite rocks as in Czech Republic. The results from the performed analysis are presented and discussed in the paper.

  11. Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

    Science.gov (United States)

    Torriani, Giulia; Galan-Navarro, Clara; Kunz, Stefan

    2017-02-15

    Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  12. The Survival Strategies of Malaria Parasite in the Red Blood Cell and Host Cell Polymorphisms

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    Gunanidhi Dhangadamajhi

    2010-01-01

    Full Text Available Parasite growth within the erythrocyte causes dramatic alterations of host cell which on one hand facilitates nutrients acquisition from extracellular environment and on other hand contributes to the symptoms of severe malaria. The current paper focuses on interactions between the Plasmodium parasite and its metabolically highly reduced host cell, the natural selection of numerous polymorphisms in the genes encoding hemoglobin and other erythrocyte proteins.

  13. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

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    Georgia Schäfer

    2015-05-01

    Full Text Available Currently, seven viruses, namely Epstein-Barr virus (EBV, Kaposi’s sarcoma-associated herpes virus (KSHV, high-risk human papillomaviruses (HPVs, Merkel cell polyomavirus (MCPyV, hepatitis B virus (HBV, hepatitis C virus (HCV and human T cell lymphotropic virus type 1 (HTLV-1, have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  14. The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

    Science.gov (United States)

    Bannister, L H

    1979-04-11

    Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the

  15. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  16. Human Cytomegalovirus Immediate Early Interaction with Host Nuclear Structures: Definition of an Immediate Transcript Environment

    Science.gov (United States)

    Ishov, Alexander M.; Stenberg, Richard M.; Maul, Gerd G.

    1997-01-01

    The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp. PMID:9214377

  17. Examination of cucurbit[7]uril and its host-guest complexes by diffusion nuclear magnetic resonance.

    Science.gov (United States)

    Wheate, Nial J; Kumar, P G Anil; Torres, Allan M; Aldrich-Wright, Janice R; Price, William S

    2008-02-28

    The self-diffusion of cucurbit[7]uril (CB[7]) and its host-guest complexes in D2O has been examined using pulsed gradient spin-echo nuclear magnetic resonance spectroscopy. CB[7] diffuses freely at a concentration of 2 mM with a diffusion coefficient (D) of 3.07 x 10(-10) m(2) s(-1). At saturation (3.7 mM), CB[7] diffuses more slowly (D = 2.82 x 10(-10) m(2) s(-1)) indicating that it partially self-associates. At concentrations between 2 and 200 mM, CsCl has no effect on the diffusion coefficient of CB[7] (1 mM). Conversely, CB[7] (2 mM) significantly affects the diffusion of 133Cs+ (1 mM), decreasing its diffusion coefficient from 1.86 to 0.83 x 10(-9) m(2) s(-1). Similar changes in the rate of diffusion of other alkali earth metal cations are observed upon the addition of CB[7]. The diffusion coefficient of 23Na+ changes from 1.26 to 0.90 x 10(-9) m(2) s(-1) and 7Li+ changes from 3.40 to 3.07 x 10(-9) m(2) s(-1). In most cases, encapsulation of a variety of inorganic and organic guests within CB[7] decreases their rates of diffusion in D2O. For instance, the diffusion coefficient of the dinuclear platinum complex trans-[[PtCl(NH3)2}2mu-dpzm](2+) (where dpzm is 4,4'-dipyrazolylmethane) decreases from 4.88 to 2.95 x 10(-10) m(2) s(-1) upon encapsulation with an equimolar concentration of CB[7].

  18. Yersinia pseudotuberculosis Spatially Controls Activation and Misregulation of Host Cell Rac1.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and

  19. Emerging functions as host cell factors - an encyclopedia of annexin-pathogen interactions.

    Science.gov (United States)

    Kuehnl, Alexander; Musiol, Agnes; Raabe, Carsten A; Rescher, Ursula

    2016-10-01

    Emerging infectious diseases and drug-resistant infectious agents call for the development of innovative antimicrobial strategies. With pathogenicity now considered to arise from the complex and bi-directional interplay between a microbe and the host, host cell factor targeting has emerged as a promising approach that might overcome the limitations of classical antimicrobial drug development and could open up novel and efficient therapeutic strategies. Interaction with and modulation of host cell membranes is a recurrent theme in the host-microbe relationship. In this review, we provide an overview of what is currently known about the role of the Ca2+ dependent, membrane-binding annexin protein family in pathogen-host interactions, and discuss their emerging functions as host cell derived auxiliary proteins in microbe-host interactions and host cell targets.

  20. 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.

  1. How stem cells speak with host immune cells in inflammatory brain diseases.

    Science.gov (United States)

    Pluchino, Stefano; Cossetti, Chiara

    2013-09-01

    Advances in stem cell biology have raised great expectations that diseases and injuries of the central nervous system (CNS) may be ameliorated by the development of non-hematopoietic stem cell medicines. Yet, the application of adult stem cells as CNS therapeutics is challenging and the interpretation of some of the outcomes ambiguous. In fact, the initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent cellular signaling between the graft and the host. Cellular signaling is the foundation of coordinated actions and flexible responses, and arises via networks of exchanging and interacting molecules that transmit patterns of information between cells. Sustained stem cell graft-to-host communication leads to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses in vivo, ultimately promoting the healing of the injured CNS. Among a number of adult stem cell types, mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their ability to signal to the immune system upon transplantation in experimental CNS diseases. Here, we focus on the main cellular signaling pathways that grafted MSCs and NPCs use to establish a therapeutically relevant cross talk with host immune cells, while examining the role of inflammation in regulating some of the bidirectionality of these communications. We propose that the identification of the players involved in stem cell signaling might contribute to the development of innovative, high clinical impact therapeutics for inflammatory CNS diseases.

  2. Epigenetic reprogramming by somatic cell nuclear transfer in primates.

    Science.gov (United States)

    Sparman, Michelle; Dighe, Vikas; Sritanaudomchai, Hathaitip; Ma, Hong; Ramsey, Cathy; Pedersen, Darlene; Clepper, Lisa; Nighot, Prashant; Wolf, Don; Hennebold, Jon; Mitalipov, Shoukhrat

    2009-06-01

    We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

  3. Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells

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    José Dijair Antonino de Souza Junior

    2017-06-01

    Full Text Available Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J, both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

  4. Nuclear area measurement on viable cells, using confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Townsend, K.M.S.; Marsden, S.J. (Medical Research Council, Harwell (United Kingdom). Radiobiological Research Unit)

    1992-04-01

    The authors describe a rapid procedure for the accurate measurement of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation. (author).

  5. Bacterial colonization of host cells in the absence of cholesterol.

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    Stacey D Gilk

    2013-01-01

    Full Text Available Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/- mouse embryonic fibroblasts (MEFs. DHCR24(-/- MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/- MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/- MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(Vβ(3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/- MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

  6. Bacterial Colonization of Host Cells in the Absence of Cholesterol

    Science.gov (United States)

    Gilk, Stacey D.; Cockrell, Diane C.; Luterbach, Courtney; Hansen, Bryan; Knodler, Leigh A.; Ibarra, J. Antonio; Steele-Mortimer, Olivia; Heinzen, Robert A.

    2013-01-01

    Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24−/− mouse embryonic fibroblasts (MEFs). DHCR24−/− MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24−/− MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24−/− MEFs. In contrast, C. burnetii entry was significantly decreased in −cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated αVβ3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24−/− MEFs lacked the CD63-postive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. PMID:23358892

  7. Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

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    Feng Gui

    2011-04-01

    Full Text Available Abstract Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

  8. The transforming parasite Theileria co-opts host cell mitotic and central spindles to persist in continuously dividing cells.

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    Conrad von Schubert

    Full Text Available The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.

  9. 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

  10. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  11. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    Science.gov (United States)

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  12. A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

    OpenAIRE

    Coleman, Bradley I.; Gubbels, Marc-Jan

    2012-01-01

    The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understo...

  13. The nuclear pore complex acts as a master switch for nuclear and cell differentiation

    Science.gov (United States)

    Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-01-01

    Cell differentiation is associated with the functional differentiation of the nucleus, in which alteration of the expression profiles of transcription factors occurs to destine cell fate. Nuclear transport machineries, such as importin-α, have also been reported as critical factors that induce cell differentiation. Using various fluorescence live cell imaging methods, including time-lapse imaging, FRAP analysis and live-cell imaging associated correlative light and electron microscopy (Live CLEM) of Tetrahymena, a unicellular ciliated protozoan, we have recently discovered that type switching of the NPC is the earliest detectable event of nuclear differentiation. Our studies suggest that this type switching of the NPC directs the fate of the nucleus to differentiate into either a macronucleus or a micronucleus. Our findings in this organism may provide new insights into the role of the NPC in controlling nuclear functions in general in eukaryotes, including controlling cell fate leading to cell differentiation in multicellular metazoa. PMID:26479399

  14. Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography.

    Science.gov (United States)

    Sisodiya, Vikram N; Lequieu, Joshua; Rodriguez, Maricel; McDonald, Paul; Lazzareschi, Kathlyn P

    2012-10-01

    Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

  15. Hepatitis C virus and host cell lipids: an intimate connection.

    Science.gov (United States)

    Alvisi, Gualtiero; Madan, Vanesa; Bartenschlager, Ralf

    2011-01-01

    Hepatitis C virus (HCV) is a major human pathogen, persistently infecting more than 170 million individuals worldwide. The recent establishment of fully permissive culture systems allowed unraveling the close link between host cell lipids and HCV, at each step of the viral replication cycle. HCV entry is triggered by the timely coordinated interaction of virus particles with cell surface receptors, including the low-density lipoprotein receptor. Viral RNA replication strictly depends on fatty acids and cholesterol biosynthesis. This process occurs on modified intracellular membranes, forming a membranous web. Their biogenesis is induced by the viral nonstructural proteins (NS) 4B and NS5A and requires the activity of cellular lipid kinases belonging to the phosphatidylinositol-4-kinase III family. A hallmark of HCV-induced membranes is thus the presence of phosphatidylinositol-4-phosphate (PI4P), which is synthesized by these kinases. Intriguingly, certain recently identified HCV dependency factors selectively bind to PI derivatives, suggesting a crucial role for PIPs in viral RNA replication and assembly. The latter occurs on the surface of lipid droplets and is tightly connected to the very low density lipoprotein pathway leading to the formation of unique lipoviro particles. Thus, HCV exploits lipid metabolism in many ways and may therefore serve as a model system to gain insights into membrane biogenesis, lipid droplet formation and lipid trafficking.

  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. 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.

  18. Cutaneous graft-versus-host disease after hematopoietic stem cell transplant - a review*

    Science.gov (United States)

    Villarreal, Cesar Daniel Villarreal; Alanis, Julio Cesar Salas; Pérez, Jose Carlos Jaime; Candiani, Jorge Ocampo

    2016-01-01

    Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplants (allo-HSCT) associated with significant morbidity and mortality. The earliest and most common manifestation is cutaneous graft-versus-host disease. This review focuses on the pathophysiology, clinical features, prevention and treatment of cutaneous graft-versus-host disease. We discuss various insights into the disease's mechanisms and the different treatments for acute and chronic skin graft-versus-host disease. PMID:27438202

  19. Host cell protein adsorption characteristics during protein A chromatography.

    Science.gov (United States)

    Tarrant, Richard D R; Velez-Suberbie, M Lourdes; Tait, Andrew S; Smales, C Mark; Bracewell, Daniel G

    2012-07-01

    Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

  20. Nuclear transfer to study the nuclear reprogramming of human stem cells.

    Science.gov (United States)

    Saito, Shigeo; Sawai, Ken; Murayama, Yoshinobu; Fukuda, Keiichi; Yokoyama, Kazunari

    2008-01-01

    Research of stem cells will enable us to understand the development and function of tissues and organs in mammals. The ability to induce regeneration of new tissues from embryonic stem (ES) cells derived from cloned blastocysts via nuclear transfer can be expected in the not-too-distant future. The fact that there is no way except nuclear cloning for the return of differentiated cells to undifferentiated cells remains an interesting problem to be solved. We describe protocols for the production of cloned calves from bovine ES cells to study nuclear reprogramming ability of stem cells. The frequency of term pregnancies for blastocysts from ES cells is higher than those of early pregnancies and maintained pregnancies after nuclear transfer with bovine somatic cells. We also describe protocols for gene introduction into bovine ES cells in vitro, particularly the human leukocyte antigens (HLA). Bovine ES cells provide a powerful tool for the generation of transgenic clonal offspring. This technique, when perfected for humans, may be critical for neural stem cell transplantation.

  1. A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

    Science.gov (United States)

    Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-02-01

    Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

  2. Life in cells, hosts, and vectors: parasite evolution across scales.

    Science.gov (United States)

    Mideo, Nicole; Acosta-Serrano, Alvaro; Aebischer, Toni; Brown, Mark J F; Fenton, Andy; Friman, Ville-Petri; Restif, Olivier; Reece, Sarah E; Webster, Joanne P; Brown, Sam P

    2013-01-01

    Parasite evolution is increasingly being recognized as one of the most important issues in applied evolutionary biology. Understanding how parasites maximize fitness whilst facing the diverse challenges of living in cells, hosts, and vectors, is central to disease control and offers a novel testing ground for evolutionary theory. The Centre for Immunity, Infection, and Evolution at the University of Edinburgh recently held a symposium to address the question "How do parasites maximise fitness across a range of biological scales?" The symposium brought together researchers whose work looks across scales and environments to understand why and how parasites 'do what they do', tying together mechanism, evolutionary explanations, and public health implications. With a broad range of speakers, our aim was to define and encourage more holistic approaches to studying parasite evolution. Here, we present a synthesis of the current state of affairs in parasite evolution, the research presented at the symposium, and insights gained through our discussions. We demonstrate that such interdisciplinary approaches are possible and identify key areas for future progress.

  3. Cancer cell: using inflammation to invade the host

    Directory of Open Access Journals (Sweden)

    Aller María-Angeles

    2007-04-01

    Full Text Available Abstract Background Inflammation is increasingly recognized as an important component of tumorigenesis, although the mechanisms involved are not fully characterized. The invasive capacity of cancers is reflected in the classic metastatic cascade: tumor (T, node (N and metastasis (M. However, this staging system for cancer would also have a tumoral biological significance. Presentation of the hypothesis To integrate the mechanisms that control the inflammatory response in the actual staging system of cancer. It is considered that in both processes of inflammation and cancer, three successive phenotypes are presented that represent the expression of trophic functional systems of increasing metabolic complexity for using oxygen. Testing the hypothesis While a malignant tumor develops it express phenotypes that also share the inflammatory response such as: an ischemic phenotype (anoxic-hypoxic, a leukocytic phenotype with anaerobic glycolysis and migration, and an angiogenic phenotype with hyperactivity of glycolytic enzymes, tumor proliferation and metastasis, and cachexia of the host. The increasing metabolic complexity of the tumor cell to use oxygen allows for it to be released, migrate and proliferate, thus creating structures of growing complexity. Implication of the hypothesis One aim of cancer gene therapy could be the induction of oxidative phosphorylation, the last metabolic step required by inflammation in order to differentiate the tissue that it produces.

  4. Host cell invasion and virulence mediated by Candida albicans Ssa1.

    Directory of Open Access Journals (Sweden)

    Jianing N Sun

    2010-11-01

    Full Text Available Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.

  5. 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.

  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. Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis

    OpenAIRE

    Penterman, Jon; Abo, Ryan P.; De Nisco, Nicole J.; Markus F F Arnold; Longhi, Renato; ZANDA, Matteo; Walker, Graham C.

    2014-01-01

    Sinorhizobium meliloti and its legume hosts establish a symbiosis in which bacterial fixed nitrogen is exchanged for plant carbon compounds. We study this symbiosis because it is agriculturally and ecologically important and to identify mechanisms used in host–microbe interactions. S. meliloti is internalized in specialized host nodule cells that then use small, cysteine-rich peptides to drive their differentiation into polyploid cells that fix nitrogen. We found that a representative host pe...

  8. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis

    Science.gov (United States)

    Quigley, Jeff; Hughitt, V. Keith; Velikovsky, Carlos A.; Mariuzza, Roy A.

    2017-01-01

    ABSTRACT The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. PMID:28270579

  9. Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

    Science.gov (United States)

    Hegde, Shrilakshmi; Gabriel, Cordula; Kragl, Martin; Chopra-Dewasthaly, Rohini

    2015-10-01

    Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

  10. Host Factors Invovled in the Entry of Coronaviruses into Mammalian Cells

    NARCIS (Netherlands)

    Burkard, C.

    2015-01-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fus

  11. Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

    Energy Technology Data Exchange (ETDEWEB)

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2016-04-19

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  12. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

    Science.gov (United States)

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2014-09-30

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  13. Human somatic cell nuclear transfer is alive and well.

    Science.gov (United States)

    Cibelli, Jose B

    2014-06-05

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well.

  14. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  15. Nuclear envelope rupture and repair during cancer cell migration

    Science.gov (United States)

    Denais, Celine M.; Gilbert, Rachel M.; Isermann, Philipp; McGregor, Alexandra L.; te Lindert, Mariska; Weigelin, Bettina; Davidson, Patricia M.; Friedl, Peter; Wolf, Katarina; Lammerding, Jan

    2016-01-01

    During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, requiring extensive deformation of the cell and its nucleus. Here, we investigated tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport-III (ESCRT-III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content, requiring efficient NE and DNA damage repair for survival. PMID:27013428

  16. From microbiology to cell biology: when an intracellular bacterium becomes part of its host cell.

    Science.gov (United States)

    McCutcheon, John P

    2016-08-01

    Mitochondria and chloroplasts are now called organelles, but they used to be bacteria. As they transitioned from endosymbionts to organelles, they became more and more integrated into the biochemistry and cell biology of their hosts. Work over the last 15 years has shown that other symbioses show striking similarities to mitochondria and chloroplasts. In particular, many sap-feeding insects house intracellular bacteria that have genomes that overlap mitochondria and chloroplasts in terms of size and coding capacity. The massive levels of gene loss in some of these bacteria suggest that they, too, are becoming highly integrated with their host cells. Understanding these bacteria will require inspiration from eukaryotic cell biology, because a traditional microbiological framework is insufficient for understanding how they work.

  17. A Systems Survey of Progressive Host-Cell Reorganization during Rotavirus Infection.

    Science.gov (United States)

    Green, Victoria A; Pelkmans, Lucas

    2016-07-13

    Pathogen invasion is often accompanied by widespread alterations in cellular physiology, which reflects the hijacking of host factors and processes for pathogen entry and replication. Although genetic perturbation screens have revealed the complexity of host factors involved for numerous pathogens, it has remained challenging to temporally define the progression of events in host cell reorganization during infection. We combine high-confidence genome-scale RNAi screening of host factors required for rotavirus infection in human intestinal cells with an innovative approach to infer the trajectory of virus infection from fixed cell populations. This approach reveals a comprehensive network of host cellular processes involved in rotavirus infection and implicates AMPK in initiating the development of a rotavirus-permissive environment. Our work provides a powerful approach that can be generalized to order complex host cellular requirements along a trajectory of cellular reorganization during pathogen invasion.

