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Sample records for actively dividing cells

  1. Divided electrochemical cell assembly

    SciTech Connect

    King, Ch. J. H.

    1985-02-19

    A divided electrochemical cell assembly comprises stacked bipolar substantially square parallel planar electrodes and membranes. The corners and edges of the electrodes with bordering insulative spacers in juxtaposition with the chamber walls define four electrolyte circulation manifolds. Anolyte and catholyte channeling means permit the separate introduction of anolyte and catholyte into two of the manifolds and the withdrawal of anolyte and catholyte separately from at least two other manifolds. The electrodes and membranes are separated from one another by the insulative spacers which are also channeling means disposed to provide electrolyte channels across the interfaces of adjacent electrodes and membranes.

  2. DNA repair mechanisms in dividing and non-dividing cells

    PubMed Central

    Iyama, Teruaki; Wilson, David M.

    2013-01-01

    DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye towards how these pathways may regulate the development of neurological disease. PMID:23684800

  3. DNA repair mechanisms in dividing and non-dividing cells.

    PubMed

    Iyama, Teruaki; Wilson, David M

    2013-08-01

    DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.

  4. Centrosome positioning in non-dividing cells.

    PubMed

    Barker, Amy R; McIntosh, Kate V; Dawe, Helen R

    2016-07-01

    Centrioles and centrosomes are found in almost all eukaryotic cells, where they are important for organising the microtubule cytoskeleton in both dividing and non-dividing cells. The spatial location of centrioles and centrosomes is tightly controlled and, in non-dividing cells, plays an important part in cell migration, ciliogenesis and immune cell functions. Here, we examine some of the ways that centrosomes are connected to other organelles and how this impacts on cilium formation, cell migration and immune cell function in metazoan cells.

  5. Dividing Cells Regulate Their Lipid Composition and Localization

    PubMed Central

    Atilla-Gokcumen, G. Ekin; Muro, Eleonora; Relat-Goberna, Josep; Sasse, Sofia; Bedigian, Anne; Coughlin, Margaret L.; Garcia-Manyes, Sergi; Eggert, Ulrike S.

    2014-01-01

    Summary Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology. PMID:24462247

  6. Flagellation of Pseudomonas aeruginosa in newly divided cells

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Lee, Calvin; Anda, Jaime; Wong, Gerard

    2015-03-01

    For monotrichous bacteria, Pseudomonas aeruginosa, after cell division, one daughter cell inherits the old flagellum from its mother cell, and the other grows a new flagellum during or after cell division. It had been shown that the new flagellum grows at the distal pole of the dividing cell when the two daughter cells haven't completely separated. However, for those daughter cells who grow new flagella after division, it still remains unknown at which pole the new flagellum will grow. Here, by combining our newly developed bacteria family tree tracking techniques with genetic manipulation method, we showed that for the daughter cell who did not inherit the old flagellum, a new flagellum has about 90% chances to grow at the newly formed pole. We proposed a model for flagellation of P. aeruginosa.

  7. Retroviral infection of non-dividing cells: Old and new perspectives

    SciTech Connect

    Yamashita, Masahiro; Emerman, Michael . E-mail: memerman@fhcrc.org

    2006-01-05

    The dependence of retroviral replication on cell proliferation was described as early as 1958, although different classes of retroviruses are able to infect non-dividing cells with different efficiencies. For example, the human immunodeficiency virus (HIV) and other lentiviruses infect most non-dividing cells nearly as well as dividing cells, while the gammaretroviruses such as the murine leukemia virus (MLV) cannot infect non-dividing cells, and other retroviruses have intermediate phenotypes. One exception to the ability of HIV to infect non-dividing cells involves resting CD4+ T cells in vitro where there are multiple restrictions. However, recent data show that there is massive infection of non-activated CD4+ T cell during acute infection which suggests that the situation is different in vivo. Finally, much work trying to explain the difference between HIV and MLV in non-dividing cells has focused on describing the ability of HIV to enter the nucleus during interphase. However, we suggest that events in the viral lifecycle other than nuclear import may be more important in determining the ability of a given retrovirus to infect non-dividing cells.

  8. Thymoquinone causes multiple effects, including cell death, on dividing plant cells.

    PubMed

    Hassanien, Sameh E; Ramadan, Ahmed M; Azeiz, Ahmed Z Abdel; Mohammed, Rasha A; Hassan, Sabah M; Shokry, Ahmed M; Atef, Ahmed; Kamal, Khalid B H; Rabah, Samar; Sabir, Jamal S M; Abuzinadah, Osama A; El-Domyati, Fotouh M; Martin, Gregory B; Bahieldin, Ahmed

    2013-01-01

    Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1mg/mL greatly reduced wheat seed germination rate, whereas 0.2mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1h of TQ treatment (0.2mg/mL), with severe cell death occurring in this zone after 2h of treatment. Light microscopy of TQ-treated (0.2mg/mL) onion hairy root tips for 1h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2mg/mL) for 1h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.

  9. Transcriptional Profiling of ParA and ParB Mutants in Actively Dividing Cells of an Opportunistic Human Pathogen Pseudomonas aeruginosa

    PubMed Central

    Bartosik, Aneta A.; Glabski, Krzysztof; Jecz, Paulina; Mikulska, Sylwia; Fogtman, Anna; Koblowska, Marta; Jagura-Burdzy, Grazyna

    2014-01-01

    Accurate chromosome segregation to progeny cells is a fundamental process ensuring proper inheritance of genetic material. In bacteria with simple cell cycle, chromosome segregation follows replication initiation since duplicated oriC domains start segregating to opposite halves of the cell soon after they are made. ParA and ParB proteins together with specific DNA sequences are parts of the segregation machinery. ParA and ParB proteins in Pseudomonas aeruginosa are important for optimal growth, nucleoid segregation, cell division and motility. Comparative transcriptome analysis of parAnull and parBnull mutants versus parental P. aeruginosa PAO1161 strain demonstrated global changes in gene expression pattern in logarithmically growing planktonic cultures. The set of genes similarly affected in both mutant strains is designated Par regulon and comprises 536 genes. The Par regulon includes genes controlled by two sigma factors (RpoN and PvdS) as well as known and putative transcriptional regulators. In the absence of Par proteins, a large number of genes from RpoS regulon is induced, reflecting the need for slowing down the cell growth rate and decelerating the metabolic processes. Changes in the expression profiles of genes involved in c-di-GMP turnover point out the role of this effector in such signal transmission. Microarray data for chosen genes were confirmed by RT-qPCR analysis. The promoter regions of selected genes were cloned upstream of the promoter-less lacZ gene and analyzed in the heterologous host E. coliΔlac. Regulation by ParA and ParB of P. aeruginosa was confirmed for some of the tested promoters. Our data demonstrate that ParA and ParB besides their role in accurate chromosome segregation may act as modulators of genes expression. Directly or indirectly, Par proteins are part of the wider regulatory network in P. aeruginosa linking the process of chromosome segregation with the cell growth, division and motility. PMID:24498062