  18. Assessing Kazakhstan´s Proposal to Host a Nuclear Fuel Bank

    Directory of Open Access Journals (Sweden)

    Caitlin A. Buckley

    2012-01-01

    Full Text Available Los defensores del control de armamentos buscan mecanismos para desincentivar la extensión de tecnologías sensibles para la proliferación. La mayoría de planes consideran situar las tecnologías del ciclo de combustible nuclear bajo un mayor control multilateral. Las propuestas para establecer uno o más bancos de combustible nuclear encajan claramente en este enfoque. Bajo este sistema, los países pueden "tomar prestado" el combustible que necesiten para sus reactores nucleares de un repositorio bajo control de la AIEA. Kazajstán ha ofrecido considerar el establecimiento de un banco internacional de combustible nuclear en su territorio. El país cuenta con ciertas características que podrían convertirlo en un buen candidato para un banco de combustible. Por ejemplo, Kazajstán es un actor destacado en el mercado internacional de uranio, tiene buenas relaciones con muchos países, y su gobierno cuenta con un historial sólido de no proliferación. Sin embargo, existe preocupación entre los observadores nacionales e internacionales acerca del ambicioso programa del gobierno kazajo, sus relaciones con países propensos a la proliferación, y la oposición de la sociedad en Kazajstán a las actividades nucleares.

  19. Modeling of Flow in Nuclear Reactor Fuel Cell Outlet

    Directory of Open Access Journals (Sweden)

    František URBAN

    2010-12-01

    Full Text Available Safe and effective load of nuclear reactor fuel cells demands qualitative and quantitative analysis of relations between coolant temperature in fuel cell outlet temperature measured by thermocouple and middle temperature of coolant in thermocouple plane position. In laboratory at Insitute of thermal power engineering of the Slovak University of Technology in Bratislava was installed an experimental physical fuel cell model of VVER 440 nuclear power plant with V 213 nuclear reactors. Objective of measurements on physical model was temperature and velocity profiles analysis in the fuel cell outlet. In this paper the measured temperature and velocity profiles are compared with the results of CFD simulation of fuel cell physical model coolant flow.

  20. [Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

    Science.gov (United States)

    Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

    2006-12-01

    Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

  1. Host-based Th2 cell therapy for prolongation of cardiac allograft viability.

    Directory of Open Access Journals (Sweden)

    Shoba Amarnath

    Full Text Available Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1 reduced the frequency of activated T cells in secondary lymphoid organs; (2 shifted post-transplant cytokines towards a Th2 phenotype; and (3 prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use "direct" host T cell therapy for prolongation of allograft viability as an alternative to "indirect" therapy mediated by donor T cell infusion.

  2. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

    Science.gov (United States)

    Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

  3. Natural variation in populations of persistently colonizing bacteria affect human host cell phenotype.

    Science.gov (United States)

    Aras, Rahul A; Lee, Yongchan; Kim, Sung-Kook; Israel, Dawn; Peek, Richard M; Blaser, Martin J

    2003-08-15

    The highly diverse bacterium Helicobacter pylori, which persistently colonizes the human stomach, provides models to study the role of genome plasticity in host adaptation. Within H. pylori populations from 2 colonized individuals, intragenomic recombination between cagA DNA repeat sequences leads to deletion or duplication of tyrosine phosphorylation sites in the CagA protein, which is injected by a type IV secretion system into host cells. Experimental coculture of gastric epithelial cells with the strains containing these naturally occurring CagA phosphorylation site variants induced markedly divergent host cell morphologic responses. Mutants were constructed in which a phosphorylation site was either added or deleted in the expressed CagA protein; coculture studies confirmed that the naturally occurring differences in CagA phosphorylation are responsible for the observed phenotypic variation. These findings indicate that within an individual host, intragenomic recombination between H. pylori repetitive DNA produces strain variants differing in their signals to host cells.

  4. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  5. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways

    Directory of Open Access Journals (Sweden)

    Renata Watanabe Costa

    2016-03-01

    Full Text Available Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

  6. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and su

  7. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and

  8. 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.

  9. Kinematics and Host-Galaxy Properties Suggest a Nuclear Origin for Calcium-Rich Supernova Progenitors

    CERN Document Server

    Foley, Ryan J

    2015-01-01

    Calcium-rich supernovae (Ca-rich SNe) are peculiar low-luminosity SNe Ib with relatively strong Ca spectral lines at ~2 months after peak brightness. This class also has an extended projected offset distribution, with several members of the class offset from their host galaxies by 30 - 150 kpc. There is no indication of any stellar population at the SN positions. Using a sample of 13 Ca-rich SNe, we present kinematic evidence that the progenitors of Ca-rich SNe originate near the centers of their host galaxies and are kicked to the locations of the SN explosions. Specifically, SNe with small projected offsets have large line-of-sight velocity shifts as determined by nebular lines, while those with large projected offsets have no significant velocity shifts. Therefore, the velocity shifts must not be primarily the result of the SN explosion. There is an excess of SNe with blueshifted velocity shifts within two isophotal radii (5/6 SNe), indicating that the SNe are moving away from their host galaxies and redsh...

  10. The host cell sulfonation pathway contributes to retroviral infection at a step coincident with provirus establishment.

    Directory of Open Access Journals (Sweden)

    James W Bruce

    2008-11-01

    Full Text Available The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV vector. This approach identified a role for 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1, one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.

  11. The host cell sulfonation pathway contributes to retroviral infection at a step coincident with provirus establishment.

    Directory of Open Access Journals (Sweden)

    James W Bruce

    2008-11-01

    Full Text Available The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV vector. This approach identified a role for 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1, one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.

  12. A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Adam Sateriale

    2011-01-01

    Full Text Available The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model.

  13. A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

    Science.gov (United States)

    Sateriale, Adam; Huston, Christopher D.

    2011-01-01

    The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

  14. 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

  15. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    Science.gov (United States)

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine.

  16. Host parasite communications-Messages from helminths for the immune system: Parasite communication and cell-cell interactions.

    Science.gov (United States)

    Coakley, Gillian; Buck, Amy H; Maizels, Rick M

    2016-07-01

    Helminths are metazoan organisms many of which have evolved parasitic life styles dependent on sophisticated manipulation of the host environment. Most notably, they down-regulate host immune responses to ensure their own survival, by exporting a range of immuno-modulatory mediators that interact with host cells and tissues. While a number of secreted immunoregulatory parasite proteins have been defined, new work also points to the release of extracellular vesicles, or exosomes, that interact with and manipulate host gene expression. These recent results are discussed in the overall context of how helminths communicate effectively with the host organism.

  17. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  18. Galvanic cell for processing of used nuclear fuel

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Diaz, Brenda L.; Martinez-Rodriguez, Michael J.; Gray, Joshua R.; Olson, Luke C.

    2017-02-07

    A galvanic cell and methods of using the galvanic cell is described for the recovery of uranium from used nuclear fuel according to an electrofluorination process. The galvanic cell requires no input energy and can utilize relatively benign gaseous fluorinating agents. Uranium can be recovered from used nuclear fuel in the form of gaseous uranium compound such as uranium hexafluoride, which can then be converted to metallic uranium or UO.sub.2 and processed according to known methodology to form a useful product, e.g., fuel pellets for use in a commercial energy production system.

  19. Transcriptional adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its susceptible wheat host.

    Science.gov (United States)

    Keon, John; Antoniw, John; Carzaniga, Raffaella; Deller, Siân; Ward, Jane L; Baker, John M; Beale, Michael H; Hammond-Kosack, Kim; Rudd, Jason J

    2007-02-01

    Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.

  20. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells.

    Science.gov (United States)

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-28

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.

  1. PARP activation promotes nuclear AID accumulation in lymphoma cells.

    Science.gov (United States)

    Tepper, Sandra; Jeschke, Julia; Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-03-15

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma.

  2. Dissecting the membrane cholesterol requirement for mycobacterial entry into host cells.

    Science.gov (United States)

    Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha

    2015-07-01

    Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-β-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry.

  3. A Stem Cell-rescue Operation in a Nuclear Devastation

    Directory of Open Access Journals (Sweden)

    Editorial

    2011-01-01

    Full Text Available Dear friends, The aftermath of one of the most devastating post earthquake- tsunamis in the history of mankind, the March 2011 post-earthquake-tsunami-hitnuclear accident in Fukushima, Japan, has brought to fore several questions throughout the world on the safety of nuclearinstallations. Such Calamities,which are far beyond our comprehension, has left all nations with such installations and those planning to expand their nuclear power plant programmes in a dilemma. However, the recent news that the Japanese authorities are considering to harvest and store bone marrow stem cells for transfusion to radiation-exposed workers has ushered in a new ray of hope. The hematopoietic stem cells will be harvested before exposure and in case of bone marrow depletion after radiation exposure; these cells can be immediately retrieved from storage and transfused to the radiation-exposed worker so that they have the potential to re-constitute the entire bone marrow in the patient. This will be a great life-saver and game-changer as bone marrow depletion is one of the major consequences of high levels of radiation exposure. This strategy can be prescribed as a routine procedure in all the countries with such nuclear installations, especially for workers at very high risk of radiation exposure. In future, such preventive strategies should be considered for implementation to not only the workers in the facility, but also the rescue team and the vulnerable population in the radiation zone.The regenerative potential of hematopoietic stem cell (HSC is the key factor to be considered now. This is high time that the studies on optimal in vitro expansion of HSCs and ways of successful engraftment of the transfused cells to a niche of radiation-exposed host environment should be attempted in all earnestness by the regenerative medicine community at present. Natural calamities like this help man to realise that the millions of scientific discoveries and inventions can

  4. Recent Progress of Somatic Cell Nuclear Transfer in Pigs

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaoming; Dou Zhongying

    2005-01-01

    Research in the field of somatic cell nuclear transfer (SCNT) and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low (<1% ). Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

  5. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tonatiuh Barrios-García

    2016-06-01

    Full Text Available Tristetraprolin (TTP is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα, which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR, glucocorticoid receptor (GR and androgen receptor (AR. In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

  6. Galactose/N-acetylgalactosamine lectin: the coordinator of host cell killing

    Indian Academy of Sciences (India)

    Douglas R Boettner; Christopher Huston; William A Petri Jr

    2002-11-01

    Entamoeba histolytica is an enteric parasite that can kill host cells via a contact-dependent mechanism. This killing involves the amoebic surface protein referred to as the Gal/GalNAc lectin. The Gal/GalNAc lectin binds galactose and N-acetylgalactosamine allowing the adherence of amoebas to host cells. Involvement of the lectin in the pathogenesis of E. histolytica infection will be reviewed in this paper. The lectin has been shown to have very specific and substantial effects on adherence, cytotoxicity, and encystation. There is also possible involvement of the lectin in phagocytosis and caspase activation in host cells.

  7. Chew on this: Amoebic trogocytosis and host cell killing by Entamoeba histolytica

    Science.gov (United States)

    Ralston, Katherine S.

    2015-01-01

    Entamoeba histolytica was named “histolytica” (histo-: tissue; lytic-: dissolving) for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. Here we review this process, termed “amoebic trogocytosis” (trogo-: nibble), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. “Nibbling” processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange. PMID:26070402

  8. In situ regeneration of skeletal muscle tissue through host cell recruitment.

    Science.gov (United States)

    Ju, Young Min; Atala, Anthony; Yoo, James J; Lee, Sang Jin

    2014-10-01

    Standard reconstructive procedures for restoring normal function after skeletal muscle defects involve the use of existing host tissues such as muscular flaps. In many instances, this approach is not feasible and delays the rehabilitation process and restoration of tissue function. Currently, cell-based tissue engineering strategies have been used for reconstruction; however, donor tissue biopsy and ex vivo cell manipulation are required prior to implantation. The present study aimed to overcome these limitations by demonstrating mobilization of muscle cells into a target-specific site for in situ muscle regeneration. First, we investigated whether host muscle cells could be mobilized into an implanted scaffold. Poly(l-lactic acid) (PLLA) scaffolds were implanted in the tibialis anterior (TA) muscle of rats, and the retrieved scaffolds were characterized by examining host cell infiltration in the scaffolds. The host cell infiltrates, including Pax7+ cells, gradually increased with time. Second, we demonstrated that host muscle cells could be enriched by a myogenic factor released from the scaffolds. Gelatin-based scaffolds containing a myogenic factor were implanted in the TA muscle of rats, and the Pax7+ cell infiltration and newly formed muscle fibers were examined. By the second week after implantation, the Pax7+ cell infiltrates and muscle formation were significantly accelerated within the scaffolds containing insulin-like growth factor 1 (IGF-1). Our data suggest an ability of host stem cells to be recruited into the scaffolds with the capability of differentiating to muscle cells. In addition, the myogenic factor effectively promoted host cell recruitment, which resulted in accelerating muscle regeneration in situ.

  9. Calculation of financial compensation due of municipalities hosting nuclear waste deposit

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Renata A. da, E-mail: renata.amaral@ufrj.b [Instituto de Engenharia Nuclear (IEN/CNEN-RJ), Rio de Janeiro, RJ (Brazil); Simoes, Francisco Fernando L.; Martins, Vivian B., E-mail: flamego@ien.gov.b [Instituto de Engenharia Nuclear (LIMA/IEN/CNEN-RJ), Rio de Janeiro, RJ (Brazil). Lab. Impactos Ambientais

    2011-07-01

    The present work evaluates the math from monthly financial transfers to municipalities with technical viability for building of initial or intermediate repository for storing of radioactivity nuclear waste: gloves, sneakers, mask, resins and filters came from thermonuclear facilities. Several aspects have been considered as the geological factors of the site as presence of capable faults, groundwater vulnerability, infiltration of seawater. Also, it was take into account socioeconomic factors: population density, costs for construction, maintenance and operation of repository; size and activity of waste; among others. Hereafter, we have presented the key features of low and average activity repository and high activity repository even as initial, intermediate and final repository and the possible environment impact. The methodology for calculation of financial compensation of municipalities was established by CNEN will be applied for a specific assumed municipality. The analysis of financial compensation due to the specific nuclear waste deposit and the possible guidelines for the use of that compensation by the municipality will be analyzed. In addiction, it will be compared the model for compensation used for nuclear wastes with other plants receiving permanent wastes from cemeteries and sanitary landfills, where the land should not be allowed for the human activities the same as: crops, livestock and buildings. Also, comparison with royalties and indemnities were paid by facilities of energy production as hydroelectric dams as well as petroleum and gas exploration plants. The destination of financial compensation transfer to the municipality is in charge of the city administration. The compensation could be applied of investments in education and culture, health, sanitation works, improvement of public transport, environment, among others. It will be discussed the cost-benefit relation for the assumed municipality. (author)

  10. 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.

  11. Nuclear Factor-Kappa B Activity in the Host-Tumor Microenvironment of Ovarian Cancer

    Science.gov (United States)

    2014-10-01

    Holson EB, Khabele D, Hiebert SW. HDAC3 is essential for the maintenance of chromatin structure and genome stability. Cancer Cell 18: 436-47, 2010... aspirin (ASA) in COX-1 positive ovarian cancer cells through augmentation of p21. Cancer Biol. Ther. 9: 928-35, 2010. PMID: 20404564 11. Wilson AJ, Chueh...elucidate effects of disrupting activity of COX-1 in ovarian cancer cells and platelets via aspirin on progression of ovarian cancer. The ability of

  12. Hot-cell for dismantling of nuclear gauges

    CERN Document Server

    Reis, L C A

    2000-01-01

    This work objectives the design of a hot-cell that will be used for dismantling of nuclear gauges. In the hot-cell, nuclear gauges received as radioactive waste at the Centro de Desenvolvimento da Tecnologia Nuclear - CDTN will be dismantled, in order to decrease the volume of radioactive waste to be stored at the Center. Sources originally conditioned as special form radioactive material will be tested and in case do not present leakage, the respective gauges will be disposable for reusing by radioisotope users. The remaining sources will be taken off the original shielding and conditioned in special packages adequate for storage and disposal. All steps of work, the hot-cell design and methodology for conditioning are also described.

  13. 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.

  14. The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses

    Energy Technology Data Exchange (ETDEWEB)

    Terrier, Olivier; Moules, Vincent; Carron, Coralie; Cartet, Gaeelle [Equipe VirCell, Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Frobert, Emilie [Laboratoire de Virologie, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, 59 boulevard Pinel, F-69677 Bron Cedex, Lyon (France); Yver, Matthieu; Traversier, Aurelien [Equipe VirCell, Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Wolff, Thorsten [Division of Influenza/Respiratory Viruses, Robert Koch Institute, Nordufer 20, D-13353 Berlin (Germany); Riteau, Beatrice [Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Naffakh, Nadia [Institut Pasteur, Unite de Genetique Moleculaire des Virus Respiratoires, URA CNRS 3015, EA302 Universite Paris Diderot, Paris (France); and others

    2012-10-10

    Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

  15. 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.

  16. 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.

  17. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    Science.gov (United States)

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    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. PMID:22174816

  18. Comprehensive assessment of host responses to ionizing radiation by nuclear factor-κB bioluminescence imaging-guided transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Chung-Ta Chang

    Full Text Available The aim of this study was to analyze the host responses to ionizing radiation by nuclear factor-κB (NF-κB bioluminescence imaging-guided transcriptomic tool. Transgenic mice carrying the NF-κB-driven luciferase gene were exposed to a single dose of 8.5 Gy total-body irradiation. In vivo imaging showed that a maximal NF-κB-dependent bioluminescent intensity was observed at 3 h after irradiation and ex vivo imaging showed that liver, intestine, and brain displayed strong NF-κB activations. Microarray analysis of these organs showed that irradiation altered gene expression signatures in an organ-specific manner and several pathways associated with metabolism and immune system were significantly altered. Additionally, the upregulation of fatty acid binding protein 4, serum amyloid A2, and serum amyloid A3 genes, which participate in both inflammation and lipid metabolism, suggested that irradiation might affect the cross pathways of metabolism and inflammation. Moreover, the alteration of chemokine (CC-motif ligand 5, chemokine (CC-motif ligand 20, and Jagged 1 genes, which are involved in the inflammation and enterocyte proliferation, suggested that these genes might be involved in the radiation enteropathy. In conclusion, this report describes the comprehensive evaluation of host responses to ionizing radiation. Our findings provide the fundamental information about the in vivo NF-κB activity and transcriptomic pattern after irradiation. Moreover, novel targets involved in radiation injury are also suggested.

  19. Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes†

    Science.gov (United States)

    Trinks, Daniela; Rajeswaran, R.; Shivaprasad, P. V.; Akbergenov, Rashid; Oakeley, Edward J.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing. PMID:15681452

  20. Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

    Science.gov (United States)

    Trinks, Daniela; Rajeswaran, R; Shivaprasad, P V; Akbergenov, Rashid; Oakeley, Edward J; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-02-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

  1. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  2. Interaction of the host immune system with tumor cells in human papillomavirus associated diseases

    OpenAIRE

    Sauer, Madeleine

    2016-01-01

    Human papillomaviruses (HPV) are very common in the sexually active population and contribute to 610,000 cancers per year occurring at different locations. The initial step of HPV-related carcinogenesis is the induction of transforming processes in the host cells mediated by the viral oncoproteins E6 and E7 that interfere with critical host cell pathways. The transforming infection is highlighted by overexpression of the tumor suppressor protein p16INK4a. Only a small number of precancerous l...

  3. The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro

    OpenAIRE

    Stadelmann, Britta; Hanevik, Kurt; Andersson, Mattias; Bruserud, Øystein; Staffan G Svärd

    2013-01-01

    Background: Arginine is a conditionally essential amino acid important in growing individuals and under nonhomeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and argini...

  4. Study of community leaders in a nuclear host community: local issues, expectations and support and opposition

    Energy Technology Data Exchange (ETDEWEB)

    Bronfman, B. H.

    1977-08-01

    A study of community leaders was undertaken in Hartsville, Tennessee, site of the TVA Hartsville Nuclear Power Plant currently under construction. Leaders were found to be extremely supportive of the plant and of TVA's efforts to mitigate impacts expected to result from construction. Like their citizen counterparts, leaders expect economic benefits and some growth-related disruption to occur as a result of the plant, while environmental impacts are seen as extremely unlikely to occur. Plant-related issues, such as housing availability and traffic congestion, dominate leaders' thinking about current issues. These issues are expected to continue to be important in the future, and new issues dealing with growth and planning, employment and taxation are expected to arise.

  5. Nuclear responses to depletion of mitochondrial DNA in human cells.

    Science.gov (United States)

    Li, K; Neufer, P D; Williams, R S

    1995-11-01

    The derivation of human cell lines devoid of mitochondrial (mt) DNA (rho 0) provides an opportunity to study nuclear responses to a chronic impairment of mitochondrial oxidative phosphorylation. Expression of several nuclear genes is induced in human rho 0 cells, including those encoding integral proteins of the mitochondrial inner membrane, intermediate filaments, and ribosomes. In contrast to conditions in which mitochondrial respiration is altered acutely, expression of heat shock proteins and immediate early genes is not induced. Mitochondria from rho 0 cells maintain a transmembrane electrochemical potential and are distributed within the cytoplasm of these cells in a manner indistinguishable from that of wild-type cells. We conclude that a chronic deficiency of mitochondrial oxidative phosphorylation produced by elimination of mtDNA is associated with a different pattern of gene induction than that provoked by other acute or subacute conditions that impair mitochondrial respiration or create energy demands in excess of mitochondrial respiratory capacity.

  6. Molecular structure and biological function of proliferating cell nuclear antigen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote.As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.

  7. 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.

  8. Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells

    Science.gov (United States)

    Deiser, Katrin; Stoycheva, Diana; Bank, Ute; Blankenstein, Thomas; Schüler, Thomas

    2016-01-01

    The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols. PMID:27447484

  9. Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells.

    Directory of Open Access Journals (Sweden)

    Katrin Deiser

    Full Text Available The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7 is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+ host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7 therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.

  10. Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells.

    Science.gov (United States)

    Deiser, Katrin; Stoycheva, Diana; Bank, Ute; Blankenstein, Thomas; Schüler, Thomas

    2016-01-01

    The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.

  11. Resetting the T Cell Repertoire in Prostate Cancer Bearing Host

    Science.gov (United States)

    2009-03-01

    Immunol 15: 535 562. 16. Kronenberg M, Gapin L (2002) The unconventional lifestyle of NKT cells. Nat Rev Immunol 2: 557 568. 17. Matsuda JL, Gapin L...cells. Curr Opin Immunol 14: 250 254. 24. Gapin L, Matsuda JL, Surh CD, Kronenberg M (2001) NKT cells derive from double-positive thymocytes that are

  12. CotH3 mediates fungal invasion of host cells during mucormycosis.

    Science.gov (United States)

    Gebremariam, Teclegiorgis; Liu, Mingfu; Luo, Guanpingsheng; Bruno, Vincent; Phan, Quynh T; Waring, Alan J; Edwards, John E; Filler, Scott G; Yeaman, Michael R; Ibrahim, Ashraf S

    2014-01-01

    Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.

  13. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer

    Directory of Open Access Journals (Sweden)

    J You

    2014-01-01

    Full Text Available Background: Nonsmall cell lung cancer (NSCLC is the major cause of cancer death worldwide. Increasing evidence shows that noncoding RNAs (ncRNAs are widely involved in the development and progression of NSCLC. ncRNA small nucleolar RNA host gene 1 (SNHG1 has not been studied in cancer, especially its role in lung cancer remains unknown. Our studies were designed to investigate the expression and biological significance of SNHG1 in lung cancer. SNHG1 may be a novel ncRNA in early diagnosis in lung cancer. Methods: Noncoding RNA SNHG1 expression in 7 lung cancer cell lines was measured by quantitative real-time polymerase chain reaction. RNA interference approaches were used to find the biological functions of SNHG1. The effect of SNHG1 on proliferation was evaluated by cell count and crystal violet stains. Results: Noncoding RNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells. In addition, in vitro assays our results indicated that knockdown of SNHG1 inhibited cell proliferation. Conclusions: Our data indicated that ncRNA SNHG1 is significantly upregulated in NSCLC cell lines and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.

  14. COS-1 cells as packaging host for production of lentiviruses.

    Science.gov (United States)

    MacKenzie, Crystal J; Shioda, Toshi

    2011-03-01

    We present a protocol for in vitro production of recombinant lentiviruses using COS-1 African green monkey kidney epithelial cells and HEK293T human embryonic kidney epithelial cells as packaging cells. COS-1 and HEK293T express SV40 large T antigen, amplifying transfected circular plasmids harboring SV40 replication origin. Support protocols for evaluation of transfection efficiency by in situ β-galactosidase enzyme activity assay and titer of infection-capable virions are also provided. Advantages of using COS-1 packaging cells over the standard HEK293T cells for contamination-sensitive applications or automated processing are discussed.

  15. Cif type III effector protein: a smart hijacker of the host cell cycle.

    Science.gov (United States)

    Samba-Louaka, Ascel; Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2009-09-01

    During coevolution with their hosts, bacteria have developed functions that allow them to interfere with the mechanisms controlling the proliferation of eukaryotic cells. Cycle inhibiting factor (Cif) is one of these cyclomodulins, the family of bacterial effectors that interfere with the host cell cycle. Acquired early during evolution by bacteria isolated from vertebrates and invertebrates, Cif is an effector protein of type III secretion machineries. Cif blocks the host cell cycle in G1 and G2 by inducing the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). The x-ray crystal structure of Cif reveals it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases. This review summarizes and discusses what we know about Cif, from the bacterial gene to the host target.

  16. Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells.

    Science.gov (United States)

    Shera, K A; Shera, C A; McDougall, J K

    2001-12-01

    More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, providing insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of fundamental mechanisms regulating eukaryotic gene expression.

  17. Trypanosoma cruzi extracellular amastigotes and host cell signaling: more pieces to the puzzle

    Directory of Open Access Journals (Sweden)

    Éden Ramalho Ferreira

    2012-11-01

    Full Text Available Among the different infective stages that Trypanosoma cruzi employs to invade cells, extracellular amastigotes have recently gained attention by our group. This is true primarily because these amastigotes are able to infect cultured cells and animals, establishing a sustainable infective cycle. Extracellular amastigotes are thus an excellent means of adaptation and survival for T. cruzi, whose different infective stages each utilize unique mechanisms for attachment and penetration. Here we discuss some features of host cell invasion by extracellular amastigotes and the associated host cell signaling events that occur as part of the process.

  18. 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

  19. From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?

    Science.gov (United States)

    Yamada, Mitsutoshi; Byrne, James; Egli, Dieter

    2015-10-01

    Nuclear transfer has seen a remarkable comeback in the past few years. Three groups have independently reported the derivation of stem cell lines by somatic cell nuclear transfer, from either adult, neonatal or fetal cells. Though the ability of human oocytes to reprogram somatic cells to stem cells had long been anticipated, success did not arrive on a straightforward path. Little was known about human oocyte biology, and nuclear transfer protocols developed in animals required key changes to become effective with human eggs. By overcoming these challenges, human nuclear transfer research has contributed to a greater understanding of oocyte biology, provided a point of reference for the comparison of induced pluripotent stem cells, and delivered a method for the generation of personalized stem cells with therapeutic potential.

  20. The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes

    Science.gov (United States)

    Gamradt, Pia; Xu, Yun; Gratz, Nina; Duncan, Kellyanne; Kobzik, Lester; Högler, Sandra; Decker, Thomas

    2016-01-01

    Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen. PMID:27973535

  1. Helicobacter pylori Infection Induces Genetic Instability of Nuclear and Mitochondrial DNA in Gastric Cells

    DEFF Research Database (Denmark)

    Machado, Ana Manuel; Figueiredo, Ceu; Touati, Eliette;

    2009-01-01

    Purpose: Helicobacter pylori is a major cause of gastric carcinoma. To investigate a possible link between bacterial infection and genetic instability of the host genome, we examined the effect of H. pylori infection on known cellular repair pathways in vitro and in vivo. Moreover, various types...... of genetic instabilities in the nuclear and mitochondrial DNA (mtDNA) were examined. Experimental Design: We observed the effects of H pylori infection on a gastric cell line (AGS), on C57BL/6 mice, and on individuals with chronic gastritis. In AGS cells, the effect of H pylori infection on base excision...... cells and chronic gastritis tissue were determined by PCR, single-stranded conformation polymorphism, and sequencing. H pylori vacA and cagA genotyping was determined by multiplex PCR and reverse hybridization. Results: Following H pylori infection, the activity and expression of base excision repair...

  2. Identification of host proteins associated with HIV-1 preintegration complexes isolated from infected CD4+ cells.

    Science.gov (United States)

    Raghavendra, Nidhanapati K; Shkriabai, Nikolozi; Graham, Robert Lj; Hess, Sonja; Kvaratskhelia, Mamuka; Wu, Li

    2010-08-11

    An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to

  3. Trypanosoma cruzi: Entry into Mammalian Host Cells and Parasitophorous Vacuole Formation

    Science.gov (United States)

    Barrias, Emile Santos; de Carvalho, Tecia Maria Ulisses; De Souza, Wanderley

    2013-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are the metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. The recognition between the parasite and mammalian host cell, involves numerous molecules present in both cell types, and similar to several intracellular pathogens, T. cruzi is internalized by host cells via multiple endocytic pathways. Morphological studies demonstrated that after the interaction of the infective forms of T. cruzi with phagocytic or non-phagocytic cell types, plasma membrane (PM) protrusions can form, showing similarity with those observed during canonical phagocytosis or macropinocytic events. Additionally, several molecules known to be molecular markers of membrane rafts, macropinocytosis, and phagocytosis have been demonstrated to be present at the invasion site. These events may or may not depend on the host cell lysosomes and cytoskeleton. In addition, after penetration, components of the host endosomal-lysosomal system, such as early endosomes, late endosomes, and lysosomes, participate in the formation of the nascent parasitophorous vacuole (PV). Dynamin, a molecule involved in vesicle formation, has been shown to be involved in the PV release from the host cell PM. This review focuses on the multiple pathways that T. cruzi can use to enter the host cells until complete PV formation. We will describe different endocytic processes, such as phagocytosis, macropinocytosis, and endocytosis using membrane microdomains and clathrin-dependent endocytosis and show results that are consistent with their use by this smart parasite. We will also discuss others mechanisms that have been described, such as active penetration and the process that takes advantage of cell membrane wound repair. PMID:23914186

  4. Trypanosoma cruzi: Entry Into Mammalian Host Cells and Parasitophorous Vacuole Formation

    Directory of Open Access Journals (Sweden)

    Emile Santos Barrias

    2013-08-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are the metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. The recognition between the parasite and mammalian host cell, involves numerous molecules present in both cell types, and similar to several intracellular pathogens, T.cruzi is internalized by host cells via multiple endocytic pathways. Morphological studies demonstrated that after the interaction of the infective forms of T.cruzi with phagocytic or non-phagocytic cell types, plasma membrane protrusions can form, showing similarity with those observed during canonical phagocytosis or macropinocytic events. Additionally, several molecules known to be molecular markers of membrane rafts, macropinocytosis and phagocytosis have been demonstrated to be present at the invasion site. These events may or may not depend on the host cell lysosomes and cytoskeleton. In addition, after penetration, components of the host endosomal-lysosomal system, such as early endosomes, late endosomes and lysosomes, participate in the formation of the nascent parasithophorous vacuole (VP. Dynamin, a molecule involved in vesicle formation, has been shown to be involved in the parasitophorous vacuole release from the host cell plasma membrane. This review focuses on the multiple pathways that T.cruzi can use to enter the host cells until complete VP formation. We will describe different endocytic processes, such as phagocytosis, macropinocytosis, endocytosis using membrane microdomains and clathrin-dependent endocytosis and show results that are consistent with their use by this smart parasite. We will also discuss other mechanisms that have been described, such as active penetration and the process that takes advantage of cell membrane wound repair.

  5. Intracellular Theileria annulata promote invasive cell motility through kinase regulation of the host actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Min Ma

    2014-03-01

    Full Text Available The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva or Tropical Theileriosis (T. annulata. These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.

  6. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  7. 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.

  8. Host Cell Factors in Filovirus Entry: Novel Players, New Insights

    Directory of Open Access Journals (Sweden)

    Stefan Pöhlmann

    2012-11-01

    Full Text Available Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1 might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism.

  9. Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells

    Directory of Open Access Journals (Sweden)

    Weikai Zhang

    2011-03-01

    Full Text Available Weikai Zhang1, Ya Zhang1, Marian Löbler2, Klaus-Peter Schmitz2, Aqeel Ahmad3, Ilmari Pyykkö1, Jing Zou11Department of Otolaryngology, University of Tampere, Medical School, Tampere, Finland; 2University of Rostock, Institute for Biomedical Engineering, Rostock, Germany; 3Department of Biomedical Engineering and Computational Science, Aalto University, Espoo, FinlandBackground: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs for gene delivery to cochlear targets.Methods: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats' round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration.Results: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine

  10. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Science.gov (United States)

    Mitchell, Jennifer A; Clay, Ieuan; Umlauf, David; Chen, Chih-Yu; Moir, Catherine A; Eskiw, Christopher H; Schoenfelder, Stefan; Chakalova, Lyubomira; Nagano, Takashi; Fraser, Peter

    2012-01-01

    In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  11. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  12. The role of microsporidian polar tube protein 4 (PTP4 in host cell infection.

    Directory of Open Access Journals (Sweden)

    Bing Han

    2017-04-01

    Full Text Available Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4 from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA, enzyme-linked immunosorbent (ELISA and fluorescence-activated cell sorting (FACS assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1 as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

  13. Cell-fusion method to visualize interphase nuclear pore formation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

    In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods.

  14. Bacterial toxins can inhibit host cell autophagy through cAMP generation.

    Science.gov (United States)

    Shahnazari, Shahab; Namolovan, Anton; Mogridge, Jeremy; Kim, Peter K; Brumell, John H

    2011-09-01

    Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of 'camouflage' proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.

  15. A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors.

    Science.gov (United States)

    Kondorosi, Eva; Mergaert, Peter; Kereszt, Attila

    2013-01-01

    Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.

  16. 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.

  17. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  18. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

    Science.gov (United States)

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-01

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

  19. Alphavirus Infection: Host Cell Shut-Off and Inhibition of Antiviral Responses.

    Science.gov (United States)

    Fros, Jelke J; Pijlman, Gorben P

    2016-06-11

    Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus-host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly.

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

    Science.gov (United States)

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-01-01

    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. PMID:22069713

  1. Cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells.

    Science.gov (United States)

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-04-01

    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.

  2. 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.

  3. IFN-inducible GTPases in host cell defense.

    Science.gov (United States)

    Kim, Bae-Hoon; Shenoy, Avinash R; Kumar, Pradeep; Bradfield, Clinton J; MacMicking, John D

    2012-10-18

    From plants to humans, the ability to control infection at the level of an individual cell-a process termed cell-autonomous immunity-equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell's interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn's colitis.

  4. Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

    Science.gov (United States)

    de Souza, Wanderley; de Carvalho, Tecia M. Ulisses

    2013-01-01

    In the present short review, we analyze past experiments that addressed the interactions of intracellular pathogenic protozoa (Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium) with host cells and the initial use of the term active penetration to indicate that a protozoan “crossed the host cell membrane, penetrating into the cytoplasm.” However, the subsequent use of transmission electron microscopy showed that, for all of the protozoans and cell types examined, endocytosis, classically defined as involving the formation of a membrane-bound vacuole, took place during the interaction process. As a consequence, the recently penetrated parasites are always within a vacuole, designated the parasitophorous vacuole (PV). PMID:23355838

  5. 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.

  6. Stem cell-paved biobridges facilitate stem transplant and host brain cell interactions for stroke therapy.

    Science.gov (United States)

    Duncan, Kelsey; Gonzales-Portillo, Gabriel S; Acosta, Sandra A; Kaneko, Yuji; Borlongan, Cesar V; Tajiri, Naoki

    2015-10-14

    Distinguished by an infarct core encased within a penumbra, stroke remains a primary source of mortality within the United States. While our scientific knowledge regarding the pathology of stroke continues to improve, clinical treatment options for patients suffering from stroke are extremely limited. Tissue plasminogen activator (tPA) remains the sole FDA-approved drug proven to be helpful following stroke. However, due to the need to administer the drug within 4.5h of stroke onset its usefulness is constrained to less than 5% of all patients suffering from ischemic stroke. One experimental therapy for the treatment of stroke involves the utilization of stem cells. Stem cell transplantation has been linked to therapeutic benefit by means of cell replacement and release of growth factors; however the precise means by which this is accomplished has not yet been clearly delineated. Using a traumatic brain injury model, we recently demonstrated the ability of transplanted mesenchymal stromal cells (MSCs) to form a biobridge connecting the area of injury to the neurogenic niche within the brain. We hypothesize that MSCs may also have the capacity to create a similar biobridge following stroke; thereby forming a conduit between the neurogenic niche and the stroke core and peri-infarct area. We propose that this biobridge could assist and promote interaction of host brain cells with transplanted stem cells and offer more opportunities to enhance the effectiveness of stem cell therapy in stroke. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. A host-parasite model for a two-type cell population

    CERN Document Server

    Alsmeyer, Gerold

    2012-01-01

    A host-parasite model is considered for a population of cells that can be of two types, A or B, and exhibits unilateral reproduction: while a B-cell always splits into two cells of the same type, the two daughter cells of an A-cell can be of any type. The random mechanism that describes how parasites within a cell multiply and are then shared into the daughter cells is allowed to depend on the hosting mother cell as well as its daughter cells. Focusing on the subpopulation of A-cells and its parasites, the model differs from the single-type model recently studied by Bansaye (2008) in that the sharing mechanism may be biased towards one of the two types. Main results are concerned with the nonextinctive case and provide information on the behavior, as $n\\to\\infty$, of the number A-parasites in generation n and the relative proportion of A- and B-cells in this generation which host a given number of parasites. As in (Bansaye,2008), proofs will make use of a so-called random cell line which, when conditioned to ...

  8. Enhancement and abrogation : modifications of host immune status influence IL-2 and LAK cell immunotherapy

    NARCIS (Netherlands)

    E.P. Steller (Erick)

    1988-01-01

    textabstractThis thesis will discuss the role immune cells and the host immune system can play in enhancement and abrogation of this novel immunotherapy with interleukin 2 and lymphokine-activated killer cells. Chapter 3 and 4 will discuss the scoring methods in this intraperitoneal cancer and immun

  9. Ammonium secretion by Colletotrichum coccodes activates host NADPH oxidase activity enhancing host cell death and fungal virulence in tomato fruits.