  10. Effect of protistan grazing on the frequency of dividing cells in bacterioplankton assemblages.

    PubMed

    Sherr, B F; Sherr, E B; McDaniel, J

    1992-08-01

    Grazing by phagotrophic flagellates and ciliates is a major source of mortality for bacterioplankton in both marine and freshwater systems. Recent studies have demonstrated a positive relationship between clearance rate and prey size for bacterivorous protists. We tested the idea that, by selectively grazing the larger (more actively growing or dividing) cells in a bacterial assemblage, protists control bacterial standing stock abundances by directly cropping bacterial production. Samples of estuarine water were passed through 0.8-mum-pore-size filters (bacteria only) or 20-mum-mesh screens (bacteria and bacterivorous protists) and placed in dialysis tubing suspended in 7 liters of unfiltered water. Changes in total bacterial biovolume per milliliter (bacterial biomass), frequency of dividing cells (FDC), and average per cell biovolume were followed over a period of 24 h. In three experiments, the FDC increased more rapidly and attained higher values in water passed through 0.8-mum-pore-size filters (average, 5.1 to 8.9%; maximum, 15.5%) compared with FDC values in water passed through 20-mum-mesh screens (average, 2.7 to 5.3%; maximum, 6.7%). Increases in bacterial biomass per milliliter lagged behind increases in FDC by about 4 to 6 h. Grazed bacterial assemblages were characterized by lower total biomasses and smaller average cell sizes compared with those of cells in nongrazed assemblages. We conclude that bacterivorous protists control bacterial standing stock abundances partly by preferentially removing dividing cells. Selective grazing of the more actively growing cells may also explain, in part, the ability of slow-growing cells to persist in bacterioplankton assemblages.

  11. Three-dimensional structure of Drosophila testis tip: the spatial relation between dividing cells.

    PubMed

    Lee, Kyung Eun; Han, Sung Sik; Jeon, Hyesung

    2013-08-01

    At the apical tip of Drosophila testis, there is a stem cell niche known as the proliferation center, where the stem cells are maintained by hub cell cluster for the regulation of differentiation and proliferation. Germline stem cells go through mitosis four times from one primary spermatogonial cell to the 16-cell stage before the maturation. The cells derived from the same germline stem cell are located within one cyst, an enclosed system by two cyst cells, and they are connected by the intercellular bridges called ring canals. In this study, the three-dimensional (3D) structure of Drosophila testis tip was reconstructed from serial sections. The size of cells at each stage was compared in volume from the 3D structure. The stages of cells in a cyst could be distinguishable exactly by counting the cells linked with intercellular bridges in 3D-reconstructed structure. The cysts containing the same stage cells appeared in the horizontal plane. Both the germline stem cell directly attached to the hub cell and the spermatogonial cells detached from the hub cell were divided at the almost perpendicular direction to the spermatogonial cell layers. The dividing phase in one cyst was delayed gradually through the cytoplasmic region of intercellular bridge.

  12. Cell-cycle regulation in green algae dividing by multiple fission.

    PubMed

    Bišová, Kateřina; Zachleder, Vilém

    2014-06-01

    Green algae dividing by multiple fission comprise unrelated genera but are connected by one common feature: under optimal growth conditions, they can divide into more than two daughter cells. The number of daughter cells, also known as the division number, is relatively stable for most species and usually ranges from 4 to 16. The number of daughter cells is dictated by growth rate and is modulated by light and temperature. Green algae dividing by multiple fission can thus be used to study coordination of growth and progression of the cell cycle. Algal cultures can be synchronized naturally by alternating light/dark periods so that growth occurs in the light and DNA replication(s) and nuclear and cellular division(s) occur in the dark; synchrony in such cultures is almost 100% and can be maintained indefinitely. Moreover, the pattern of cell-cycle progression can be easily altered by differing growth conditions, allowing for detailed studies of coordination between individual cell-cycle events. Since the 1950s, green algae dividing by multiple fission have been studied as a unique model for cell-cycle regulation. Future sequencing of algal genomes will provide additional, high precision tools for physiological, taxonomic, structural, and molecular studies in these organisms.

  13. Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio.

    PubMed

    Reugels, Alexander M; Boggetti, Barbara; Scheer, Nico; Campos-Ortega, José A

    2006-04-01

    In the neural plate and tube of the zebrafish embryo, cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, whilst in the neural keel and rod, the spindle is oriented perpendicular to it. This change is achieved by a 90 degrees rotation of the mitotic spindle. We cloned zebrafish homologues of the gene for the Drosophila cell fate determinant Numb, and analyzed the localization of EGFP fusion proteins in vivo in dividing neuroepithelial cells during neurulation. Whereas Numb isoform 3 and the related protein Numblike are localized in the cytoplasm, Numb isoform 1 is localized to the cell membrane. Time-lapse analyses showed that Numb 1 is distributed uniformly around the cell cortex in dividing cells during plate and keel stages, but becomes localized at the basolateral membrane of some dividing cells during the transition from neural rod to tube. Using in vitro mutagenesis and Numb:EGFP deletion constructs, we showed that the first 196 amino acids of Numb are sufficient for this localization. Furthermore, we found that an 11-amino acid insertion in the PTB domain is essential for localization to the cortex, whereas amino acids 2-12 mediate the basolateral localization in the neural tube stage.

  14. Brain activity during divided and selective attention to auditory and visual sentence comprehension tasks

    PubMed Central

    Moisala, Mona; Salmela, Viljami; Salo, Emma; Carlson, Synnöve; Vuontela, Virve; Salonen, Oili; Alho, Kimmo

    2015-01-01

    Using functional magnetic resonance imaging (fMRI), we measured brain activity of human participants while they performed a sentence congruence judgment task in either the visual or auditory modality separately, or in both modalities simultaneously. Significant performance decrements were observed when attention was divided between the two modalities compared with when one modality was selectively attended. Compared with selective attention (i.e., single tasking), divided attention (i.e., dual-tasking) did not recruit additional cortical regions, but resulted in increased activity in medial and lateral frontal regions which were also activated by the component tasks when performed separately. Areas involved in semantic language processing were revealed predominantly in the left lateral prefrontal cortex by contrasting incongruent with congruent sentences. These areas also showed significant activity increases during divided attention in relation to selective attention. In the sensory cortices, no crossmodal inhibition was observed during divided attention when compared with selective attention to one modality. Our results suggest that the observed performance decrements during dual-tasking are due to interference of the two tasks because they utilize the same part of the cortex. Moreover, semantic dual-tasking did not appear to recruit additional brain areas in comparison with single tasking, and no crossmodal inhibition was observed during intermodal divided attention. PMID:25745395

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

    NASA Astrophysics Data System (ADS)

    Dalhaimer, Paul

    2013-06-01

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

  16. Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia

    PubMed Central

    O’Brien, Jason M.; Beal, Marc A.; Yauk, Carole L.; Marchetti, Francesco

    2016-01-01

    Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number of mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). MutaMouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia, and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, P < .01) and spermatogonial stem cells (2.1-fold at highest dose, P < .01). No significant increases in MF were detected in cauda sperm or seminiferous tubule cells collected 3 days post-exposure. Dose-response modelling suggested that the mutational response in male germ cells to BaP is sub-linear at low doses. Our results demonstrate that oral exposure to BaP causes spermatogonial stem cell mutations, that different phases of spermatogenesis exhibit varying sensitivities to BaP, with dividing spermatogonia representing a window of peak sensitivity, and that sampling spermatogenic cells from the seminiferous tubules at earlier time-points may underestimate germ cell mutagenicity. This information is critical to optimize the use of the international test guideline for transgenic rodent mutation assays for detecting germ cell mutagens. PMID:27208087

  17. Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia.