    Science.gov (United States)

    Alkan, Noam; Davydov, Olga; Sagi, Moshe; Fluhr, Robert; Prusky, Dov

    2009-12-01

    Colletotrichum pathogens of fruit and leaves are known ammonium secretors. Here, we show that Colletotrichum coccodes virulence, as measured by tomato (Solanum lycopersicum cv. Motelle) fruit tissue necrosis, correlates with the amount of ammonium secreted. Ammonium application to fruit tissue induced hydrogen peroxide (H(2)O(2)) accumulation. To examine whether the tomato NADPH oxidase, SlRBOH, is a source for the ammonium-induced H(2)O(2), wild-type and antisense lines abrogated for SlRBOH (SlRBOH-AS) were examined. Wild-type lines produced 7.5-fold more reactive oxygen species when exposed to exogenous ammonium than did SlRBOH-AS lines. C. coccodes colonization of wild-type tomato lines resulted in higher H(2)O(2) production and faster fungal growth rate compared with colonization in the SlRBOH-AS mutant, although the amount of ammonium secreted by the fungi was similar in both cases. Enhanced ion leakage and cell death of fruit tissue were correlated with H(2)O(2) accumulation, and treatment with the reactive oxygen scavenger N-acetyl-l-cysteine decreased H(2)O(2) production, ion leakage, and cell death. Importantly, the activation of reactive oxygen species production by ammonium was positively affected by an extracellular pH increase from 4 to 9, implying that ammonium exerts its control via membrane penetration. Our results show that C. coccodes activates host reactive oxygen species and H(2)O(2) production through ammonium secretion. The resultant enhancement in host tissue decay is an important step in the activation of the necrotrophic process needed for colonization.

  10. Hosting the plant cells in vitro: recent trends in bioreactors.

    Science.gov (United States)

    Georgiev, Milen I; Eibl, Regine; Zhong, Jian-Jiang

    2013-05-01

    Biotechnological production of high-value metabolites and therapeutic proteins by plant in vitro systems has been considered as an attractive alternative of classical technologies. Numerous proof-of-concept studies have illustrated the feasibility of scaling up plant in vitro system-based processes while keeping their biosynthetic potential. Moreover, several commercial processes have been established so far. Though the progress on the field is still limited, in the recent years several bioreactor configurations has been developed (e.g., so-called single-use bioreactors) and successfully adapted for growing plant cells in vitro. This review highlights recent progress and limitations in the bioreactors for plant cells and outlines future perspectives for wider industrialization of plant in vitro systems as "green cell factories" for sustainable production of value-added molecules.

  11. A combined gas cooled nuclear reactor and fuel cell cycle

    Science.gov (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  12. 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

  13. Somatic cell nuclear transfer in horses.

    Science.gov (United States)

    Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna

    2008-07-01

    The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.

  14. 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

  15. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    Science.gov (United States)

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

    2015-01-01

    ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural

  16. Memory CD4+ T cells do not induce graft-versus-host disease.

    Science.gov (United States)

    Anderson, Britt E; McNiff, Jennifer; Yan, Jun; Doyle, Hester; Mamula, Mark; Shlomchik, Mark J; Shlomchik, Warren D

    2003-07-01

    Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). Donor T cells that accompany stem cell grafts cause GVHD by attacking recipient tissues; therefore, all patients receive GVHD prophylaxis by depletion of T cells from the allograft or through immunosuppressant drugs. In addition to providing a graft-versus-leukemia effect, donor T cells are critical for reconstituting T cell-mediated immunity. Ideally, immunity to infectious agents would be transferred from donor to host without GVHD. Most donors have been exposed to common pathogens and have an increased precursor frequency of memory T cells against pathogenic antigens. We therefore asked whether memory CD62L-CD44+ CD4+ T cells would induce less GVHD than unfractionated or naive CD4+ T cells. Strikingly, we found that memory CD4 cells induced neither clinical nor histologic GVHD. This effect was not due to the increased number of CD4+CD25+ regulatory T cells found in the CD62L-CD44+ fraction because memory T cells depletion of these cells did not cause GVHD. Memory CD4 cells engrafted and responded to antigen both in vivo and in vitro. If these murine results are applicable to human alloSCT, selective administration of memory T cells could greatly improve post-transplant immune reconstitution.

  17. Dynamic Behavior and Function of Foxp3+ Regulatory T Cells in Tumor Bearing Host

    Institute of Scientific and Technical Information of China (English)

    F. Xiao-Feng Qin

    2009-01-01

    Regulatory T cells (Tregs) expressing forkhead/winged-helix transcription factor Foxp3 represent a distinct lineage of lymphocytes which play a central role in protecting the host from autoimmune diseases. However, Tregs also pose a major problem to anti-tumor immunity. Growing body of evidence from both laboratory and clinical investigations has demonstrated that expansion and accumulation of these immunosuppressive cells correlates with advanced tumor growth and predicts poor disease prognosis. How tumor development subverts normal self-tolerance function of Tregs thereby thwarts host anti-tumor immunity remains elusive. This review will discuss our current knowledge in understanding the dynamics and plasticity of Foxp3+ Treg activation and induction in tumor bearing hosts and their interaction with various antigen presenting cells (APCs) in tumor microenvironment leading to the establishment of active local and systemic immune suppression.

  18. 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.

  19. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

    Science.gov (United States)

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

    2016-11-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

  20. African swine fever virus uses macropinocytosis to enter host cells.

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    Elena G Sánchez

    Full Text Available African swine fever (ASF is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV, which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V, and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+/H(+ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

  1. Adult human mesenchymal stromal cells and the treatment of graft versus host disease

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    Herrmann RP

    2014-02-01

    Full Text Available Richard P Herrmann, Marian J Sturm Cell and Tissue Therapies, Western Australia, Royal Perth Hospital, Wellington Street, Perth, WA, Australia Abstract: Graft versus host disease is a difficult and potentially lethal complication of hematopoietic stem cell transplantation. It occurs with minor human leucocyte antigen (HLA mismatch and is normally treated with corticosteroid and other immunosuppressive therapy. When it is refractory to steroid therapy, mortality approaches 80%. Mesenchymal stromal cells are rare cells found in bone marrow and other tissues. They can be expanded in culture and possess complex and diverse immunomodulatory activity. Moreover, human mesenchymal stromal cells carry low levels of class 1 and no class 2 HLA antigens, making them immunoprivileged and able to be used without HLA matching. Their use in steroid-refractory graft versus host disease was first described in 2004. Subsequently, they have been used in a number of Phase I and II trials in acute and chronic graft versus host disease trials with success. We discuss their mode of action, the results, their production, and potential dangers with a view to future application. Keywords: mesenchymal stromal cells, graft versus host disease, acute, chronic

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

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    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.

  3. Cloning of ES cells and mice by nuclear transfer.

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    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  4. Recent advancements in cloning by somatic cell nuclear transfer.

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    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  5. A genetic screen to isolate Toxoplasma gondii host-cell egress mutants.

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    Coleman, Bradley I; Gubbels, Marc-Jan

    2012-02-08

    The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca(2+), a signal that is also critical for host cell invasion. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology, only recently has genetic mapping of mutations underlying the phenotypes become routine. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants

  6. Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

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    Tecia Maria Ulisses Carvalho

    2013-01-01

    Full Text Available A significant number of scientific groups working on several countries have made efforts to better understand the process of invasion of several types of host cells by Trypanosoma cruzi, the etiologic agent of Chagas disease. In this mini-review we analyze the two mechanisms of invasion considered to be relevant: active penetration and endocytosis. The term active penetration is considered in view of its original description by Dvorak and co-workers. Taking into consideration all results obtained we conclude that endocytosis, with its many variations, is the only mechanism used by T. cruzi to invade host cells.

  7. Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

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    João Antonio Chaves de Souza

    2012-04-01

    Full Text Available Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.

  8. Know your neighbor: Microbiota and host epithelial cells interact locally to control intestinal function and physiology.

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    Sommer, Felix; Bäckhed, Fredrik

    2016-05-01

    Interactions between the host and its associated microbiota differ spatially and the local cross talk determines organ function and physiology. Animals and their organs are not uniform but contain several functional and cellular compartments and gradients. In the intestinal tract, different parts of the gut carry out different functions, tissue structure varies accordingly, epithelial cells are differentially distributed and gradients exist for several physicochemical parameters such as nutrients, pH, or oxygen. Consequently, the microbiota composition also differs along the length of the gut, but also between lumen and mucosa of the same intestinal segment, and even along the crypt-villus axis in the epithelium. Thus, host-microbiota interactions are highly site-specific and the local cross talk determines intestinal function and physiology. Here we review recent advances in our understanding of site-specific host-microbiota interactions and discuss their functional relevance for host physiology.

  9. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    Science.gov (United States)

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.

  10. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

    Science.gov (United States)

    Sateriale, Adam; Miller, Peter

    2016-01-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  11. ERK5 and cell proliferation: nuclear localization is what matters

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    Nestor Gomez

    2016-09-01

    Full Text Available ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumour growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote

  12. Nuclear lamins and oxidative stress in cell proliferation and longevity.

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    Shimi, Takeshi; Goldman, Robert D

    2014-01-01

    In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidative stress. The major cause of oxidative stress is the production of reactive oxygen species (ROS), which is critically important for cell proliferation and longevity. Moderate increases in ROS act to initiate signaling pathways involved in cell proliferation and differentiation, whereas excessive increases in ROS cause oxidative stress, which in turn induces cell death and/or senescence. In this review, we cover current findings about the role of lamins in regulating cell proliferation and longevity through oxidative stress responses and ROS signaling pathways. We also speculate on the involvement of lamins in tumor cell proliferation through the control of ROS metabolism.

  13. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

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    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  14. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

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    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  15. The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

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    David Hare

    2016-11-01

    Full Text Available Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.

  16. IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

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    Copenhaver, Alan M.; Casson, Cierra N.; Nguyen, Hieu T.; Duda, Matthew M.; Shin, Sunny

    2015-01-01

    The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response. PMID:26034289

  17. Adaptation of HIV-1 Depends on the Host-Cell Environment

    Science.gov (United States)

    van Opijnen, Tim; de Ronde, Anthony; Boerlijst, Maarten C.; Berkhout, Ben

    2007-01-01

    Many viruses have the ability to rapidly develop resistance against antiviral drugs and escape from the host immune system. To which extent the host environment affects this adaptive potential of viruses is largely unknown. Here we show that for HIV-1, the host-cell environment is key to the adaptive potential of the virus. We performed a large-scale selection experiment with two HIV-1 strains in two different T-cell lines (MT4 and C8166). Over 110 days of culture, both virus strains adapted rapidly to the MT4 T-cell line. In contrast, when cultured on the C8166 T-cell line, the same strains did not show any increase in fitness. By sequence analyses and infections with viruses expressing either yellow or cyan fluorescent protein, we were able to show that the absence of adaptation was linked to a lower recombination rate in the C8166 T-cell line. Our findings suggest that if we can manipulate the host-cellular factors that mediate viral evolution, we may be able to significantly retard viral adaptability. PMID:17342205

  18. Signalome-wide assessment of host cell response to hepatitis C virus.

    Science.gov (United States)

    Haqshenas, Gholamreza; Wu, Jianmin; Simpson, Kaylene J; Daly, Roger J; Netter, Hans J; Baumert, Thomas F; Doerig, Christian

    2017-05-08

    Host cell signalling during infection with intracellular pathogens remains poorly understood. Here we report on the use of antibody microarray technology to detect variations in the expression levels and phosphorylation status of host cell signalling proteins during hepatitis C virus (HCV) replication. Following transfection with HCV RNA, the JNK and NF-κB pathways are suppressed, while the JAK/STAT5 pathway is activated; furthermore, components of the apoptosis and cell cycle control machineries are affected in the expression and/or phosphorylation status. RNAi-based hit validation identifies components of the JAK/STAT, NF-κB, MAPK and calcium-induced pathways as modulators of HCV replication. Selective chemical inhibition of one of the identified targets, the JNK activator kinase MAP4K2, does impair HCV replication. Thus this study provides a comprehensive picture of host cell pathway mobilization by HCV and uncovers potential therapeutic targets. The strategy of identifying targets for anti-infective intervention within the host cell signalome can be applied to any intracellular pathogen.

  19. Obtaining control of cell surface functionalizations via Pre-targeting and Supramolecular host guest interactions.

    Science.gov (United States)

    Rood, Mark T M; Spa, Silvia J; Welling, Mick M; Ten Hove, Jan Bart; van Willigen, Danny M; Buckle, Tessa; Velders, Aldrik H; van Leeuwen, Fijs W B

    2017-01-06

    The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or "training" of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; KD = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

  20. Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells.

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    Younes Smani

    Full Text Available Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs were identified as fibronectin binding proteins (FBPs: OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells. Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells.

  1. Mg2Si As Li-Intercalation Host For Li Cells

    Science.gov (United States)

    Huang, Chen-Kuo; Surampudi, Subbarao; Attia, Alan; Halpert, Gerald

    1993-01-01

    Compound Mg2Si shows promise as lithium-intercalation host for ambient-temperature rechargeable lithium electrochemical cells. As anode reactant material, LiXMg2Si chemically stable in presence of organic electrolyte used in such cells and stores large amounts of lithium. Intercalation reactions highly reversible at room temperature. Also retains sufficient mechanical strength during charge/discharge cycling. Lithium cells containing LixMg2Si anodes prove useful in spacecraft, military, communications, automotive, and other applications in which high energy-storage densities of lithium cells in general and rechargeability of cells needed.

  2. Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

    Science.gov (United States)

    Long, Charles R; Westhusin, Mark E; Golding, Michael C

    2014-02-01

    Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

  3. NUCLEAR REPROGRAMMING AND ITS EPIGENETIC CONSEQUENCES IN SOMATIC CELL NUCLEAR TRANSFER DERIVED ANIMALS

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    Meirelles FV

    2015-12-01

    Full Text Available Somatic cell nuclear transfer (SCNT is a powerful tool to understand the mechanisms driving the cellular reprogramming, as well as addressing fundamental questions on developmental biology. Hundreds of animals have already been generated through this technology, however its large-scale use is still hampered by the low efficiency and disrupted phenotypes reported in SCNT derived newborns. Therefore, this review aims to expose some of the consequences often seen in cloned animals, as well as to discuss and propose some possibilities and perspectives to avoid epigenetic failures during reprogramming.

  4. Nuclear microprobe imaging of gallium nitrate in cancer cells

    Science.gov (United States)

    Ortega, Richard; Suda, Asami; Devès, Guillaume

    2003-09-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 μm diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material.

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

    Directory of Open Access Journals (Sweden)

    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

  6. Cloned calves produced by nuclear transfer from cultured cumulus cells

    Institute of Scientific and Technical Information of China (English)

    AN; Xiaorong(安晓荣); GOU; Kemian(苟克勉); ZHU; Shien(朱士恩); GUAN; Hong(关宏); HOU; Jian(侯健); LIN; Aixing(林爱星); ZENG; Shenming(曾申明); TIAN; Jianhui(田见辉); CHEN; Yongfu(陈永福)

    2002-01-01

    Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

  7. Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

    Science.gov (United States)

    Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric

    2017-01-01

    ABSTRACT   Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. PMID:28119472

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

    Directory of Open Access Journals (Sweden)

    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. In situ tissue engineering of functional small-diameter blood vessels by host circulating cells only

    NARCIS (Netherlands)

    Talacua, Hanna; Smits, Anthal I P M; Muylaert, Dimitri E P; Van Rijswijk, Jan Willem; Vink, Aryan; Verhaar, Marianne C.; Driessen-Mol, Anita; Van Herwerden, Lex A.; Bouten, Carlijn V C; Kluin, Jolanda; Baaijens, Frank P T

    2015-01-01

    Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent

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

    Science.gov (United States)

    Dirk, Brennan S.; Van Nynatten, Logan R.; Dikeakos, Jimmy D.

    2016-01-01

    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. PMID:27775563

  11. In situ tissue engineering of functional small-diameter blood vessels by host circulating cells only

    NARCIS (Netherlands)

    Talacua, Hanna; Smits, Anthal I P M; Muylaert, Dimitri E P; Van Rijswijk, Jan Willem; Vink, Aryan; Verhaar, Marianne C.; Driessen-Mol, Anita; Van Herwerden, Lex A.; Bouten, Carlijn V C; Kluin, Jolanda; Baaijens, Frank P T

    2015-01-01

    Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent

  12. Intracellular accommodation of rhizobia in legume host cell: the fine-tuning of the endomembrane system

    NARCIS (Netherlands)

    Gavrin, A.Y.

    2015-01-01

    The symbiosis of legumes with rhizobia leads to the formation of root nodules. Rhizobia which are hosted inside specialized infected cells are surrounded by hostderived membranes, forming symbiosomes. Although it is known that symbiosome formation involves proliferation of membranes and changing of

  13. Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

    Energy Technology Data Exchange (ETDEWEB)

    Schnorr, Kirk; Kramer, Randall

    2017-03-28

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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 heat-inact

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

    Science.gov (United States)

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D; Mörgelin, Matthias; Karpman, Diana

    2015-02-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry.

    Science.gov (United States)

    Schmid, Maximilian; Dufner, Bianca; Dürk, Julius; Bedal, Konstanze; Stricker, Kristina; Prokoph, Lukas Ali; Koch, Christoph; Wege, Anja K; Zirpel, Henner; van Zandbergen, Ger; Ecker, Rupert; Boghiu, Bogdan; Ritter, Uwe

    2015-01-01

    Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.

  20. Novel insights into host-fungal pathogen interactions derived from live-cell imaging.

    Science.gov (United States)

    Bain, Judith; Gow, Neil A R; Erwig, Lars-Peter

    2015-03-01

    The theoretical physicist and Nobel laureate Richard Feynman outlined in his 1959 lecture, "There's plenty of room at the bottom", the enormous possibility of producing and visualising things at smaller scales. The advent of advanced scanning and transmission electron microscopy and high-resolution microscopy has begun to open the door to visualise host-pathogen interactions at smaller scales, and spinning disc confocal and two-photon microscopy has improved our ability to study these events in real time in three dimensions. The aim of this review is to illustrate some of the advances in understanding host-fungal interactions that have been made in recent years in particular those relating to the interactions of live fungal pathogens with phagocytes. Dynamic imaging of host-pathogen interactions has recently revealed novel detail and unsuspected mechanistic insights, facilitating the dissection of the phagocytic process into its component parts. Here, we will highlight advances in our knowledge of host-fungal pathogen interactions, including the specific effects of fungal cell viability, cell wall composition and morphogenesis on the phagocytic process and try to define the relative contributions of neutrophils and macrophages to the clearance of fungal pathogens in vitro and the infected host.

  1. Conventional NK cells can produce IL-22 and promote host defense in Klebsiella pneumoniae pneumonia.

    Science.gov (United States)

    Xu, Xin; Weiss, Ido D; Zhang, Hongwei H; Singh, Satya P; Wynn, Thomas A; Wilson, Mark S; Farber, Joshua M

    2014-02-15

    It was reported that host defense against pulmonary Klebsiella pneumoniae infection requires IL-22, which was proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22(-/-) mice had decreased survival compared with wild-type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2(-/-) mice did not differ from wild-type mice in survival or levels of IL-22 in the lungs postinfection with K. pneumoniae. In contrast, K. pneumoniae-infected Rag2(-/-)Il2rg(-/-) mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells in host defense and production of IL-22. Unlike NK cell-like innate lymphoid cells that produce IL-22 and display a surface phenotype of NK1.1(-)NKp46(+)CCR6(+), lung NK cells showed the conventional phenotype, NK1.1(+)NKp46(+)CCR6(-). Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver, compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within 1 d postinfection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild-type and Rag2(-/-) mice. Our data suggest that, during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.

  2. Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis.

    Science.gov (United States)

    Penterman, Jon; Abo, Ryan P; De Nisco, Nicole J; Arnold, Markus F F; Longhi, Renato; Zanda, Matteo; Walker, Graham C

    2014-03-04

    The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called "nodule-specific cysteine-rich" (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA, and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

  3. Regulatory T-Cell Therapy for Graft-versus-host Disease

    OpenAIRE

    Heinrichs, Jessica; Bastian, David; Veerapathran, Anandharaman; Anasetti, Claudio; Betts, Brain; Yu, Xue-Zhong

    2016-01-01

    Graft-versus-host disease (GVHD) is a significant cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Existing strategies to prevent and treat GVHD are incomplete, where a significant portion of allo-HCT recipients developed this complication. Despite this, one such therapy has emerged involving the use of regulatory T cells (Tregs) to control GVHD. The use of natural Tregs (nTregs) yielded positive pre-clinical results and are actively under investi...

  4. Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

    Directory of Open Access Journals (Sweden)

    Stephanie N Bartley

    Full Text Available We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

  5. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    Science.gov (United States)

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  6. TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm.

    Science.gov (United States)

    De Iaco, Alberto; Santoni, Federico; Vannier, Anne; Guipponi, Michel; Antonarakis, Stylianos; Luban, Jeremy

    2013-02-15

    Despite intensive investigation the mechanism by which HIV-1 reaches the host cell nucleus is unknown. TNPO3, a karyopherin mediating nuclear entry of SR-proteins, was shown to be required for HIV-1 infectivity. Some investigators have reported that TNPO3 promotes HIV-1 nuclear import, as would be expected for a karyopherin. Yet, an equal number of investigators have failed to obtain evidence that supports this model. Here, a series of experiments were performed to better elucidate the mechanism by which TNPO3 promotes HIV-1 infectivity. To examine the role of TNPO3 in HIV-1 replication, the 2-LTR circles that are commonly used as a marker for HIV-1 nuclear entry were cloned after infection of TNPO3 knockdown cells. Potential explanation for the discrepancy in the literature concerning the effect of TNPO3 was provided by sequencing hundreds of these clones: a significant fraction resulted from autointegration into sites near the LTRs and therefore were not bona fide 2-LTR circles. In response to this finding, new techniques were developed to monitor HIV-1 cDNA, including qPCR reactions that distinguish 2-LTR circles from autointegrants, as well as massive parallel sequencing of HIV-1 cDNA. With these assays, TNPO3 knockdown was found to reduce the levels of 2-LTR circles. This finding was puzzling, though, since previous work has shown that the HIV-1 determinant for TNPO3-dependence is capsid (CA), an HIV-1 protein that forms a mega-dalton protein lattice in the cytoplasm. TNPO3 imports cellular splicing factors via their SR-domain. Attention was therefore directed towards CPSF6, an SR-protein that binds HIV-1 CA and inhibits HIV-1 nuclear import when the C-terminal SR-domain is deleted. The effect of 27 HIV-1 capsid mutants on sensitivity to TNPO3 knockdown was then found to correlate strongly with sensitivity to inhibition by a C-terminal deletion mutant of CPSF6 (R2 = 0.883, p HIV-1 replication. Additionally, targeting CPSF6 to the nucleus by fusion to a

  7. IgE and mast cells in host defense against parasites and venoms.

    Science.gov (United States)

    Mukai, Kaori; Tsai, Mindy; Starkl, Philipp; Marichal, Thomas; Galli, Stephen J

    2016-09-01

    IgE-dependent mast cell activation is a major effector mechanism underlying the pathology associated with allergic disorders. The most dramatic of these IgE-associated disorders is the fatal anaphylaxis which can occur in some people who have developed IgE antibodies to otherwise innocuous antigens, such as those contained in certain foods and medicines. Why would such a highly "maladaptive" immune response develop in evolution and be retained to the present day? Host defense against parasites has long been considered the only beneficial function that might be conferred by IgE and mast cells. However, recent studies have provided evidence that, in addition to participating in host resistance to certain parasites, mast cells and IgE are critical components of innate (mast cells) and adaptive (mast cells and IgE) immune responses that can enhance host defense against the toxicity of certain arthropod and animal venoms, including enhancing the survival of mice injected with such venoms. Yet, in some people, developing IgE antibodies to insect or snake venoms puts them at risk for having a potentially fatal anaphylactic reaction upon subsequent exposure to such venoms. Delineating the mechanisms underlying beneficial versus detrimental innate and adaptive immune responses associated with mast cell activation and IgE is likely to enhance our ability to identify potential therapeutic targets in such settings, not only for reducing the pathology associated with allergic disorders but perhaps also for enhancing immune protection against pathogens and animal venoms.

  8. Nuclear morphometry in canine acanthomatous ameloblastomas and squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    M Martano

    2009-06-01

    Full Text Available The aim of this study was to evaluate whether morphometrical analysis can be of diagnostic value for canine acanthomatous ameloblastoma. We calculated, by means of an automated image analyser, some morphometric nuclear parameters, in particular: mean nuclear area (MNA, mean nuclear perimeter (MNP, maximum and minimum diameters (MDx and MDm coefficient of variation of the nuclear area (NACV, largest to smallest dimension ratio (LS ratio, and form factor (FF, in 8 canine acanthomatous ameloblastomas, and we compared these morphometric data to those of 13 squamous cell carcinomas of canine gingiva. The results indicated a progressive increase of the MNA, NACV, MNP and MDm proceeding from acanthomatous ameloblastomas (MNA: 42.11±8.74; NACV: 28,36±7,23; MNP: 24.18± 2.68; MDm: 5.69±0.49 to squamous cell carcinomas (MNA:49,69±9,10; NACV: 30,89±7,75; MNP: 25.63±2.54; MDm: 6.64±0.73. On the contrary, the LS ratio and the FF resulted greater in acanthomatous ameloblastomas (LS ratio: 1,63±0,12; FF: 1,13±0,002 than in SCCs (LS ratio: 1,40±0,12; FF:0.91±0.38. Moreover, the MNA, MNP,MDx and MDm resulted similar (MNA: p=0.89; MNP: p=0,65; MDm: p=0,16; MDx: p=0,13 in a subset of four acanthomatous ameloblastomas with cellular atypia (MNA:49,01±6,88; MNP: 26,28±1,99; MDm: 6.08±0.41; MDx: 10.18±0.88 and in squamous cell carcinomas (MNA:49.69±9,10; MNP: 25.63±2.54; MDm: 6.64±0.73; MDx: 9.26±1.05. While the NACV values resulted higher in typical acanthomatous ameloblastoma (29,99±6,06 than in atypical acanthomatous ameloblastoma (26,74±8,84 and similar to those of the SCCs (30,89±7,75. These results seem to confirm that acanthomatous ameloblastoma is a malignant or potentially malignant lesion and emphasizes that nuclear morphometry analysis can be an useful diagnostic and prognostic method in canine oral pathology.

  9. Serratia marcescens induces apoptotic cell death in host immune cells via a lipopolysaccharide- and flagella-dependent mechanism.

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

    2012-10-19

    Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH(2)-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.

  10. Nuclear Membrane-Targeted Gold Nanoparticles Inhibit Cancer Cell Migration and Invasion.

    Science.gov (United States)

    Ali, Moustafa R K; Wu, Yue; Ghosh, Deepraj; Do, Brian H; Chen, Kuangcai; Dawson, Michelle R; Fang, Ning; Sulchek, Todd A; El-Sayed, Mostafa A

    2017-03-27

    Most cancer patients die from metastasis. Recent studies have shown that gold nanoparticles (AuNPs) can slow down the migration/invasion speed of cancer cells and suppress metastasis. Since nuclear stiffness of the cell largely decreases cell migration, our hypothesis is that targeting AuNPs to the cell nucleus region could enhance nuclear stiffness, and therefore inhibit cell migration and invasion. Our results showed that upon nuclear targeting of AuNPs, the ovarian cancer cell motilities decrease significantly, compared with nontargeted AuNPs. Furthermore, using atomic force microscopy, we observed an enhanced cell nuclear stiffness. In order to understand the mechanism of cancer cell migration/invasion inhibition, the exact locations of the targeted AuNPs were clearly imaged using a high-resolution three-dimensional imaging microscope, which showed that the AuNPs were trapped at the nuclear membrane. In addition, we observed a greatly increased expression level of lamin A/C protein, which is located in the inner nuclear membrane and functions as a structural component of the nuclear lamina to enhance nuclear stiffness. We propose that the AuNPs that are trapped at the nuclear membrane both (1) add to the mechanical stiffness of the nucleus and (2) stimulate the overexpression of lamin A/C located around the nuclear membrane, thus increasing nuclear stiffness and slowing cancer cell migration and invasion.

  11. A Coevolutionary Arms Race between Hosts and Viruses Drives Polymorphism and Polygenicity of NK Cell Receptors.

    Science.gov (United States)

    Carrillo-Bustamante, Paola; Keşmir, Can; de Boer, Rob J

    2015-08-01

    Natural killer cell receptors (NKRs) monitor the expression of major histocompatibility class I (MHC-I) and stress molecules to detect unhealthy tissue, such as infected or tumor cells. The NKR gene family shows a remarkable genetic diversity, containing several genes encoding receptors with activating and inhibiting signaling, and varying in gene content and allelic polymorphism. The expansion of the NKR genes is species-specific, with different species evolving alternative expanded NKR genes, which encode structurally different proteins, yet perform comparable functions. So far, the biological function of this expansion within the NKR cluster has remained poorly understood. To study the evolution of NKRs, we have developed an agent-based model implementing a coevolutionary scenario between hosts and herpes-like viruses that are able to evade the immune response by downregulating the expression of MHC-I on the cell surface. We show that hosts evolve specific inhibitory NKRs, specialized to particular MHC-I alleles in the population. Viruses in our simulations readily evolve proteins mimicking the MHC molecules of their host, even in the absence of MHC-I downregulation. As a result, the NKR locus becomes polygenic and polymorphic, encoding both specific inhibiting and activating receptors to optimally protect the hosts from coevolving viruses.

  12. Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

    Science.gov (United States)

    Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

    2014-04-01

    Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants.

  13. The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis.

    Science.gov (United States)

    Chavez, Carolina Varela; Jubelin, Grégory; Courties, Gabriel; Gomard, Aurélie; Ginibre, Nadège; Pages, Sylvie; Taïeb, Frédéric; Girard, Pierre-Alain; Oswald, Eric; Givaudan, Alain; Zumbihl, Robert; Escoubas, Jean-Michel

    2010-12-01

    Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G(2)/M phase transition in human cell lines. We report here the first direct functional analysis of Cif(Pl), from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif(Pl) gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif(Pl) was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif(Pl) inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G(2)/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif(Pl) catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.

  14. Role of nuclear receptors in breast cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Alessio; Papi; Marina; Orlandi

    2016-01-01

    The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.

  15. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    Science.gov (United States)

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

  16. Host and viral factors contributing to CD8+ T cell failure in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Christoph Neumann-Haefelin; Hans Christian Spangenberg; Hubert E Blum; Robert Thimme

    2007-01-01

    Virus-specific CD8+ T cells are thought to be the major anti-viral effector cells in hepatitis C virus (HCV)infection. Indeed, viral clearance is associated with vigorous CD8+ T cell responses targeting multiple epitopes. In the chronic phase of infection, HCV-specific CD8+ T cell responses are usually weak, narrowly focused and display often functional defects regarding cytotoxicity, cytokine production, and proliferative capacity. In the last few years, different mechanisms which might contribute to the failure of HCV-specific CD8+ T cells in chronic infection have been identified,including insufficient CD4+ help, deficient CD8+ T cell differentiation, viral escape mutations, suppression by viral factors, inhibitory cytokines, inhibitory ligands, and regulatory T cells. In addition, host genetic factors such as the host's human leukocyte antigen (HLA) background may play an important role in the efficiency of the HCVspecific CD8+ T cell response and thus outcome of infection. The growing understanding of the mechanisms contributing to T cell failure and persistence of HCV infection will contribute to the development of successful immunotherapeutical and -prophylactical strategies.

  17. Coxiella burnetii Infects Primary Bovine Macrophages and Limits Their Host Cell Response.

    Science.gov (United States)

    Sobotta, Katharina; Hillarius, Kirstin; Mager, Marvin; Kerner, Katharina; Heydel, Carsten; Menge, Christian

    2016-06-01

    Although domestic ruminants have long been recognized as the main source of human Q fever, little is known about the lifestyle that the obligate intracellular Gram-negative bacterium Coxiella burnetii adopts in its animal host. Because macrophages are considered natural target cells of the pathogen, we established primary bovine monocyte-derived macrophages (MDM) as an in vitro infection model to study reservoir host-pathogen interactions at the cellular level. In addition, bovine alveolar macrophages were included to take cell type peculiarities at a host entry site into account. Cell cultures were inoculated with the virulent strain Nine Mile I (NMI; phase I) or the avirulent strain Nine Mile II (NMII; phase II). Macrophages from both sources internalized NMI and NMII. MDM were particularly permissive for NMI internalization, but NMI and NMII replicated with similar kinetics in these cells. MDM responded to inoculation with a general upregulation of Th1-related cytokines such as interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha (TNF-α) early on (3 h postinfection). However, inflammatory responses rapidly declined when C. burnetii replication started. C. burnetii infection inhibited translation and release of IL-1β and vastly failed to stimulate increased expression of activation markers, such as CD40, CD80, CD86, and major histocompatibility complex (MHC) molecules. Such capability of limiting proinflammatory responses may help Coxiella to protect itself from clearance by the host immune system. The findings provide the first detailed insight into C. burnetii-macrophage interactions in ruminants and may serve as a basis for assessing the virulence and the host adaptation of C. burnetii strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. The CSN hosts the Second International Conference on Nuclear Security in Madrid; El CSN acoge en Madrid la Segunda Conferencia Internacional de Seguridad Fisica Nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Scialdone Garcia, A.

    2016-08-01

    The second International Conference on Nuclear Security was held in Madrid between May 9th and 13th last. The acting Minister of the Interior announced that the number of officers of the Guardia Civil watching over the Spanish nuclear power plants will be doubled. (Author)

  19. Applications of somatic cell nuclear transfer in goats

    Directory of Open Access Journals (Sweden)

    Ton Yoisungnern

    2014-06-01

    Full Text Available A number of animals with genetically identical appearance can be produced by somatic cell nuclear transfer (SCNT. From current advancement of SCNT and molecular techniques, production of a transgenic animal becomes easier. Although cloning efficiency in goat is low, the ability to propagate genetically identical animals, with a gene or genes of interest, would be important for increasing productivity and ultimately the economic livelihood. In this paper, the potential applications and uses of SCNT technology like production of transgenic goat for production of quality milk and meat are discussed.

  20. Different serotypes of dengue viruses differently regulate the expression of the host cell antigen processing machinery.

    Science.gov (United States)

    Gan, Chye Sheng; Yusof, Rohana; Othman, Shatrah

    2015-09-01

    Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

  1. 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.

  2. Comparative Analysis of Host Cell Entry of Ebola Virus From Sierra Leone, 2014, and Zaire, 1976.

    Science.gov (United States)

    Hofmann-Winkler, Heike; Gnirß, Kerstin; Wrensch, Florian; Pöhlmann, Stefan

    2015-10-01

    The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors.

  3. Leishmania Interferes with Host Cell Signaling to Devise a Survival Strategy

    Directory of Open Access Journals (Sweden)

    Suvercha Bhardwaj

    2010-01-01

    Full Text Available The protozoan parasite Leishmania spp. exists as extracellular promastigotes in its vector whereas it resides and replicates as amastigotes within the macrophages of its mammalian host. As a survival strategy, Leishmania modulates macrophage functions directly or indirectly. The direct interference includes prevention of oxidative burst and the effector functions that lead to its elimination. The indirect effects include the antigen presentation and modulation of T cell functions in such a way that the effector T cells help the parasite survive by macrophage deactivation. Most of these direct and indirect effects are regulated by host cell receptor signaling that occurs through cycles of phosphorylation and dephosphorylation in cascades of kinases and phosphatases. This review highlights how Leishmania selectively manipulates the different signaling pathways to ensure its survival.

  4. Biochemical and cellular mechanisms regulating Acanthamoeba castellanii adherence to host cells.

    Science.gov (United States)

    Soto-Arredondo, K J; Flores-Villavicencio, L L; Serrano-Luna, J J; Shibayama, M; Sabanero-López, M

    2014-04-01

    Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.

  5. Chlamydia inhibit host cell apoptosis by degradation of proapoptotic BH3-only proteins.

    Science.gov (United States)

    Fischer, Silke F; Vier, Juliane; Kirschnek, Susanne; Klos, Andreas; Hess, Simone; Ying, Songmin; Häcker, Georg

    2004-10-04

    Chlamydia are obligate intracellular bacteria that replicate in a vacuole inside a host cell. Chlamydial infection has been shown to protect the host cell against apoptotic stimuli. This is likely important for the ability of Chlamydia to reproduce in human cells. Here we show that resistance to apoptosis is conveyed by the destruction of the proapoptotic BH3-only proteins Bim/Bod, Puma, and Bad during infection. Apoptotic stimuli were blocked upstream of the mitochondrial activation of Bax/Bak. During infection with both species, Chlamydia trachomatis and Chlamydia pneumoniae, Bim protein gradually disappeared without noticeable changes in Bim mRNA. The disappearance was blocked by inhibitors of the proteasome. Infected cells retained sensitivity to Bim expressed by transfection, indicating functional relevance of the Bim disappearance. Fusion to Bim targeted the green fluorescent protein for destruction during infection. Analysis of truncation mutants showed that a short region of Bim containing the BH3 domain was sufficient for destruction during chlamydial infection. Like Bim, Puma and Bad proteins disappeared during infection. These results reveal a novel way by which microbes can interfere with the host cell's apoptotic machinery, and provide a molecular explanation of the cellular resistance to apoptosis during infection with Chlamydia.

  6. Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus.