    PubMed

    O'Brien, Jason M; Beal, Marc A; Yauk, Carole L; Marchetti, Francesco

    2016-08-01

    Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number of mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). MutaMouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia, and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, P < .01) and spermatogonial stem cells (2.1-fold at highest dose, P < .01). No significant increases in MF were detected in cauda sperm or seminiferous tubule cells collected 3 days post-exposure. Dose-response modelling suggested that the mutational response in male germ cells to BaP is sub-linear at low doses. Our results demonstrate that oral exposure to BaP causes spermatogonial stem cell mutations, that different phases of spermatogenesis exhibit varying sensitivities to BaP, with dividing spermatogonia representing a window of peak sensitivity, and that sampling spermatogenic cells from the seminiferous tubules at earlier time-points may underestimate germ cell mutagenicity. This information is critical to optimize the use of the international test guideline for transgenic rodent mutation assays for detecting germ cell mutagens. PMID:27208087

  18. A survey of cellulose microfibril patterns in dividing, expanding, and differentiating cells of Arabidopsis thaliana.

    PubMed

    Fujita, Miki; Wasteneys, Geoffrey O

    2014-05-01

    Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.

  19. Particle-in-cell simulations of electron beam control using an inductive current divider

    DOE PAGES

    Swanekamp, S. B.; Angus, J. R.; Cooperstein, G.; Ottinger, P. F.; Richardson, A. S.; Schumer, J. W.; Weber, B. V.

    2015-11-18

    Kinetic, time-dependent, electromagnetic, particle-in-cell simulations of the inductive current divider are presented. The inductive current divider is a passive method for controlling the trajectory of an intense, hollow electron beam using a vacuum structure that inductively splits the beam’s return current. The current divider concept was proposed and studied theoretically in a previous publication [Phys. Plasmas 22, 023107 (2015)] A central post carries a portion of the return current (I1) while the outer conductor carries the remainder (I2) with the injected beam current given by Ib=I1+I2. The simulations are in agreement with the theory which predicts that the total forcemore » on the beam trajectory is proportional to (I2-I1) and the force on the beam envelope is proportional to Ib. For a fixed central post, the beam trajectory is controlled by varying the outer conductor radius which changes the inductance in the return-current path. The simulations show that the beam emittance is approximately constant as the beam propagates through the current divider to the target. As a result, independent control over both the current density and the beam angle at the target is possible by choosing the appropriate return-current geometry.« less

  20. Particle-in-cell simulations of electron beam control using an inductive current divider

    SciTech Connect

    Swanekamp, S. B.; Angus, J. R.; Cooperstein, G.; Ottinger, P. F.; Richardson, A. S.; Schumer, J. W.; Weber, B. V.

    2015-11-18

    Kinetic, time-dependent, electromagnetic, particle-in-cell simulations of the inductive current divider are presented. The inductive current divider is a passive method for controlling the trajectory of an intense, hollow electron beam using a vacuum structure that inductively splits the beam’s return current. The current divider concept was proposed and studied theoretically in a previous publication [Phys. Plasmas 22, 023107 (2015)] A central post carries a portion of the return current (I1) while the outer conductor carries the remainder (I2) with the injected beam current given by Ib=I1+I2. The simulations are in agreement with the theory which predicts that the total force on the beam trajectory is proportional to (I2-I1) and the force on the beam envelope is proportional to Ib. For a fixed central post, the beam trajectory is controlled by varying the outer conductor radius which changes the inductance in the return-current path. The simulations show that the beam emittance is approximately constant as the beam propagates through the current divider to the target. As a result, independent control over both the current density and the beam angle at the target is possible by choosing the appropriate return-current geometry.

  1. The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus.

    PubMed

    Panteris, E; Adamakis, I-D S; Chanoumidou, K

    2013-01-01

    Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co-localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co-localising with the phragmoplast. In early post-cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co-localised with the fern-specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.

  2. Particle-in-cell simulations of electron beam control using an inductive current divider

    SciTech Connect

    Swanekamp, S. B.; Angus, J. R.; Cooperstein, G.; Ottinger, P. F.; Richardson, A. S.; Schumer, J. W.; Weber, B. V.

    2015-11-15

    Kinetic, time-dependent, electromagnetic, particle-in-cell simulations of the inductive current divider are presented. The inductive current divider is a passive method for controlling the trajectory of an intense, hollow electron beam using a vacuum structure that inductively splits the beam's return current. The current divider concept was proposed and studied theoretically in a previous publication [Swanekamp et al., Phys. Plasmas 22, 023107 (2015)]. A central post carries a portion of the return current (I{sub 1}), while the outer conductor carries the remainder (I{sub 2}) with the injected beam current given by I{sub b} = I{sub 1} + I{sub 2}. The simulations are in agreement with the theory which predicts that the total force on the beam trajectory is proportional to (I{sub 2}−I{sub 1}) and the force on the beam envelope is proportional to I{sub b}. Independent control over both the current density and the beam angle at the target is possible by choosing the appropriate current-divider geometry. The root-mean-square (RMS) beam emittance (ε{sub RMS}) varies as the beam propagates through the current divider to the target. For applications where control of the beam trajectory is desired and the current density at the target is similar to the current density at the entrance foil, there is a modest 20% increase in ε{sub RMS} at the target. For other applications where the beam is pinched to a current density ∼5 times larger at the target, ε{sub RMS} is 2–3 times larger at the target.

  3. Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

    SciTech Connect

    Rutten, A.A.; Beems, R.B.; Wilmer, J.W.; Feron, V.J.

    1988-09-01

    The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated (methyl-3H)-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating (methyl-/sup 3/H)thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration.

  4. Ultrastructural studies on the Ca2+ localization in the dividing cells of the maize root tip.

    PubMed

    Sakai-Wada, A; Yagi, S

    1993-12-01

    The distribution of Ca2+ in dividing cells of the maize root tip was examined by potassium pyroantimonate precipitation and EGTA treatment methods. Ca2+ was found in most of the cell organelles, such as the matrix of the mitochondria, the thylakoid membrane of the proplastid and the Golgi vesicles, and on the plasma membrane. Ca2+ was also distributed throughout the cytoplasmic ground matrix and attractoplasm, inside the vacuoles, in the granular zone of the nucleolus in the interphasic nucleus and in the regenerated nucleolus in the telophasic nucleus. The amounts of Ca2+ distributed in the cytoplasmic ground matrix, the vacuole and the nucleolus varied during nuclear division. From the results of the present experiment, the following considerations on the role of Ca2+ and the regulation site of Ca2+ in dividing plant cells were drawn: 1) Ca2+ may play a role in the construction of the granular form of the ribosome. 2) Ca2+ may be an essential ion in the regeneration of nucleolus. 3) Vacuoles may act as the regulatory site of the Ca2+ concentration in the cytoplasm and attractoplasm in plant cells. Spindle microtubules and phragmo-microtubules are probably surrounded by other ions, such as Mg2+.

  5. Phosphate ions in root-tip dividing cells: a combined trapping and squash method with implications for nuclear transcription.