    Science.gov (United States)

    Cardoso, Rita; Nolasco, Sofia; Gonçalves, João; Cortes, Helder C; Leitão, Alexandre; Soares, Helena

    2014-09-01

    Besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

  7. SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

    Science.gov (United States)

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin; Mund, Andreas; Wimmer, Peter; Schubert, Tobias; Groitl, Peter; Will, Hans; Dobner, Thomas

    2013-01-01

    Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, 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 functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic 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 cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming

  8. Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

    Science.gov (United States)

    Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

    2017-10-15

    The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ((30)LKKVMRR(36)) in the human enzyme. Substituting the residues KK(31,32) and RR(35,36) with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. CELL SCALE HOST-PATHOGEN MODELING: ANOTHER BRANCH IN THE EVOLUTION OF CONSTRAINT-BASED METHODS

    Directory of Open Access Journals (Sweden)

    Neema eJamshidi

    2015-10-01

    Full Text Available Constraint-based models have become popular methods for systems biology as they enable the integration of complex, disparate datasets in a biologically cohesive framework that also supports the description of biological processes in terms of basic physicochemical constraints and relationships. The scope, scale, and application of genome scale models have grown from single cell bacteria to multi-cellular interaction modeling; host-pathogen modeling represents one of these examples at the current horizon of constraint-based methods. There are now a small number of examples of host-pathogen constraint-based models in the literature, however there has not yet been a definitive description of the methodology required for the functional integration of genome scale models in order to generate simulation capable host-pathogen models. Herein we outline a systematic procedure to produce functional host-pathogen models, highlighting steps which require debugging and iterative revisions in order to successfully build a functional model. The construction of such models will enable the exploration of host-pathogen interactions by leveraging the growing wealth of omic data in order to better understand mechanism of infection and identify novel therapeutic strategies.

  10. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  11. The topologic and chronologic patterns of hematopoietic cell seeding in host femoral bone marrow after transplantation.

    Science.gov (United States)

    Askenasy, Nadir; Stein, Jeremiah; Yaniv, Isaac; Farkas, Daniel L

    2003-08-01

    The early stages of homing, seeding, and engraftment of hematopoietic stem and progenitor cells are poorly characterized. We have developed an optical technique that allows in vivo tracking of transplanted, fluorescent-tagged cells in the host femurs. In this study we used fluorescence microscopy to monitor the topologic and chronologic patterns of hematopoietic cell seeding in the femoral bone marrow (BM) of mice. PKH-labeled cells homed to the femur within minutes after injection into a peripheral vein. Most cells drifted within the marrow space and gradually seeded in clusters close to the endosteal surface of the epiphyseal cortex. Three days after transplantation 85% to 94% (14%) of PKH-labeled cells in the femoral marrow were located within 100 microm of the epiphyseal bone surface (P <.001 versus the more central cells), whereas labeled cells were absent in the femoral diaphysis. Primary seeding of juxtaendosteal, epiphyseal marrow occurred independently of recipient conditioning (myeloablated and nonconditioned hosts), donor-recipient antigen disparity, or the phenotype of the injected cells (whole BM and lineage-negative cells) and was consistently observed in secondary recipients of BM-homed cells. Seeding in regions close to the epiphyseal bone was also observed in freshly excised femurs perfused ex vivo and in femurs assessed without prior placement of optical windows, indicating that the site of primary seeding was not affected by surgical placement of optical windows. Four to 5 days after transplantation, cellular clusters appeared in the more central regions of the epiphyses and in the diaphyses. Centrally located cells showed decreased PKH fluorescence, suggesting that they were progeny of the seeding cells, and brightly fluorescent cells (quiescent first-generation seeding cells) were observed close to the bone surface for as long as 24 days after transplantation. These data indicate that the periphery of the femoral marrow hosts primary seeding

  12. 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.

  13. Towards identifying host cell-type specific response patterns to bacterial endosymbiosis

    DEFF Research Database (Denmark)

    Gavrilovic, Srdjan

    of view, available techniques have relied heavily on whole organ analyses that disregard specificities of individual cell types. To address this issue we aimed to develop a technology for comparative global analysis of mature mRNA and small RNA populations at the cell type specific level in the model...... plant Lotus japonicus. A powerful approach referred to here as Defined Expression and RNA Affinity co-Purification (DERAP) was developed to study gene expression and small RNA populations in the host roots during early phases of signal exchange at the cell-type level. As a basis for DERAP analysis...

  14. [Influence of human gastrointestinal tract bacterial pathogens on host cell apoptosis].

    Science.gov (United States)

    Wronowska, Weronika; Godlewska, Renata; Jagusztyn-Krynicka, Elzbieta Katarzyna

    2005-01-01

    Several pathogenic bacteria are able to trigger apoptosis in the host cell, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Induction and/or blockage of programmed cell death upon infection is a result of complex interaction of bacterial proteins with cellular proteins involved in signal transduction and apoptosis. In this review we focus on pro/anti-apoptotic activities exhibited by two enteric pathogens Salmonella enterica, Yersinia spp. and gastric pathogen Helicobacter pylori. We present current knowledge on how interaction between mammalian and bacterial cell relates to the molecular pathways of apoptosis, and what is the role of apoptosis in pathogenesis.

  15. Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells.

    Science.gov (United States)

    Markwell, M A; Fredman, P; Svennerholm, L

    1984-08-01

    Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es

    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.

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

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    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. Alphavirus Infection: Host Cell Shut-Off and Inhibition of Antiviral Responses

    Directory of Open Access Journals (Sweden)

    Jelke J. Fros

    2016-06-01

    Full Text Available Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus–host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly.

  19. Salmonella Typhimurium Enzymatically Landscapes the Host Intestinal Epithelial Cell (IEC) Surface Glycome to Increase Invasion.

    Science.gov (United States)

    Park, Dayoung; Arabyan, Narine; Williams, Cynthia C; Song, Ting; Mitra, Anupam; Weimer, Bart C; Maverakis, Emanual; Lebrilla, Carlito B

    2016-12-01

    Although gut host-pathogen interactions are glycan-mediated processes, few details are known about the participating structures. Here we employ high-resolution mass spectrometric profiling to comprehensively identify and quantitatively measure the exact modifications of native intestinal epithelial cell surface N-glycans induced by S. typhimurium infection. Sixty minutes postinfection, select sialylated structures showed decreases in terms of total number and abundances. To assess the effect of cell surface mannosylation, we selectively rerouted glycan expression on the host using the alpha-mannosidase inhibitor, kifunensine, toward overexpression of high mannose. Under these conditions, internalization of S. typhimurium significantly increased, demonstrating that bacteria show preference for particular structures. Finally, we developed a novel assay to measure membrane glycoprotein turnover rates, which revealed that glycan modifications occur by bacterial enzyme activity rather than by host-derived restructuring strategies. This study is the first to provide precise structural information on how host N-glycans are altered to support S. typhimurium invasion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens

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    Simona John Von Freyend

    2017-04-01

    Full Text Available Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

  1. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

    Science.gov (United States)

    John Von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

    2017-04-21

    Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

  2. Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells.

    Science.gov (United States)

    Stuart, Elizabeth S; Webley, Wilmore C; Norkin, Leonard C

    2003-07-01

    Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.

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

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    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. Host cell apoptosis induced by infection with duck swollen head hemorrhagic disease virus

    Institute of Scientific and Technical Information of China (English)

    Chuanfeng Li; Xiaoyue Chen; Anchun Cheng; Mingshu Wang; Chanjuan Shen; Na Zhang; Yi Zhou; Dekang Zhu; Qihui Luo; Renyong Jia

    2009-01-01

    This study aimed to examine the host cell apoptosis in the tissues of Peking ducks infected with duck swollen head hemorrhagic dis-ease virus (DSHDV).The dynamic changes associated with apoptosis occurring in the internal tissues were evaluated at different time points postinoculation (PI) by performing hematoxylin and eosin (HE) staining,followed by light microscopy,terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay,and transmission electron microscopy (TEM).The results showed that DSHDV infection could induce apoptosis in host cells,including those of the bursa of Fabricius (BF),thymus,spleen,liver,intestinal tract,kidney,and esophagus.The apoptotic index (AI) values increased with time from 2 to 72 h PI,and the highest values were recorded at 72 h PI.Further,cell death due to classic necrosis was observed in the dying or deceased ducks after 72 h PI.In conclusion,host cell apoptosis can be induced by DSHDV and may play an important role in the pathogenesis of duck viral swollen head hemorrhagic disease (DVSHD).

  5. 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.

  6. A pore-forming toxin enables Serratia a nonlytic egress from host cells.

    Science.gov (United States)

    Di Venanzio, Gisela; Lazzaro, Martina; Morales, Enrique S; Krapf, Darío; García Véscovi, Eleonora

    2017-02-01

    Several pathogens co-opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome-like vacuoles. These Serratia-containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore-forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA-dependent, Ca(2)(+) local increase was shown in SeCVs tight proximity, intracellular Ca(2)(+) sequestration prevented Serratia exit. Accordingly, a Ca(2)(+) surge rescued a ShlA-deficient strain exit capacity, demonstrating that Ca(2)(+) mobilization is essential for egress. As opposed to wild-type-SeCV, the mutant strain-vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild-type Serratia escape, while increased intracellular Ca(2)(+) reorganized the mutant strain-SeCV actin distribution, restoring wild-type-SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca(2)(+) signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic-like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.

  7. Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii.

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    Wassim Daher

    Full Text Available The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.

  8. Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells

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    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.

  9. Campylobacter jejuni FlpA binds fibronectin and is required for maximal host cell adherence.

    Science.gov (United States)

    Konkel, Michael E; Larson, Charles L; Flanagan, Rebecca C

    2010-01-01

    Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a approximately 250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (approximately 45 amino acids), type II (approximately 60 amino acids), and type III (approximately 90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.

  10. Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

    Science.gov (United States)

    Verplaetse, Emilie; Slamti, Leyla; Gohar, Michel

    2015-01-01

    ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host’s death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment. PMID:25922389

  11. Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

    Science.gov (United States)

    Sipkens, Jessica A; Hahn, Nynke; van den Brand, Carlien S; Meischl, Christof; Cillessen, Saskia A G M; Smith, Desirée E C; Juffermans, Lynda J M; Musters, René J P; Roos, Dirk; Jakobs, Cornelis; Blom, Henk J; Smulders, Yvo M; Krijnen, Paul A J; Stehouwer, Coen D A; Rauwerda, Jan A; van Hinsbergh, Victor W M; Niessen, Hans W M

    2013-11-01

    Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

  12. Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells.

    Science.gov (United States)

    Sobotta, Katharina; Wilsky, Steffi; Althof, Nadine; Wiesener, Nadine; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection

    Science.gov (United States)

    Elahi, Shokrollah; Ertelt, James M.; Kinder, Jeremy M.; Jiang, Tony T.; Zhang, Xuzhe; Xin, Lijun; Chaturvedi, Vandana; Strong, Beverly S.; Qualls, Joseph E.; Steinbrecher, Kris A.; Kalfa, Theodosia A.; Shaaban, Aimen F.; Way, Sing Sing

    2013-12-01

    Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71+ erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71+ cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71+ cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71+ cell-mediated susceptibility to infection is counterbalanced by CD71+ cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71+ cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these

  14. Nuclear reprogramming by interphase cytoplasm of 2-cell mouse embryos

    Science.gov (United States)

    Kang, Enugu; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P.; Schöler, Hans; Mitalipov, Shoukhrat

    2014-01-01

    Summary Successful mammalian cloning employing somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II-arrested (MII) oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing pluripotency in somatic cell nuclei1-3. However, these poorly defined maternal factors presumably decline sharply after fertilization since cytoplasm of pronuclear stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase (M-phase) can also support derivation of embryonic stem cells (ESCs) following SCNT6-8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in M-phase but not in interphase cytoplasm are “trapped” inside the nucleus during interphase and effectively removed during enucleation9. Here, we investigated the presence of reprogramming activity in the interphase cytoplasm of 2-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated M-phase and interphase zygotes and 2-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Then, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ESC, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ESCs capable of contributing to traditional germline and tetraploid chimeras. In addition, direct transfer of cloned embryos, reconstructed with ESC nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to utilize interphase cytoplasm in SCNT could impact efforts to generate autologous human ESCs for

  15. 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

  16. Viral protease cleavage of inhibitor of κBα triggers host cell apoptosis

    Science.gov (United States)

    Zaragoza, Carlos; Saura, Marta; Padalko, Elizaveta Y.; Lopez-Rivera, Ester; Lizarbe, Tania R.; Lamas, Santiago; Lowenstein, Charles J.

    2006-01-01

    Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3Cpro cleaves inhibitor of κBα (IκBα). A proteolytic fragment of IκBα then forms a stable complex with NF-κB, translocates to the nucleus, and inhibits NF-κB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IκBα expression are more susceptible to viral infection, with less apoptosis and more viral replication. IκBα thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection. PMID:17138672

  17. 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.

  18. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    Science.gov (United States)

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

  19. Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression

    OpenAIRE

    de Klerk, Nele; Maudsdotter, Lisa; Gebreegziabher, Hanna; Sunil D Saroj; Eriksson, Beatrice; Eriksson, Olaspers Sara; Roos, Stefan; Lindén, Sara; Sjölinder, Hong; Jonsson, Ann-Beth

    2016-01-01

    The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit ad...

  20. Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling

    Science.gov (United States)

    Warncke, Jan D.; Vakonakis, Ioannis

    2016-01-01

    SUMMARY During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family. PMID:27582258

  1. Simvastatin inhibits Staphylococcus aureus host cell invasion through modulation of isoprenoid intermediates

    OpenAIRE

    Horn, Mary P.; Knecht, Sharmon M.; Rushing, Frances L.; Birdsong, Julie; Siddall, C. Parker; Johnson, Charron M.; Abraham, Terri N.; Brown, Amy; Volk, Catherine B.; Gammon, Kelly; Bishop, Derron L.; McKillip, John L.; McDowell, Susan A.

    2008-01-01

    Patients on a statin regimen are at a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small-guanosine triphosphatases (GTPases) CDC42, Rac, and RhoB. Actin stress fiber disassembl...

  2. Voriconazole-Induced Periostitis Mimicking Chronic Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation.

    Science.gov (United States)

    Sweiss, Karen; Oh, Annie; Rondelli, Damiano; Patel, Pritesh

    2016-01-01

    Voriconazole is an established first-line agent for treatment of invasive fungal infections in patients undergoing allogeneic stem cell transplantation (ASCT). It is associated with the uncommon complication of periostitis. We report this complication in a 58-year-old female undergoing HSCT. She was treated with corticosteroids with minimal improvement. The symptoms related to periostitis can mimic chronic graft-versus-host disease in patients undergoing HSCT and clinicians should differentiate this from other diagnoses and promptly discontinue therapy.

  3. Leishmania Interferes with Host Cell Signaling to Devise a Survival Strategy

    OpenAIRE

    Suvercha Bhardwaj; Neetu Srivastava; Raki Sudan; Bhaskar Saha

    2010-01-01

    The protozoan parasite Leishmania spp. exists as extracellular promastigotes in its vector whereas it resides and replicates as amastigotes within the macrophages of its mammalian host. As a survival strategy, Leishmania modulates macrophage functions directly or indirectly. The direct interference includes prevention of oxidative burst and the effector functions that lead to its elimination. The indirect effects include the antigen presentation and modulation of T cell functions in such a wa...

  4. Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae

    Directory of Open Access Journals (Sweden)

    Kempf Volkhard AJ

    2011-04-01

    Full Text Available Abstract Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA, the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (VirB/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail.

  5. Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae.

    Science.gov (United States)

    Franz, Bettina; Kempf, Volkhard A J

    2011-04-13

    Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail.

  6. 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...... 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....

  7. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    Science.gov (United States)

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. 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.

  9. 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.

  10. Liver Graft versus Host Disease after Allogeneic Peripheral Stem Cell Transplantation: Update on Etiopathogenesis and Diagnosis.

    Science.gov (United States)

    Mihăilă, R-G

    2016-01-01

    Graft versus host disease (GVHD) is the main complication of allogeneic hematopoietic cell transplantation and is more frequent after peripheral stem cell transplants. Graft versus leukemia or lymphoma component of them is beneficial to eradicate residual tumor mass after previous treatment and conditioning regimen. A severe GVHD may endanger the patient's life. The most important liver manifestations of GVHD are increased serum alkaline phosphatase and bilirubin values. The last allows to estimate the GVHD severity. Sometimes, an increase of aminotransferases can mimic an acute hepatitis. Donor-derived hematopoietic cells appeared to turn in mesenchymal liver cells. Activated CD4(+) T cells, humoral and complement activation, a large number of cytokines and cytokine receptors are involved in GVHD development. Correct and early recognition of GVHD and its differentiation from the other liver diseases are essential for the medical practice.

  11. Host cell poly(ADP-ribose glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

    Directory of Open Access Journals (Sweden)

    Salomé C Vilchez Larrea

    Full Text Available Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose glycohydrolase in a trypanosomatid (TcPARG. In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl pyrrolidinediol or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

  12. 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

    2015-12-01

    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.

  13. Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

    Science.gov (United States)

    O’Connor, Roddy S.; Thangavelu, Govindarajan; Lovitch, Scott B.; Dandamudi, Durga Bhavani; Vincent, Benjamin G.; Tkachev, Victor; Pawlicki, Jan M.; Furlan, Scott N.; Kean, Leslie S.; Aoyama, Kazutoshi; Taylor, Patricia A.; Panoskaltsis-Mortari, Angela; Foncea, Rocio; Ranganathan, Parvathi; Devine, Steven M.; Burrill, Joel S.; Guo, Lili; Sacristan, Catarina; Snyder, Nathaniel W.; Blair, Ian A.; Milone, Michael C.; Dustin, Michael L.; Riley, James L.; Bernlohr, David A.; Murphy, William J.; Fife, Brian T.; Munn, David H.; Miller, Jeffrey S.; Serody, Jonathan S.; Freeman, Gordon J.; Sharpe, Arlene H.; Turka, Laurence A.

    2016-01-01

    Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD. PMID:27294527

  14. 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.

  15. Interactions between mycoplasma lipid-associated membrane proteins and the host cells

    Institute of Scientific and Technical Information of China (English)

    YOU Xiao-xing; ZENG Yan-hua; WU Yi-mou

    2006-01-01

    Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also considered to be cofactors in the progression of AIDS.

  16. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  17. Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse.

    Science.gov (United States)

    Markoulaki, Styliani; Meissner, Alexander; Jaenisch, Rudolf

    2008-06-01

    Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct--through genetic manipulations--degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body--characteristics that are essential for transplantation therapy or any other in vivo application.

  18. IL-2-targeted therapy ameliorates the severity of graft-versus-host disease: ex vivo selective depletion of host-reactive T cells and in vivo therapy.

    Science.gov (United States)

    Yarkoni, Shai; Prigozhina, Tatyana B; Slavin, Shimon; Askenasy, Nadir

    2012-04-01

    T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.

  19. Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.

  20. A20 deletion in T cells modulates acute graft-versus-host disease in mice.

    Science.gov (United States)

    Fischer, Julius C; Otten, Vera; Steiger, Katja; Schmickl, Martina; Slotta-Huspenina, Julia; Beyaert, Rudi; van Loo, Geert; Peschel, Christian; Poeck, Hendrik; Haas, Tobias; Spoerl, Silvia

    2017-08-22

    The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4(+) and CD8(+) donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20(+/+) T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20(+/+) T cells, highlighting that A20 balances both, T cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T cell mediated inflammatory diseases like GVHD. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

    Directory of Open Access Journals (Sweden)

    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  2. Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Epstein, A.L.; Clevenger, C.V.; Huberman, E.

    1985-02-01

    The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin DT induced the cells to acquire a phenotype that resembled that of granulocytes and monocytesmacrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. The authors suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells. 40 references, 2 figures, 1 table.

  3. Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Chen Zhaojun; Li Huanrong; Zhang Guanglin [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Li Feng [The First Renmin Hospital, Houma, Shanxi 043000 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036 (China)

    2012-03-01

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 {mu}M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 {mu}M) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 {mu}M and 10 {mu}M BaP-treated groups, 2 in the 0.1 {mu}M and 10 {mu}M BaP-treated groups, 4 in the 0.1 {mu}M and 1 {mu}M BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical-host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.

  4. Science Signaling Podcast for 16 May 2017: Vibrio rewires host cells.

    Science.gov (United States)

    De Nisco, Nicole J; Orth, Kim; VanHook, Annalisa M

    2017-05-16

    This Podcast features a conversation with Kim Orth and Nicole De Nisco, authors of a Research Resource that appears in the 16 May 2017 issue of Science Signaling, about how the marine bacterium Vibrio parahaemolyticus rewires host cell signaling networks. V. parahaemolyticus thrives in warm brackish waters and infects both shellfish and finfish. This bacterium causes gastroenteritis when humans consume contaminated seafood that is raw or undercooked. V. parahaemolyticus delivers virulence factors into host cells through two different type 3 secretion systems (T3SSes). Whereas T3SS2 mediates gastroenteritis, T3SS1 is required for the bacterium to survive in its natural environment and delivers virulence factors that target conserved cellular processes. De Nisco et al examined transcriptional changes in human cells infected with a strain of V. parahaemolyticus that lacked T3SS2 but had an intact T3SS1. They found that the virulence factors delivered through T3SS1 initially induced transcriptional changes that promoted cell survival, then later repressed prosurvival signaling to induce cell death.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

  5. Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA.

    Science.gov (United States)

    Alzhanov, Damir; Barnes, Jennifer; Hruby, Dennis E; Rockey, Daniel D

    2004-07-01

    Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells. Cells transfected with plasmids expressing Chlamydophila caviae incA were not productively infected by C. caviae. Expression of C. caviae incA also reduced inclusion formation by Chlamydia trachomatis, but not to the degree seen for C. caviae. Chlamydia trachomatis incA did not block development of either C. trachomatis or C. caviae. Deletion mutagenesis was used to demonstrate that plasmids encoding either the amino or carboxy-terminal regions of the protein, as well as the changing of a single amino acid within IncA (serine 17) could not block C. caviae infection. Immunoblot analysis of truncated IncA in a Vaccinia virus system provided evidence that serine 17 of C. caviae IncA is a target for phosphorylation. These experiments provide insight into the interaction of Inc proteins with the host cell and introduce a model system where these interactions can be explored further.

  6. Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells

    Directory of Open Access Journals (Sweden)

    Hensel Michael

    2010-10-01

    Full Text Available Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

  7. Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA

    Directory of Open Access Journals (Sweden)

    Barnes Jennifer

    2004-07-01

    Full Text Available Abstract Background Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells. Results Cells transfected with plasmids expressing Chlamydophila caviae incA were not productively infected by C. caviae. Expression of C. caviae incA also reduced inclusion formation by Chlamydia trachomatis, but not to the degree seen for C. caviae. Chlamydia trachomatis incA did not block development of either C. trachomatis or C. caviae. Deletion mutagenesis was used to demonstrate that plasmids encoding either the amino or carboxy-terminal regions of the protein, as well as the changing of a single amino acid within IncA (serine 17 could not block C. caviae infection. Immunoblot analysis of truncated IncA in a Vaccinia virus system provided evidence that serine 17 of C. caviae IncA is a target for phosphorylation. Conclusions These experiments provide insight into the interaction of Inc proteins with the host cell and introduce a model system where these interactions can be explored further.

  8. Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Soumya Mitra, Oleg Mironov, Thomas H. Foster

    2012-01-01

    Full Text Available We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH-mediated photodynamic therapy (PDT of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

  9. 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.

  10. Towards system-level understanding of baculovirus host cell interactions: from molecular fundamental studies to large-scale proteomics approaches

    Directory of Open Access Journals (Sweden)

    Francisca eMonteiro

    2012-11-01

    Full Text Available Baculoviruses are insect viruses extensively exploited as eukaryotic protein expression vectors. Molecular biology studies have provided exciting discoveries on virus-host interactions, but the application of omic high throughput techniques on the baculovirus-insect cell system has been hampered by the lack of host genome sequencing. While a broader, systems level analysis of biological responses to infection is urgently needed, recent advances on proteomic studies have yielded new insights on the impact of infection on the host cell. These works are reviewed and critically assessed in the light of current biological knowledge of the molecular biology of baculoviruses and insect cells.

  11. A Comprehensive Nuclear Receptor Network for Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ralf Kittler

    2013-02-01

    Full Text Available In breast cancer, nuclear receptors (NRs play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

  12. Cobalt distribution in keratinocyte cells indicates nuclear and peri-nuclear accumulation and interaction with magnesium and zinc homeostasis

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, R.; Seznec, H.; Moretto, Ph. [Univ Bordeaux 1, CNRS, IN2P3, Ctr Etud Nucl Bordeaux Gradignan, F-33175 Gradignan, (France); Bresson, C.; Sandre, C.; Gombert, C.; Moulin, Ch. [CEA, DEN, SECR, Lab Speciat Radionucleides et Mol, F-91191 Gif Sur Yvette, (France); Tabarant, M. [CEA, DEN, SCP, Lab React Surfaces et Interfaces, F-91191 Gif Sur Yvette, (France); Bleuet, P. [European Synchrotron Radiat Facil, F-38043 Grenoble, (France); Simionovici, A. [Univ Grenoble 1, LGIT, OSUG, CNRS, F-38041 Grenoble, (France)

    2009-07-01

    Cobalt is known to be toxic at high concentration, to induce contact dermatosis, and occupational radiation skin damage because of its use in nuclear industry. We investigated the intracellular distribution of cobalt in HaCaT human keratinocytes as a model of skin cells, and its interaction with endogenous trace elements. Direct micro-chemical imaging based on ion beam techniques was applied to determine the quantitative distribution of cobalt in HaCaT cells. In addition, synchrotron radiation X-ray fluorescence microanalysis in tomography mode was performed, for the first time on a single cell, to determine the 3D intracellular distribution of cobalt. Results obtained with these micro-chemical techniques were compared to a more classical method based on cellular fractionation followed by inductively coupled plasma atomic emission spectrometry (ICP-AES) measurements. Cobalt was found to accumulate in the cell nucleus and in peri-nuclear structures indicating the possible direct interaction with genomic DNA, and nuclear proteins. The peri-nuclear accumulation in the cytosol suggests that cobalt could be stored in the endoplasmic reticulum or the Golgi apparatus. The multi-elemental analysis revealed that cobalt exposure significantly decreased magnesium and zinc content, with a likely competition of cobalt for magnesium and zinc binding sites in proteins. Overall, these data suggest a multiform toxicity of cobalt related to interactions with genomic DNA and nuclear proteins, and to the alteration of zinc and magnesium homeostasis. (authors)

  13. Role of Fc Gamma Receptors in Triggering Host Cell Activation and Cytokine Release by Borrelia burgdorferi

    Science.gov (United States)

    Talkington, Jeffrey; Nickell, Steven P.

    2001-01-01

    Borrelia burgdorferi, the spirochetal bacterium that causes human Lyme disease, encodes numerous lipoproteins which have the capacity to trigger the release of proinflammatory cytokines from a variety of host cell types, and it is generally believed that these cytokines contribute to the disease process in vivo. We previously reported that low-passage-number infectious B. burgdorferi spirochetes express a novel lipidation-independent activity which induces secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) by the mouse MC/9 mast cell line. Using RNase protection assays, we determined that mast cells exposed in vitro to low-passage-number, but not high-passage-number, B. burgdorferi spirochetes show increased expression of additional mRNAs representing several chemokines, including macrophage-inflammatory protein 1α (MIP-1α), MIP-1β, and TCA3, as well as the proinflammatory cytokine interleukin-6. Furthermore, mast cell TNF-α secretion can be inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin and also by preincubation with purified mouse immunoglobulin G1 (IgG1) and IgG2a, but not mouse IgG3, and by a mouse Fc gamma receptor II and III (FcγRII/III)-specific rat monoclonal antibody, suggesting the likely involvement of host FcγRIII in B. burgdorferi-mediated signaling. A role for passively adsorbed rabbit or bovine IgG or serum components in B. burgdorferi-mediated FcγR signaling was excluded in control experiments. These studies confirm that low-passage-number B. burgdorferi spirochetes express a novel activity which upregulates the expression of a variety of host cell chemokine and cytokine genes, and they also establish a novel antibody-independent role for FcγRs in transduction of activation signals by bacterial products. PMID:11119532

  14. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    Energy Technology Data Exchange (ETDEWEB)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  15. TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells

    Science.gov (United States)

    McCoy, James M.; Whitehead, Lachlan; van Dooren, Giel G.; Tonkin, Christopher J.

    2012-01-01

    The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca2+-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca2+ ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K+ concentration of the host cell. Our results therefore suggest that TgCDPK3's role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca2+ signaling in specific ionic environments to upregulate multiple processes required for gliding motility. PMID:23226109

  16. Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection*

    Science.gov (United States)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen. PMID:23970565

  17. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection.

    Science.gov (United States)

    Nakayasu, Ernesto S; Tempel, Rebecca; Cambronne, Xiaolu A; Petyuk, Vladislav A; Jones, Marcus B; Gritsenko, Marina A; Monroe, Matthew E; Yang, Feng; Smith, Richard D; Adkins, Joshua N; Heffron, Fred

    2013-11-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen.

  18. Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion

    Science.gov (United States)

    Clemente, Tatiana Mordente; Cortez, Cristian; Novaes, Antônio da Silva; Yoshida, Nobuko

    2016-01-01

    Background The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. Methodology/Principal Findings Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. Conclusion/Significance Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion. PMID:27483135

  19. Essential domains of Anaplasma phagocytophilum invasins utilized to infect mammalian host cells.

    Directory of Open Access Journals (Sweden)

    David Seidman

    2015-02-01

    Full Text Available Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLe(x-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLe(x sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLe(x but express the structurally similar glycan, 6-sulfo-sLe(x, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLe(x antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLe(x antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding

  20. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  1. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  2. The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

    Directory of Open Access Journals (Sweden)

    Patricia Gorocica

    2009-05-01

    Full Text Available Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The ±-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the ²-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

  3. Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection.

    Science.gov (United States)

    Gillespie, Alyssa Lundgren; Teoh, Jeffrey; Lee, Heather; Prince, Jessica; Stadnisky, Michael D; Anderson, Monique; Nash, William; Rival, Claudia; Wei, Hairong; Gamache, Awndre; Farber, Charles R; Tung, Kenneth; Brown, Michael G

    2016-02-01

    The MHC class I D(k) molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D(k), are required to control viral spread. The extent of D(k)-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D(k)-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.

  4. Global Reorganization of the Nuclear Landscape in Senescent Cells

    Directory of Open Access Journals (Sweden)

    Tamir Chandra

    2015-02-01

    Full Text Available Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF. However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs, somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation.

  5. Global reorganization of the nuclear landscape in senescent cells.

    Science.gov (United States)

    Chandra, Tamir; Ewels, Philip Andrew; Schoenfelder, Stefan; Furlan-Magaril, Mayra; Wingett, Steven William; Kirschner, Kristina; Thuret, Jean-Yves; Andrews, Simon; Fraser, Peter; Reik, Wolf

    2015-02-03

    Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. M062 is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host SAMD9 in human cells.

    Science.gov (United States)

    Liu, Jia; Wennier, Sonia; Zhang, Leiliang; McFadden, Grant

    2011-04-01

    Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.

  7. Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.

    Science.gov (United States)

    Ferraris, Pauline; Beaumont, Elodie; Uzbekov, Rustem; Brand, Denys; Gaillard, Julien; Blanchard, Emmanuelle; Roingeard, Philippe

    2013-04-01

    Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.

  8. Early shutoff of host protein synthesis in cells infected with herpes simplex viruses.

    Science.gov (United States)

    Matis, J; Kúdelová, M

    2001-01-01

    Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.

  9. 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.

  10. HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.

    Directory of Open Access Journals (Sweden)

    Frédéric Catez

    Full Text Available Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1 latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.. Latent HSV-1 genomes display two major patterns, called "Single" and "Multiple", which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs as viral DNA-containing PML-NBs (DCP-NBs. 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i., infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. "Single" and "Multiple" patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the "Multiple" pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including

  11. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  12. Nuclear vasohibin-2 promotes cell proliferation by inducing G0/G1 to S phase progression.

    Science.gov (United States)

    Ge, Qianqian; Zhou, Jia; Tu, Min; Xue, Xiaofeng; Li, Zhanjun; Lu, Zipeng; Wei, Jishu; Song, Guoxin; Chen, Jianmin; Guo, Feng; Jiang, Kuirong; Miao, Yi; Gao, Wentao

    2015-09-01

    As a member of the vasohibin (VASH2) family, VASH2 is localized intracellularly as a nuclear and cytoplasmic type. Cytoplasmic VASH2 is associated with carcinoma angiogenesis and malignant transformation and promotes cancer growth. However, the function of nuclear VASH2 has yet to be investigated. The aim of the present study was to detect the nuclear VASH2 expression profile in human organs and tissues by protein microarray technique. To examine the function of nuclear VASH2, we analyzed the relationship between nuclear VASH2 and Ki-67, and stably constructed VASH2 overexpression and knockdown in LO2 and HepG2 cell lines, based on a previous study in hepatic cells. The study was conducted using bromodeoxyuridine, immunofluorescent staining, western blot analysis and flow cytometry. Nuclear VASH2 was highly expressed in actively dividing cells in normal and cancer tissues. There was a significant positive correlation between nuclear VASH2 and Ki-67, indicating that nuclear VASH2 positively correlated with cell proliferation in normal and cancer tissues. The bromodeoxyuridine (BrdU) proliferation test showed that nuclear VASH2 increased the S-phase population and promoted cell proliferation, while VASH2 knockdown reduced BrdU absorbance. Cell cycle analysis revealed that nuclear VASH2 overexpression increased the S-phase population in LO2 and HepG2 cells, while nuclear VASH2 knockdown reduced the S-phase population and increased the G0/G1 population. The findings of this study challenge the classic view of VASH2, which was previously reported as an angiogenesis factor. Furthermore, to the best of our knowledge, these results are the first clinical data indicating that nuclear VASH2, but not cytoplasmic VASH2, promotes cell proliferation by driving the cell cycle from the G0/G1 to S phase.

  13. Interleukin-10 spot-forming cells as a novel biomarker of chronic graft-versus-host disease

    Science.gov (United States)

    Hirayama, Masahiro; Azuma, Eiichi; Nakagawa-Nakazawa, Atsuko; Kumamoto, Tadashi; Iwamoto, Shotaro; Amano, Keishiro; Tamaki, Shigehisa; Usui, Eiji; Komada, Yoshihiro

    2013-01-01

    Although there are National Institutes of Health consensus criteria for the global assessment of chronic graft-versus-host disease, no validated biomarkers have been established for this disease. Furthermore, whereas the role of T cells, B cells, and dendritic cells in chronic graft-versus-host disease has been established, the contribution of monocytes has not been clearly addressed. Using an enzyme-linked immunospot assay, we measured the spot-forming cells for interferon-γ, interleukin-4, interleukin-10, and interleukin-17 in unstimulated peripheral blood of patients following allogeneic hematopoietic stem cell transplantation. Other immunological examinations, including skin biopsy, were also done. Fifty-seven patients were enrolled. Interleukin-10 spot-forming cells were evaluable for therapeutic monitoring in 16 patients with chronic graft-versus-host disease. The number of interleukin-10 spot-forming cells in patients with active chronic graft-versus-host disease was significantly higher than the number in those with no or inactive chronic graft-versus-host disease. Interleukin-10 was predominantly produced by monocytes. CD29 expression on monocytes in patients with active chronic graft-versus-host disease was elevated. The level of plasma fibronectin, a ligand of CD29, correlated with the number of interleukin-10 spot-forming cells. Immunohistochemical analysis of the skin in active chronic graft-versus-host disease showed that infiltrating CD29+ monocytes might produce interleukin-10. A novel biomarker, interleukin-10 spot-forming cells, shows promise as both a diagnostic and prognostic indicator for chronic graft-versus-host disease, and may allow for early intervention prior to the onset of the disease. Measurement of interleukin-10 spot-forming cells would be helpful in clinical trials and in patients' management. PMID:22733028

  14. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    Energy Technology Data Exchange (ETDEWEB)

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: corinne.chadeneau@univ-poitiers.fr [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)

    2014-11-28

    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  15. Carcinoma cells misuse the host tissue damage response to invade the brain

    Science.gov (United States)

    Chuang, Han-Ning; van Rossum, Denise; Sieger, Dirk; Siam, Laila; Klemm, Florian; Bleckmann, Annalen; Bayerlová, Michaela; Farhat, Katja; Scheffel, Jörg; Schulz, Matthias; Dehghani, Faramarz; Stadelmann, Christine; Hanisch, Uwe-Karsten; Binder, Claudia; Pukrop, Tobias

    2013-01-01

    The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis. PMID:23832647

  16. Effector T cells require fatty acid metabolism during murine graft-versus-host disease.

    Science.gov (United States)

    Byersdorfer, Craig A; Tkachev, Victor; Opipari, Anthony W; Goodell, Stefanie; Swanson, Jacob; Sandquist, Stacy; Glick, Gary D; Ferrara, James L M

    2013-10-31

    Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5'-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell-mediated immune diseases.

  17. Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

    Science.gov (United States)

    Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

    2015-01-01

    The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery.

  18. Host cell phosphatidylcholine is a key mediator of malaria parasite survival during liver stage infection.

    Science.gov (United States)

    Itoe, Maurice A; Sampaio, Júlio L; Cabal, Ghislain G; Real, Eliana; Zuzarte-Luis, Vanessa; March, Sandra; Bhatia, Sangeeta N; Frischknecht, Friedrich; Thiele, Christoph; Shevchenko, Andrej; Mota, Maria M

    2014-12-10

    During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.

  19. 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.

  20. Effects of LncRNA-HOST2 on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma cell line SMMC-7721.

    Science.gov (United States)

    Liu, Run-Tian; Cao, Jing-Lin; Yan, Chang-Qing; Wang, Yang; An, Cong-Jing; Lv, Hai-Tao

    2017-01-31

    This study explored the effect of LncRNA-HOST2 on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721. HCC tissues and adjacent normal tissues from 162 HCC patients were collected. The HCC cell lines were assigned into the control group (regular culture), negative control group (NC, transfected with siRNA) and experimental group (transfected with Lnc-HOST2 siRNA). qRT-PCR was used to detect the expression of LncRNA-HOST2. Cell proliferation was detected by CCK-8 and colony-forming assays, cell apoptosis by flow cytometry and cell migration by scratch test. Transwell assay was used to evaluate cell migration and invasion abilities. LncRNA-HOST2 expression in the HCC tissues increased 2 to 10 times than that in the adjacent normal tissues. Compared with the HL-7702 cell line, LncRNA-HOST2 expression in HepG2, SMMC7721 and Huh7 cell lines was all up-regulated, but the SMMC-7721 cell had the highest Lnc-HOST2 expression. The LncRNA-HOST2 expression in the experimental group was down-regulated as compared to the control and NC groups. In comparison with the control and NC groups, cloned cells reduced, cell apoptosis increased, clone-forming ability weakened and inhibitory rate of colony formation increased in the experimental group. The cells migrating and penetrating into transwell chamber were fewer in the experimental group than those in the control and NC groups. The experimental group exhibited slow wound-healing and decreased cell migration area after 48 h. These findings indicate that LncRNA-HOST2 can promote cell proliferation, migration and invasion and inhibit cell apoptosis in human HCC cell line SMMC-7721.