    PubMed

    Tandler, C J; Rios, H

    2001-01-01

    We examined the pattern of inorganic orthophosphate (PPi) ion distribution in dividing cells of Zea mays root-tips. Unfixed and paraformaldehyde- or glutaraldehyde-vapor fixed tissues were immersed in lead acetate, glutaraldehyde, and cacodylate buffer to capture PPi as insoluble orthophosphate lead hydroxyapatite. Excess lead ions were removed with sodium citrate, then permeabilized in ammonia. Precipitates were stained with potassium sulfide, washed with distilled water and squashed in a drop of glycerin. The accumulation of PPi ions was cyclic in the cytoplasm during mitosis and they surrounded all chromosomes during metaphase and anaphase. Partition between dividing cells started with a high concentration of PPi ions at sites where plasma membrane and cell walls formed. Small daughter cells and those in G1 phase had PPi concentrated in the nucleolus, with lower levels elsewhere in the nucleus. Later in the cell cycle, there were greater amounts of PPi ions associated with condensed chromatin in larger nuclei. In Xenopus laevis oocytes, PPi was concentrated in the nucleus, mainly in the active central core of multiple nucleoli. These results and others indicate that compartmentalization of PPi occurs in the intact cell and correlates with the rate of transcription in distinct functional domains within the nucleus. PMID:11871744

  6. Whole cell cryo-electron tomography suggests mitochondria divide by budding.

    PubMed

    Hu, Guo-Bin

    2014-08-01

    Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding. PMID:24870811

  7. Phenolic Compounds of Pomegranate Byproducts (Outer Skin, Mesocarp, Divider Membrane) and Their Antioxidant Activities.

    PubMed

    Ambigaipalan, Priyatharini; de Camargo, Adriano Costa; Shahidi, Fereidoon

    2016-08-31

    Pomegranate peel was separated into outer leathery skin (PS), mesocarp (PM), and divider membrane (PD), and its phenolic compounds were extracted as free (F), esterified (E), and insoluble-bound (B) forms for the first time. The total phenolic content followed the order PD > PM > PS. ABTS(•+), DPPH, and hydroxyl radical scavenging activities and metal chelation were evaluated. In addition, pomegranate peel extracts showed inhibitory effects against α-glucosidase activity, lipase activity, and cupric ion-induced LDL-cholesterol oxidation as well as peroxyl and hydroxyl radical-induced DNA scission. Seventy-nine phenolic compounds were identified using HPLC-DAD-ESI-MS(n) mainly in the form of insoluble-bound. Thirty compounds were identified for the first time. Gallic acid was the major phenolic compound in pomegranate peel, whereas kaempferol 3-O-glucoside was the major flavonoid. Moreover, ellagic acid and monogalloyl-hexoside were the major hydrolyzable tannins, whereas the dominant proanthocyanidin was procyanidin dimers. Proanthocyanidins were detected for the first time. PMID:27509218

  8. The divide within: Older active ICT users position themselves against different 'Others'.

    PubMed

    Kania-Lundholm, Magdalena; Torres, Sandra

    2015-12-01

    Although research into older people's internet usage patterns is rapidly growing, their understandings of digital technologies, particularly in relation to how these are informed by their understandings of aging and old age, remain unexplored. This is the case because research on older active ICT users tends to regard old age as an empirically interesting part of the life-course as opposed to a theoretically profuse source of information about why and how older people engage with digital technologies. This article explores - through focus group interviews with 30 older adults (aged 66-89) - the ways in which the social position of old age is used by older active ICT users in order to make sense of how and why they engage with these technologies. In this article, positioning theory is used to shed light on how the older people interviewed positioned themselves as 'active older users' in the interviews. The analysis brings to the fore the divide that older people themselves create as they discursively position themselves against different types of ICT users and non-users (young and old) when describing how and why they engage with digital technologies. PMID:26568212

  9. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    NASA Astrophysics Data System (ADS)

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-12-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo.

  10. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    PubMed Central

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-01-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo. PMID:26632877

  11. Combined uranous nitrate production consisting of undivided electrolytic cell and divided electrolytic cell (Electrolysis → Electrolytic cell)

    SciTech Connect

    Yuan, Zhongwei; Yan, Taihong; Zheng, Weifang; Li, Xiaodong; Yang, Hui; Xian, Liang

    2013-07-01

    The electrochemical reduction of uranyl nitrate is a green, mild way to make uranous ions. Undivided electrolyzers whose maintenance is less but their conversion ratio and current efficiency are low, have been chosen. However, at the beginning of undivided electrolysis, high current efficiency can also be maintained. Divided electrolyzers' conversion ratio and current efficiency is much higher because the re-oxidation of uranous on anode is avoided, but their maintenance costs are more, because in radioactive environment the membrane has to be changed after several operations. In this paper, a combined method of uranous production is proposed which consists of 2 stages: undivided electrolysis (early stage) and divided electrolysis (late stage) to benefit from the advantages of both electrolysis modes. The performance of the combined method was tested. The results show that in combined mode, after 200 min long electrolysis (80 min undivided electrolysis and 120 min divided electrolysis), U(IV) yield can achieve 92.3% (500 ml feed, U 199 g/l, 72 cm{sup 2} cathode, 120 mA/cm{sup 2}). Compared with divided mode, about 1/3 working time in divided electrolyzer is reduced to achieve the same U(IV) yield. If 120 min long undivided electrolysis was taken, more than 1/2 working time can be reduced in divided electrolyzer, which means that about half of the maintenance cost can also be reduced. (authors)

  12. Why do bacteria divide?

    PubMed

    Norris, Vic

    2015-01-01

    The problem of not only how but also why cells divide can be tackled using recent ideas. One idea from the origins of life - Life as independent of its constituents - is that a living entity like a cell is a particular pattern of connectivity between its constituents. This means that if the growing cell were just to get bigger the average connectivity between its constituents per unit mass - its cellular connectivity - would decrease and the cell would lose its identity. The solution is division which restores connectivity. The corollary is that the cell senses decreasing cellular connectivity and uses this information to trigger division. A second idea from phenotypic diversity - Life on the Scales of Equilibria - is that a bacterium must find strategies that allow it to both survive and grow. This means that it has learnt to reconcile the opposing constraints that these strategies impose. The solution is that the cell cycle generates daughter cells with different phenotypes based on sufficiently complex equilibrium (E) and non-equilibrium (NE) cellular compounds and structures appropriate for survival and growth, respectively, alias 'hyperstructures.' The corollary is that the cell senses both the quantity of E material and the intensity of use of NE material and then uses this information to trigger the cell cycle. A third idea from artificial intelligence - Competitive Coherence - is that a cell selects the active subset of elements that actively determine its phenotype from a much larger set of available elements. This means that the selection of an active subset of a specific size and composition must be done so as to generate both a coherent cell state, in which the cell's contents work together harmoniously, and a coherent sequence of cell states, each coherent with respect to itself and to an unpredictable environment. The solution is the use of a range of mechanisms ranging from hyperstructure dynamics to the cell cycle itself. PMID:25932025

  13. Why do bacteria divide?

    PubMed

    Norris, Vic

    2015-01-01

    The problem of not only how but also why cells divide can be tackled using recent ideas. One idea from the origins of life - Life as independent of its constituents - is that a living entity like a cell is a particular pattern of connectivity between its constituents. This means that if the growing cell were just to get bigger the average connectivity between its constituents per unit mass - its cellular connectivity - would decrease and the cell would lose its identity. The solution is division which restores connectivity. The corollary is that the cell senses decreasing cellular connectivity and uses this information to trigger division. A second idea from phenotypic diversity - Life on the Scales of Equilibria - is that a bacterium must find strategies that allow it to both survive and grow. This means that it has learnt to reconcile the opposing constraints that these strategies impose. The solution is that the cell cycle generates daughter cells with different phenotypes based on sufficiently complex equilibrium (E) and non-equilibrium (NE) cellular compounds and structures appropriate for survival and growth, respectively, alias 'hyperstructures.' The corollary is that the cell senses both the quantity of E material and the intensity of use of NE material and then uses this information to trigger the cell cycle. A third idea from artificial intelligence - Competitive Coherence - is that a cell selects the active subset of elements that actively determine its phenotype from a much larger set of available elements. This means that the selection of an active subset of a specific size and composition must be done so as to generate both a coherent cell state, in which the cell's contents work together harmoniously, and a coherent sequence of cell states, each coherent with respect to itself and to an unpredictable environment. The solution is the use of a range of mechanisms ranging from hyperstructure dynamics to the cell cycle itself.

  14. A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells

    PubMed Central

    Moriwaki, Takahito; Kato, Yuichi; Nakamura, Chihiro; Ishikawa, Satoru; Zhang-Akiyama, Qiu-Mei

    2015-01-01

    DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death. PMID:26413217

  15. Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants

    PubMed Central

    Hepworth, Shelley R.; Pautot, Véronique A.

    2015-01-01

    The initiation of plant lateral organs from the shoot apical meristem (SAM) is closely associated with the formation of specialized domains of restricted growth known as the boundaries. These zones are required in separating the meristem from the growing primordia or adjacent organs but play a much broader role in regulating stem cell activity and shoot patterning. Studies have revealed a network of genes and hormone pathways that establish and maintain boundaries between the SAM and leaves. Recruitment of these pathways is shown to underlie a variety of processes during the reproductive phase including axillary meristems production, flower patterning, fruit development, and organ abscission. This review summarizes the role of conserved gene modules in patterning boundaries throughout the life cycle. PMID:26697027

  16. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum.

    PubMed

    Swahari, Vijay; Nakamura, Ayumi; Baran-Gale, Jeanette; Garcia, Idoia; Crowther, Andrew J; Sons, Robert; Gershon, Timothy R; Hammond, Scott; Sethupathy, Praveen; Deshmukh, Mohanish

    2016-01-12

    Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells. PMID:26748703

  17. Chinese hamster ovary cell performance enhanced by a rational divide-and-conquer strategy for chemically defined medium development.

    PubMed

    Liu, Yaya; Zhang, Weiyan; Deng, Xiancun; Poon, Hong Fai; Liu, Xuping; Tan, Wen-Song; Zhou, Yan; Fan, Li

    2015-12-01

    Basal medium design is considered one of the most important steps in process development. To optimize chemically defined (CD) media efficiently and effectively for the biopharmaceutical industry, a two-step rational strategy was applied to optimize four antibody producing Chinese hamster ovary (CHO) cell lines. In the first step, 48 of 52 components of our in-house medium were divided into three groups according to their characteristics. In the next step, these groups were optimized by spent medium analysis, response surface methodology and mixture design. Because these steps in our strategy involved dividing medium components into groups and subsequently adjusting the concentration of the components, we termed this medium development strategy "divide and conquer". By applying the strategy, we were able to improve the titers of CHO-S, CHO-DG44 and two CHO-K1 cell lines 1.92, 1.86, 2.92 and 1.62-fold, respectively, in 8 weeks with fewer than 60 tests. This divide-and-conquer strategy was efficient, effective, scalable and universal in our current study and offered a new approach to CD media development.

  18. Influence of the Nutrient Medium on the Recovery of Dividing Cells from Tobacco Protoplasts 12

    PubMed Central

    Uchimiya, Hirofumi; Murashige, Toshio

    1976-01-01

    Systematic tests resulted in a nutrient solution containing the following, in milligrams per liter, for the culture of protoplasts isolated from Nicotiana tabacum L. callus cells: Murashige and Skoog salts (T. Murashige and F. Skoog, 1962. Physiol. Plant. 15: 473-497); sucrose, 15,000; mannitol, 110,000; α-naphthaleneacetic acid, 0.6; kinetin, 0-0.1; thiamine·HCl, 10; pyridoxine·HCl, 10; nicotinic acid, 5; myo-inositol, 100; and glycine, 2. In this medium, regeneration of cell wall has been observed in 85% and resumption of cell division among 35% of the protoplast isolates. PMID:16659496

  19. Valley Divide

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Context image for PIA03664 Valley Divide

    These small channels join to become Sabis Vallis.

    Image information: VIS instrument. Latitude -35.3N, Longitude 159.3E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  20. Bridging the Divide: The Role of Perceived Control in Mediating Reasoning and Activism.

    ERIC Educational Resources Information Center

    Laird, Philip G.

    2003-01-01

    Reviews the Defining Issues Test and Spheres of Control results of students involved in the pro-choice or pro-life movements. Rates participation of student involvement in on-campus activities. Reveals abortion activists more frequently endorsed moral issues and scored higher on sociopolitical issues. Discusses results based on relationships among…

  1. Electrochemical treatment of reverse osmosis concentrate on boron-doped electrodes in undivided and divided cell configurations.

    PubMed

    Bagastyo, Arseto Y; Batstone, Damien J; Kristiana, Ina; Escher, Beate I; Joll, Cynthia; Radjenovic, Jelena

    2014-08-30

    An undivided electrolytic cell may offer lower electrochlorination through reduction of chlorine/hypochlorite at the cathode. This study investigated the performance of electrooxidation of reverse osmosis concentrate using boron-doped diamond electrodes in membrane-divided and undivided cells. In both cell configurations, similar extents of chemical oxygen demand and dissolved organic carbon removal were obtained. Continuous formation of chlorinated organic compounds was observed regardless of the membrane presence. However, halogenation of the organic matter did not result in a corresponding increase in toxicity (Vibrio fischeri bioassay performed on extracted samples), with toxicity decreasing slightly until 10AhL(-1), and generally remaining near the initial baseline-toxicity equivalent concentration (TEQ) of the raw concentrate (i.e., ∼2mgL(-1)). The exception was a high range toxicity measure in the undivided cell (i.e., TEQ=11mgL(-1) at 2.4AhL(-1)), which rapidly decreased to 4mgL(-1). The discrepancy between the halogenated organic matter and toxicity patterns may be a consequence of volatile and/or polar halogenated by-products formed in oxidation by OH electrogenerated at the anode. The undivided cell exhibited lower energy compared to the divided cell, 0.25kWhgCOD(-1) and 0.34kWhgCOD(-1), respectively, yet it did not demonstrate any improvement regarding by-products formation.

  2. Electrochemical treatment of reverse osmosis concentrate on boron-doped electrodes in undivided and divided cell configurations.