  1. Nuclear actin is partially associated with Cajal bodies in human cells in culture and relocates to the nuclear periphery after infection of cells by adenovirus 5.

    Science.gov (United States)

    Gedge, L J E; Morrison, E E; Blair, G E; Walker, J H

    2005-02-15

    Cajal bodies are intra-nuclear structures enriched in proteins involved in transcription and mRNA processing. In this study, immunofluorescence microscopy experiments using a highly specific antibody to actin revealed nuclear actin spots that colocalized in part with p80 coilin-positive Cajal bodies. Actin remained associated with Cajal bodies in cells extracted to reveal the nuclear matrix. Adenovirus infection, which is known to disassemble Cajal bodies, resulted in loss of actin from these structures late in infection. In infected cells, nuclear actin was observed to relocate to structures at the periphery of the nucleus, inside the nuclear envelope. Based on these findings, it is suggested that actin may play an important role in the organization or function of the Cajal body.

  2. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification

    Science.gov (United States)

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the MetacoreTM database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection. PMID:27406444

  3. Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

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    Li Xianyao

    2010-07-01

    Full Text Available Abstract Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1 causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi. Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB, cell cycle regulation (cyclin B2, CDK1, and CKI3, matrix metalloproteinases (MMPs and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR. A bioinformatics tool (Ingenuity Pathway Analysis used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections.

  4. Unconventional Specimen Preparation Techniques Using High Resolution Low Voltage Field Emission Scanning Electron Microscopy to Study Cell Motility, Host Cell Invasion, and Internal Cell Structures in Toxoplasma gondii

    Science.gov (United States)

    Schatten, Heide; Ris, Hans

    2002-04-01

    Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

  5. Quantification of nanoscale nuclear refractive index changes during the cell cycle

    Science.gov (United States)

    Bista, Rajan K.; Uttam, Shikhar; Wang, Pin; Staton, Kevin; Choi, Serah; Bakkenist, Christopher J.; Hartman, Douglas J.; Brand, Randall E.; Liu, Yang

    2011-07-01

    Intrigued by our recent finding that the nuclear refractive index is significantly increased in malignant cells and histologically normal cells in clinical histology specimens derived from cancer patients, we sought to identify potential biological mechanisms underlying the observed phenomena. The cell cycle is an ordered series of events that describes the intervals of cell growth, DNA replication, and mitosis that precede cell division. Since abnormal cell cycles and increased proliferation are characteristic of many human cancer cells, we hypothesized that the observed increase in nuclear refractive index could be related to an abundance or accumulation of cells derived from cancer patients at a specific point or phase(s) of the cell cycle. Here we show that changes in nuclear refractive index of fixed cells are seen as synchronized populations of cells that proceed through the cell cycle, and that increased nuclear refractive index is strongly correlated with increased DNA content. We therefore propose that an abundance of cells undergoing DNA replication and mitosis may explain the increase in nuclear refractive index observed in both malignant and histologically normal cells from cancer patients. Our findings suggest that nuclear refractive index may be a novel physical parameter for early cancer detection and risk stratification.

  6. Leptin Protects Host Cells from Entamoeba histolytica Cytotoxicity by a STAT3-Dependent Mechanism

    Science.gov (United States)

    Verkerke, Hans P.; Paul, Shom N.; Mackey, Aaron J.; Petri, William A.

    2012-01-01

    The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection. PMID:22331430

  7. 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

  8. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  9. 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.

  10. Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages.

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    Ludovic Tailleux

    Full Text Available BACKGROUND: Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. METHODOLOGY/PRINCIPAL FINDINGS: In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. CONCLUSIONS/SIGNIFICANCE: This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

  11. Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

    Science.gov (United States)

    He, Wei; Racine, Jeremy J; Johnston, Heather F; Li, Xiaofan; Li, Nainong; Cassady, Kaniel; Liu, Can; Deng, Ruishu; Martin, Paul; Forman, Stephen; Zeng, Defu

    2014-07-01

    We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.

  12. Propagation of elite rescue dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Oh, Hyun Ju; Choi, Jin; Kim, Min Jung; Kim, Geon A; Jo, Young Kwang; Choi, Yoo Bin; Lee, Byeong Chun

    2016-01-01

    The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.

  13. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    Science.gov (United States)

    Niemann, H; Lucas-Hahn, A

    2012-08-01

    Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

  14. Amino acid chloramine damage to proliferating cell nuclear antigen in mammalian cells.

    Science.gov (United States)

    Salama, Samir A; Snapka, Robert M

    2012-01-01

    Amino acid chloramines (AACLs) are reactive secondary products of activated neutrophils. To understand AACL damage in cell nuclei, we exploited proliferating cell nuclear antigen (PCNA) as a nuclear protein damage reporter, using western blotting and mass spectrometry. Chloramines of proline, arginine, and glycine caused significant damage to PCNA in cells. Chloramines of taurine and histidine caused slight damage to PCNA in cells. Other AACLs caused no PCNA damage in intact cells. Evidence supports a sulfonamide, sulfinamide, or sulfenamide crosslinking mechanism involving cysteine 148 at the PCNA subunit interface, methionine sulfoxide formation as the basis of electrophoretic mobility shifting, and tyrosine and/or methionine residues as the likely targets of AACL damage to the PCNA antibody epitope. An interstitial fluid model experiment showed that physiological amino acids can mediate HOCl damage to PCNA in the presence of proteins that would otherwise completely quench the HOCl. PCNA is a sensitive biomarker of AACL damage in cell nuclei. Arginine chloramine and proline chloramine, or reactive species derived from them, were shown to enter cells and damage PCNA. Amino acids were shown to have at least two different mechanisms for suppressing PCNA damage in cells by their corresponding AACLs. Cysteine 148 was shown to be essential for PCNA subunit crosslinking by AACLs, and a crosslinking mechanism was proposed.

  15. DMPD: The role of type I interferon production by dendritic cells in host defense. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17544561 The role of type I interferon production by dendritic cells in host defens...e. Fitzgerald-Bocarsly P, Feng D. Biochimie. 2007 Jun-Jul;89(6-7):843-55. Epub 2007 May 8. (.png) (.svg) (.html) (.csml) Show The rol...e of type I interferon production by dendritic cells in host defense. PubmedID 17544561 Title The role

  16. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Yang, Hanchun, E-mail: yanghanchun1@cau.edu.cn [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Hu, Hongbo, E-mail: hongbo@cau.edu.cn [College of Food Science and Nutritional Engineering, China Agricultural University, Beijing (China)

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  17. Glomerular diseases associated with chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation: case reports.

    Science.gov (United States)

    Chanswangphuwana, C; Townamchai, N; Intragumtornchai, T; Bunworasate, U

    2014-12-01

    Chronic graft-versus-host disease (cGVHD) is the major complication following allogeneic stem cell transplantation (allo-SCT). Nephrotic syndrome (NS) and other types of glomerulonephritis have been proposed to be the very rare forms of renal cGVHD. From 1991 to 2011, 253 patients underwent allo-SCT at our center. We report here 4 cases (1.6%) presenting with varieties of glomerular manifestations associated with cGVHD. The first case was typical NS. The renal pathology showed membranous nephropathy (MN). The second case was also MN, but this patient also had the pathology of focal segmental glomerulosclrosis (FSGS) and acute tubular necrosis (ATN). The third case showed lupus nephritis-like glomerular lesions with a high anti-nuclear antibody (ANA) titer. The fourth case presented with rapidly progressive glomerulonephritis (RPGN)-like symptoms. The kidney histology in this case was not available. The patient responded well to immunosuppressive therapy, but NS later recurred. Therefore, overt glomerular diseases after allo-SCT in Thai patients are not very rare. Monitoring urinalysis during withdrawal of immunosuppressive drugs and also during follow-up of patients with cGVHD may be considered.

  18. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

    Directory of Open Access Journals (Sweden)

    Emma C Skoog

    Full Text Available Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

  19. Binding of LL-37 to model biomembranes: insight into target vs host cell recognition.

    Science.gov (United States)

    Sood, Rohit; Domanov, Yegor; Pietiäinen, Milla; Kontinen, Vesa P; Kinnunen, Paavo K J

    2008-04-01

    Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.

  20. Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.

    Science.gov (United States)

    Liehl, Peter; Zuzarte-Luís, Vanessa; Chan, Jennie; Zillinger, Thomas; Baptista, Fernanda; Carapau, Daniel; Konert, Madlen; Hanson, Kirsten K; Carret, Céline; Lassnig, Caroline; Müller, Mathias; Kalinke, Ulrich; Saeed, Mohsan; Chora, Angelo Ferreira; Golenbock, Douglas T; Strobl, Birgit; Prudêncio, Miguel; Coelho, Luis P; Kappe, Stefan H; Superti-Furga, Giulio; Pichlmair, Andreas; Vigário, Ana M; Rice, Charles M; Fitzgerald, Katherine A; Barchet, Winfried; Mota, Maria M

    2014-01-01

    Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

  1. Melaleuca alternifolia Concentrate Inhibits in Vitro Entry of Influenza Virus into Host Cells

    Directory of Open Access Journals (Sweden)

    Lifang Jiang

    2013-08-01

    Full Text Available Influenza virus causes high morbidity among the infected population annually and occasionally the spread of pandemics. Melaleuca alternifolia Concentrate (MAC is an essential oil derived from a native Australian tea tree. Our aim was to investigate whether MAC has any in vitro inhibitory effect on influenza virus infection and what mechanism does the MAC use to fight the virus infection. In this study, the antiviral activity of MAC was examined by its inhibition of cytopathic effects. In silico prediction was performed to evaluate the interaction between MAC and the viral haemagglutinin. We found that when the influenza virus was incubated with 0.010% MAC for one hour, no cytopathic effect on MDCK cells was found after the virus infection and no immunofluorescence signal was detected in the host cells. Electron microscopy showed that the virus treated with MAC retained its structural integrity. By computational simulations, we found that terpinen-4-ol, which is the major bioactive component of MAC, could combine with the membrane fusion site of haemagglutinin. Thus, we proved that MAC could prevent influenza virus from entering the host cells by disturbing the normal viral membrane fusion procedure.

  2. Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection.

    Science.gov (United States)

    Gladiator, André; Wangler, Nicolette; Trautwein-Weidner, Kerstin; LeibundGut-Landmann, Salomé

    2013-01-15

    IL-17-mediated immunity has emerged as a crucial host defense mechanism against fungal infections. Although Th cells are generally thought to act as the major source of IL-17 in response to Candida albicans, we show that fungal control is mediated by IL-17-secreting innate lymphoid cells (ILCs) and not by Th17 cells. By using a mouse model of oropharyngeal candidiasis we found that IL-17A and IL-17F, which are both crucial for pathogen clearance, are produced promptly upon infection in an IL-23-dependent manner, and that ILCs in the oral mucosa are the main source for these cytokines. Ab-mediated depletion of ILCs in RAG1-deficient mice or ILC deficiency in retinoic acid-related orphan receptor c(-/-) mice resulted in a complete failure to control the infection. Taken together, our data uncover the cellular basis for the IL-23/IL-17 axis, which acts right at the onset of infection when it is most needed for fungal control and host protection.

  3. The human cathelicidin LL-37--A pore-forming antibacterial peptide and host-cell modulator.

    Science.gov (United States)

    Xhindoli, Daniela; Pacor, Sabrina; Benincasa, Monica; Scocchi, Marco; Gennaro, Renato; Tossi, Alessandro

    2016-03-01

    The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of 'alarmin' that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  4. Mycobacterium leprae–host-cell interactions and genetic determinants in leprosy: an overview

    Science.gov (United States)

    Pinheiro, Roberta Olmo; de Souza Salles, Jorgenilce; Sarno, Euzenir Nunes; Sampaio, Elizabeth Pereira

    2011-01-01

    Leprosy, also known as Hansen’s disease, is a chronic infectious disease caused by Mycobacterium leprae in which susceptibility to the mycobacteria and its clinical manifestations are attributed to the host immune response. Even though leprosy prevalence has decreased dramatically, the high number of new cases indicates active transmission. Owing to its singular features, M. leprae infection is an attractive model for investigating the regulation of human immune responses to pathogen-induced disease. Leprosy is one of the most common causes of nontraumatic peripheral neuropathy worldwide. The proportion of patients with disabilities is affected by the type of leprosy and delay in diagnosis. This article briefly reviews the clinical features as well as the immunopathological mechanisms related to the establishment of the different polar forms of leprosy, the mechanisms related to M. leprae–host cell interactions and prophylaxis and diagnosis of this complex disease. Host genetic factors are summarized and the impact of the development of interventions that prevent, reverse or limit leprosy-related nerve impairments are discussed. PMID:21366421

  5. Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.

    Science.gov (United States)

    Candela, Marco; Biagi, Elena; Centanni, Manuela; Turroni, Silvia; Vici, Manuela; Musiani, Francesco; Vitali, Beatrice; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia

    2009-10-01

    The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

  6. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  7. Strategy to prime the host and cells to augment therapeutic efficacy of progenitor cells for patients with myocardial infarction

    Directory of Open Access Journals (Sweden)

    Jeehoon Kang

    2016-11-01

    Full Text Available Cell therapy in myocardial infarction (MI is an innovative strategy that is regarded as a rescue therapy to repair the damaged myocardium and to promote neovascularization for the ischemic border zone. Among several stem cell sources for this purpose, autologous progenitors from bone marrow or peripheral blood would be the most feasible and safest cell-source. Despite the theoretical benefit of cell therapy, this method is not widely adopted in the actual clinical practice due to its low therapeutic efficacy. Various methods have been used to augment the efficacy of cell therapy in MI, such as using different source of progenitors, genetic manipulation of cells, or priming of the cells or hosts (patients with agents. Among these methods, the strategy to augment the therapeutic efficacy of the autologous peripheral blood mononuclear cells by priming agents may be the most feasible and the safest method that can be applied directly to the clinic. In this review, we will discuss the current status and future directions of priming peripheral blood mononuclear cells or patients, as for cell therapy of MI.

  8. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago

    NARCIS (Netherlands)

    Gavrin, A.Y.; Kaiser, B.N.; Geiger, D.; Tyerman, S.D.; Wen, Z.; Bisseling, T.; Fedorova, E.E.

    2014-01-01

    In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cell

  9. H+ channels in embryonic Biomphalaria glabrata cell membranes: Putative roles in snail host-schistosome interactions

    Science.gov (United States)

    Wright, Brandon J.; Bickham-Wright, Utibe; Yoshino, Timothy P.; Jackson, Meyer B.

    2017-01-01

    The human blood fluke Schistosoma mansoni causes intestinal schistosomiasis, a widespread neglected tropical disease. Infection of freshwater snails Biomphalaria spp. is an essential step in the transmission of S. mansoni to humans, although the physiological interactions between the parasite and its obligate snail host that determine success or failure are still poorly understood. In the present study, the B. glabrata embryonic (Bge) cell line, a widely used in vitro model for hemocyte-like activity, was used to investigate membrane properties, and assess the impact of larval transformation proteins (LTP) on identified ion channels. Whole-cell patch clamp recordings from Bge cells demonstrated that a Zn2+-sensitive H+ channel serves as the dominant plasma membrane conductance. Moreover, treatment of Bge cells with Zn2+ significantly inhibited an otherwise robust production of reactive oxygen species (ROS), thus implicating H+ channels in the regulation of this immune function. A heat-sensitive component of LTP appears to target H+ channels, enhancing Bge cell H+ current over 2-fold. Both Bge cells and B. glabrata hemocytes express mRNA encoding a hydrogen voltage-gated channel 1 (HVCN1)-like protein, although its function in hemocytes remains to be determined. This study is the first to identify and characterize an H+ channel in non-neuronal cells of freshwater molluscs. Importantly, the involvement of these channels in ROS production and their modulation by LTP suggest that these channels may function in immune defense responses against larval S. mansoni. PMID:28319196

  10. Lethal graft-versus-host disease: modification with allogeneic cultured donor cells.

    Science.gov (United States)

    Mauch, P; Lipton, J M; Hamilton, B; Obbagy, J; Kudisch, M; Nathan, D; Hellman, S

    1984-05-01

    The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta-positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90-day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences.

  11. Voriconazole-Induced Periostitis Mimicking Chronic Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Karen Sweiss

    2016-01-01

    Full Text Available Voriconazole is an established first-line agent for treatment of invasive fungal infections in patients undergoing allogeneic stem cell transplantation (ASCT. It is associated with the uncommon complication of periostitis. We report this complication in a 58-year-old female undergoing HSCT. She was treated with corticosteroids with minimal improvement. The symptoms related to periostitis can mimic chronic graft-versus-host disease in patients undergoing HSCT and clinicians should differentiate this from other diagnoses and promptly discontinue therapy.

  12. Research resource: Comparative nuclear receptor atlas: basal and activated peritoneal B-1 and B-2 cells.

    Science.gov (United States)

    Diehl, Cody J; Barish, Grant D; Downes, Michael; Chou, Meng-Yun; Heinz, Sven; Glass, Christopher K; Evans, Ronald M; Witztum, Joseph L

    2011-03-01

    Naïve murine B cells are typically divided into three subsets based on functional and phenotypic characteristics: innate-like B-1 and marginal zone B cells vs. adaptive B-2 cells, also known as follicular or conventional B cells. B-1 cells, the innate-immune-like component of the B cell lineage are the primary source of natural antibodies and have been shown to modulate autoimmune diseases, human B-cell leukemias, and inflammatory disorders such as atherosclerosis. On the other hand, B-2 cells are the principal mediators of the adaptive humoral immune response and represent an important pharmacological target for various conditions including rheumatoid arthritis, lupus erythematosus, and lymphomas. Using the resources of the Nuclear Receptor Signaling Atlas program, we used quantitative real-time PCR to assess the complement of the 49 murine nuclear receptor superfamily expressed in quiescent and toll-like receptor (TLR)-stimulated peritoneal B-1 and B-2 cells. We report the expression of 24 nuclear receptors in basal B-1 cells and 25 nuclear receptors in basal B-2 cells, with, in some cases, dramatic changes in response to TLR 4 or TLR 2/1 stimulation. Comparative nuclear receptor profiling between B-1 and peritoneal B-2 cells reveals a highly concordant expression pattern, albeit at quantitatively dissimilar levels. We also found that splenic B cells express 23 nuclear receptors. This catalog of nuclear receptor expression in B-1 and B-2 cells provides data to be used to better understand the specific roles of nuclear receptors in B cell function, chronic inflammation, and autoimmune disease.

  13. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

    Science.gov (United States)

    Yang, Yi-Lin; Serrano, Myrna G; Sheoran, Abhineet S; Manque, Patricio A; Buck, Gregory A; Widmer, Giovanni

    2009-11-01

    The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

  14. External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

    Science.gov (United States)

    Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

    2010-07-23

    Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

  15. Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

    Institute of Scientific and Technical Information of China (English)

    汪国顺; 罗文捷; 潘惟钧; 丁明孝; 翟中和

    1994-01-01

    Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.

  16. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    Science.gov (United States)

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.