    PubMed

    Bagastyo, Arseto Y; Batstone, Damien J; Kristiana, Ina; Escher, Beate I; Joll, Cynthia; Radjenovic, Jelena

    2014-08-30

    An undivided electrolytic cell may offer lower electrochlorination through reduction of chlorine/hypochlorite at the cathode. This study investigated the performance of electrooxidation of reverse osmosis concentrate using boron-doped diamond electrodes in membrane-divided and undivided cells. In both cell configurations, similar extents of chemical oxygen demand and dissolved organic carbon removal were obtained. Continuous formation of chlorinated organic compounds was observed regardless of the membrane presence. However, halogenation of the organic matter did not result in a corresponding increase in toxicity (Vibrio fischeri bioassay performed on extracted samples), with toxicity decreasing slightly until 10AhL(-1), and generally remaining near the initial baseline-toxicity equivalent concentration (TEQ) of the raw concentrate (i.e., ∼2mgL(-1)). The exception was a high range toxicity measure in the undivided cell (i.e., TEQ=11mgL(-1) at 2.4AhL(-1)), which rapidly decreased to 4mgL(-1). The discrepancy between the halogenated organic matter and toxicity patterns may be a consequence of volatile and/or polar halogenated by-products formed in oxidation by OH electrogenerated at the anode. The undivided cell exhibited lower energy compared to the divided cell, 0.25kWhgCOD(-1) and 0.34kWhgCOD(-1), respectively, yet it did not demonstrate any improvement regarding by-products formation. PMID:25048621

  3. Physical activity profiles and selected muscular fitness variables in English schoolchildren: A north-south divide?

    PubMed

    Ingle, Lee; Stephenson, Ashlie; Sandercock, Gavin R

    2016-11-01

    The aim of the study was to compare and contrast habitual physical activity (PA) profiles and muscular fitness in schoolchildren from northern and southern regions of England. Data were collected from two secondary schools in the north east (NE) of England. The study procedures followed methods employed by the East of England Healthy Hearts Study in 10-16-year-old boys and girls based in the south east (SE) region of England and data were compared. Habitual physical activity (PAQ-A), vertical jump test, and hand-grip (HG) strength were assessed. We converted raw scores from all assessments to age- and sex-normalised z-scores. We recruited 597 children (58% boys) in the NE and compared findings to 597 age- and sex-matched boys and girls from the SE. Boys in the SE had significantly stronger HG scores, jumped higher, were more powerful (mean peak power: 2131 W vs. 1782 W; P < 0.0001), and reported being more physically active (mean PAQ-A: 2.9 vs. 2.5; P < 0.0001) than their male counterparts in the NE. In girls, the opposite trend was evident. Girls from the NE of England had a higher HG score, jumped higher, and were more powerful (mean peak power: 2114 W vs. 1839 W; P < .0001) than their peers from the SE. Regional variations in the habitual PA profiles and muscular fitness of schoolchildren from the SE and NE of England do exist. The systematic surveillance of children's PA and fitness profiles throughout England would help identify regional inequalities on a larger scale.

  4. Resuscitation promoting factor (Rpf) from Tomitella biformata AHU 1821(T) promotes growth and resuscitates non-dividing cells.

    PubMed

    Dewi Puspita, Indun; Uehara, Moe; Katayama, Taiki; Kikuchi, Yoshitomo; Kitagawa, Wataru; Kamagata, Yoichi; Asano, Kozo; Nakatsu, Cindy H; Tanaka, Michiko

    2013-01-01

    Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821(T), a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.

  5. Phosphotyrosyl phosphatase activator facilitates localization of Miranda through dephosphorylation in dividing neuroblasts.

    PubMed

    Zhang, Fan; Huang, Zhen-Xing; Bao, Hongcun; Cong, Fei; Wang, Huashan; Chai, Phing Chian; Xi, Yongmei; Ge, Wanzhong; Somers, W Gregory; Yang, Ying; Cai, Yu; Yang, Xiaohang

    2016-01-01

    The mechanism for the basal targeting of the Miranda (Mira) complex during the asymmetric division of Drosophila neuroblasts (NBs) is yet to be fully understood. We have identified conserved Phosphotyrosyl phosphatase activator (PTPA) as a novel mediator for the basal localization of the Mira complex in larval brain NBs. In mutant Ptpa NBs, Mira remains cytoplasmic during early mitosis and its basal localization is delayed until anaphase. Detailed analyses indicate that PTPA acts independent of and before aPKC to localize Mira. Mechanistically, our data show that the phosphorylation status of the T591 residue determines the subcellular localization of Mira and that PTPA facilitates the dephosphorylation of T591. Furthermore, PTPA associates with the Protein phosphatase 4 complex to mediate localization of Mira. On the basis of these results, a two-step process for the basal localization of Mira during NB division is revealed: cortical association of Mira mediated by the PTPA-PP4 complex is followed by apical aPKC-mediated basal restriction.

  6. Lymphocyte surface membrane changes in dividing cells and following regidification with mitogens.

    PubMed

    Lewis, C M; Pegrum, G D

    1977-03-01

    Iodination of surface membrane protein has allowed the dynamics of lymphocyte surface membrane protein to be examined. A comparison has been made between the rate of membrane turnover in fresh peripheral blood lymphocytes and lymphocytes in other states. When receptor redistribution is inhibited with high concentrations of mitogens, 'rigidification' of the membrane occurs and protein turnover is very much reduced. Loss of surface membrane protein is also much slower in lymphocytes which have been stimulated with mitogens and are undergoing active DNA synthesis. It is thought that these observations may relate to immunological inactivity and may have implications for pathological unresponsiveness.

  7. The Responses of Hyperglycemic Dividing Mesangial Cells to Heparin Are Mediated by the Non-reducing Terminal Trisaccharide.

    PubMed

    Wang, Christina P; Hascall, Vincent C; Zhang, Fuming; Linhardt, Robert J; Abbadi, Amina; Wang, Aimin

    2015-11-27

    Our previous studies showed: (i) that growth-arrested G0/G1 rat mesangial cells stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy and the cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division; and (ii) that heparin inhibits the intracellular hyaluronan and autophagy responses, but after completing division, induces hyaluronan synthesis at the plasma membrane with the formation of a larger monocyte-adhesive hyaluronan matrix. This study shows: (i) that the non-terminal trisaccharide of heparin is sufficient to initiate the same responses as intact heparin, (ii) that a fully sulfated tetrasaccharide isolated from bacterial heparin lyase 1 digests of heparin that contains a Δ-2S-iduronate on the non-reducing end does not initiate the same responses as intact heparin, and (iii) that removal of the Δ-2S-iduronate to expose the fully sulfated trisaccharide (GlcNS(6S)-IdoUA(2S)-GlcNS(6S)) does initiate the same responses as intact heparin. These results provide evidence that mammalian heparanase digestion of heparin and heparan sulfate exposes a cryptic motif on the non-reducing termini that is recognized by a receptor on dividing cells.

  8. Bacterioplankton in antarctic ocean waters during late austral winter: abundance, frequency of dividing cells, and estimates of production.

    PubMed

    Hanson, R B; Shafer, D; Ryan, T; Pope, D H; Lowery, H K

    1983-05-01

    Bacterioplankton productivity in Antarctic waters of the eastern South Pacific Ocean and Drake Passage was estimated by direct counts and frequency of dividing cells (FDC). Total bacterioplankton assemblages were enumerated by epifluorescent microscopy. The experimentally determined relationship between in situ FDC and the potential instantaneous growth rate constant (mu) is best described by the regression equation ln mu = 0.081 FDC - 3.73. In the eastern South Pacific Ocean, bacterioplankton abundance (2 x 10 to 3.5 x 10 cells per ml) and FDC (11%) were highest at the Polar Front (Antarctic Convergence). North of the Subantarctic Front, abundance and FDC were between 1 x 10 to 2 x 10 cells per ml and 3 to 5%, respectively, and were vertically homogeneous to a depth of 600 m. In Drake Passage, abundance (10 x 10 cells per ml) and FDC (16%) were highest in waters south of the Polar Front and near the sea ice. Subantarctic waters in Drake Passage contained 4 x 10 cells per ml with 4 to 5% FDC. Instantaneous growth rate constants ranged between 0.029 and 0.088 h. Using estimates of potential mu and measured standing stocks, we estimated productivity to range from 0.62 mug of C per liter . day in the eastern South Pacific Ocean to 17.1 mug of C per liter . day in the Drake Passage near the sea ice. PMID:16346297

  9. Evidence for Subglacial Volcanic Activity Beneath the area of the Divide of the West Antarctic Ice Sheet

    NASA Astrophysics Data System (ADS)

    Behrendt, J. C.

    2013-12-01

    There is an increasing body of aeromagnetic, radar ice-sounding, heat flow, subglacial volcanic earthquakes, several exposed active and subglacial volcanoes and other lines of evidence for volcanic activity associated with the West Antarctic Rift System (WR) since the origin (~25 Ma) of the West Antarctic Ice Sheet (WAIS), which flows through it. Exposed late Cenozoic, alkaline volcanic rocks, 34 Ma to present concentrated in Marie Byrd Land (LeMasurier and Thomson, 1990), but also exposed along the rift shoulder on the Transantarctic Mountains flank of the WR, and >1 million cubic kilometers, of mostly subglacially erupted 'volcanic centers' beneath the WAIS inferred from aeromagnetic data, have been interpreted as evidence of a magmatic plume. About 18 high relief, (~600-2000 m) 'volcanic centers' presently beneath the WAIS surface, probably were erupted subaerially when the WAIS was absent, based on the 5-km orthogonally line spaced Central West Antarctica aerogeophysical survey. All would be above sea level after ice removal and isostatic adjustment. Nine of these high relief peaks are in the general area beneath the divide of the WAIS. This high bed relief topography was first interpreted in the 1980s as the volcanic 'Sinuous Ridge ' based on a widely spaced aeromagnetic -radar ice sounding survey (Jankowski et al,. 1983). A 70-km wide, circular ring of interpreted subglacial volcanic rocks was cited as evidence of a volcanic caldera underlying the ice sheet divide based on the CWA survey (Behrendt et al., 1998). A broad magnetic 'low' surrounding the caldera area possibly is evidence of a shallow Curie isotherm. High heat flow reported from temperature logging (Clow et al., 2012) in the WAISCORE and a thick volcanic ash layer in the core (Dunbar et al., 2012) are consistent with this interpretation. A 2 km-high subaerially erupted volcano (subglacial Mt Thiel, ~78.5 degrees S, 111 degrees W) ~ 100 km north from the WAISCORE could be the source of the ash

  10. The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

    PubMed

    Collings, David A; Harper, John D I; Vaughn, Kevin C

    2003-12-01

    We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

  11. Why Cells Grow and Divide? General Growth Mechanism and How it Defines Cells’ Growth, Reproduction and Metabolic Properties

    NASA Astrophysics Data System (ADS)

    Shestopaloff, Yuri K.

    2015-02-01

    We consider a general growth mechanism, which acts at cellular level and above (organs, systems and whole organisms). Using its mathematical representation, the growth equation, we study the growth and division mechanisms of amoeba and fission yeast Schizosaccharomyces pombe. We show how this mechanism, together with biomolecular machinery, governs growth and reproduction of cells, and these organisms in particular. This mechanism provides revealing answers to fundamental questions of biology, like why cells grow and divide, why and when cells’ growth stops. It also sheds light on questions like why and how life originated and developed. Solving the growth equation, we obtain analytical expression for the growth curve of fission yeast as a function of geometrical characteristics and nutrient influxes for RNA and protein synthesis, and compare the computed growth curves with 85 experiments. Statistical evaluation shows that these growth curves correspond to experimental data significantly better than all previous approximations. Also, using the general growth mechanism, we show how metabolic characteristics of cells, their size and evolutionary traits relate, considering fission yeast. In particular, we found that fission yeast S. pombe consumes about 16-18 times more nutrients for maintenance needs than for biomass synthesis.

  12. Bisphenol A disrupts microtubules and induces multipolar spindles in dividing root tip cells of the gymnosperm Abies cephalonica.

    PubMed

    Adamakis, Ioannis-Dimosthenis S; Panteris, Emmanuel; Eleftheriou, Eleftherios P

    2016-04-01

    The effects of bisphenol A (BPA), an endocrine chemical disruptor extensively used in the plastic and epoxy resin industry, on dividing root tip cells of the gymnosperm Abies cephalonica Loudon were investigated by confocal laser scanning microscopy after tubulin and endoplasmic reticulum immunolocalization and DNA staining. Microtubule arrays of all mitotic stages were disrupted within a few hours of treatment: preprophase bands exhibited asymmetric width; prometaphase, metaphase and anaphase spindles appeared sharply pointed, sigmoid or multipolar; phragmoplast microtubules were elongated and occasionally bended toward the daughter nuclei. Depending on the mitotic stage, the chromosomes appeared condensed at prophase, as a compact mass at metaphase and anaphase, unsegregated or bridged at telophase. Endoplasmic reticulum patterns were also affected, reflecting those of the respective microtubule arrays. Recovery of the microtubules after oryzalin treatment was more effective in a BPA solution than in water. It is concluded that the plant mitotic apparatus microtubules are very sensitive to BPA, the effect of which depends on the specific cell cycle stage. The formation of multipolar spindles is reminiscent of animal cells and is ascribed to the induction of multiple microtubule nucleation sites, deriving from the centrosomal properties of gymnosperms. PMID:26855225

  13. Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE).

    PubMed

    Deleyrolle, Loic P; Rohaus, Mark R; Fortin, Jeff M; Reynolds, Brent A; Azari, Hassan

    2012-01-01

    Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia(2-8). The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma(2,7,8). The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds(9). The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division(10). CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity(2). This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry(2). The technique has served to identify

  14. Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro

    NASA Technical Reports Server (NTRS)

    Conrad, A. H.; Jaffredo, T.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

  15. Using RuO(2) anode for chlorine dioxide production in an un-divided electrochemical cell.

    PubMed

    Chandrasekara Pillai, K; Kwon, Tae Ok; Park, Bo Bae; Moon, Il Shik

    2010-01-01

    Chlorine dioxide is a well known powerful disinfectant. Although there are several chemical and electrochemical methods developed for on-line chlorine dioxide generation, the details are mostly confined as patents. We studied in this work the electrochemical generation of dissolved chlorine dioxide from an un-buffered solution of sodium chlorite and sodium chloride mixture in an un-divided electrochemical cell set-up with RuO(2)-coated-Ti anode and Pt-coated-Ti cathode under constant current mode. Various process parameters including feed flow rate (10 to 150 ml/min), feed solution pH (2.3 to 9.4), concentration of sodium chloride (0 to 170 mM), concentration of sodium chlorite (0 to 7.7 mM), and the applied current (100 to 1,200 mA) were optimized. Experiments were conducted by performing single pass experiments, with no circulation. The current efficiency and the power consumption were calculated for the optimized conditions, and compared with IrO(2) electrode of our previous investigation. PMID:20389015

  16. Hyperglycemia Diverts Dividing Osteoblastic Precursor Cells to an Adipogenic Pathway and Induces Synthesis of a Hyaluronan Matrix That Is Adhesive for Monocytes*

    PubMed Central

    Wang, Aimin; Midura, Ronald J.; Vasanji, Amit; Wang, Andrew J.; Hascall, Vincent C.

    2014-01-01

    Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21–31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone. PMID:24569987

  17. Asymmetric constriction of dividing Escherichia coli cells induced by expression of a fusion between two min proteins.

    PubMed

    Rowlett, Veronica Wells; Margolin, William

    2014-06-01

    The Min system, consisting of MinC, MinD, and MinE, plays an important role in localizing the Escherichia coli cell division machinery to midcell by preventing FtsZ ring (Z ring) formation at cell poles. MinC has two domains, MinCn and MinCc, which both bind to FtsZ and act synergistically to inhibit FtsZ polymerization. Binary fission of E. coli usually proceeds symmetrically, with daughter cells at roughly 180° to each other. In contrast, we discovered that overproduction of an artificial MinCc-MinD fusion protein in the absence of other Min proteins induced frequent and dramatic jackknife-like bending of cells at division septa, with cell constriction predominantly on the outside of the bend. Mutations in the fusion known to disrupt MinCc-FtsZ, MinCc-MinD, or MinD-membrane interactions largely suppressed bending division. Imaging of FtsZ-green fluorescent protein (GFP) showed no obvious asymmetric localization of FtsZ during MinCc-MinD overproduction, suggesting that a downstream activity of the Z ring was inhibited asymmetrically. Consistent with this, MinCc-MinD fusions localized predominantly to segments of the Z ring at the inside of developing cell bends, while FtsA (but not ZipA) tended to localize to the outside. As FtsA is required for ring constriction, we propose that this asymmetric localization pattern blocks constriction of the inside of the septal ring while permitting continued constriction of the outside portion. PMID:24682325

  18. Asymmetric Constriction of Dividing Escherichia coli Cells Induced by Expression of a Fusion between Two Min Proteins

    PubMed Central

    Rowlett, Veronica Wells

    2014-01-01

    The Min system, consisting of MinC, MinD, and MinE, plays an important role in localizing the Escherichia coli cell division machinery to midcell by preventing FtsZ ring (Z ring) formation at cell poles. MinC has two domains, MinCn and MinCc, which both bind to FtsZ and act synergistically to inhibit FtsZ polymerization. Binary fission of E. coli usually proceeds symmetrically, with daughter cells at roughly 180° to each other. In contrast, we discovered that overproduction of an artificial MinCc-MinD fusion protein in the absence of other Min proteins induced frequent and dramatic jackknife-like bending of cells at division septa, with cell constriction predominantly on the outside of the bend. Mutations in the fusion known to disrupt MinCc-FtsZ, MinCc-MinD, or MinD-membrane interactions largely suppressed bending division. Imaging of FtsZ-green fluorescent protein (GFP) showed no obvious asymmetric localization of FtsZ during MinCc-MinD overproduction, suggesting that a downstream activity of the Z ring was inhibited asymmetrically. Consistent with this, MinCc-MinD fusions localized predominantly to segments of the Z ring at the inside of developing cell bends, while FtsA (but not ZipA) tended to localize to the outside. As FtsA is required for ring constriction, we propose that this asymmetric localization pattern blocks constriction of the inside of the septal ring while permitting continued constriction of the outside portion. PMID:24682325

  19. The nondeterministic divide

    NASA Technical Reports Server (NTRS)

    Charlesworth, Arthur

    1990-01-01

    The nondeterministic divide partitions a vector into two non-empty slices by allowing the point of division to be chosen nondeterministically. Support for high-level divide-and-conquer programming provided by the nondeterministic divide is investigated. A diva algorithm is a recursive divide-and-conquer sequential algorithm on one or more vectors of the same range, whose division point for a new pair of recursive calls is chosen nondeterministically before any computation is performed and whose recursive calls are made immediately after the choice of division point; also, access to vector components is only permitted during activations in which the vector parameters have unit length. The notion of diva algorithm is formulated precisely as a diva call, a restricted call on a sequential procedure. Diva calls are proven to be intimately related to associativity. Numerous applications of diva calls are given and strategies are described for translating a diva call into code for a variety of parallel computers. Thus diva algorithms separate logical correctness concerns from implementation concerns.

  20. Fully Automatic Determination of Soil Bacterium Numbers, Cell Volumes, and Frequencies of Dividing Cells by Confocal Laser Scanning Microscopy and Image Analysis

    PubMed Central

    Bloem, J.; Veninga, M.; Shepherd, J.

    1995-01-01

    We describe a fully automatic image analysis system capable of measuring cell numbers, volumes, lengths, and widths of bacteria in soil smears. The system also determines the number of cells in agglomerates and thus provides the frequency of dividing cells (FDC). Images are acquired from a confocal laser scanning microscope. The grey images are smoothed by convolution and by morphological erosion and dilation to remove noise. The background is equalized by flooding holes in the image and is then subtracted by two top hat transforms. Finally, the grey image is sharpened by delineation, and all particles above a fixed threshold are detected. The number of cells in each detected particle is determined by counting the number of local grey-level maxima in the particle. Thus, up to 1,500 cells in 10 fields of view in a soil smear are analyzed in 30 min without human intervention. Automatic counts of cell numbers and FDC were similar to visual counts in field samples. In microcosms, automatic measurements showed significant increases in cell numbers, FDC, mean cell volume, and length-to-width ratio after amendment of the soil. Volumes of fluorescent microspheres were measured with good approximation, but the absolute values obtained were strongly affected by the settings of the detector sensitivity. Independent measurements of bacterial cell numbers and volumes by image analysis and of cell carbon by a total organic carbon analyzer yielded an average specific carbon content of 200 fg of C (mu)m(sup-3), which indicates that our volume estimates are reasonable. PMID:16534976

  1. Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

    PubMed Central

    Walsh, James C.; Angstmann, Christopher N.; Duggin, Iain G.; Curmi, Paul M. G.

    2015-01-01

    Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo. PMID:26018614

  2. Positioning the Flagellum at the Center of a Dividing Cell To Combine Bacterial Division with Magnetic Polarity

    PubMed Central

    Bennet, Mathieu; Klumpp, Stefan

    2015-01-01

    ABSTRACT Faithful replication of all structural features is a sine qua non condition for the success of bacterial reproduction by binary fission. For some species, a key challenge is to replicate and organize structures with multiple polarities. Polarly flagellated magnetotactic bacteria are the prime example of organisms dealing with such a dichotomy; they have the challenge of bequeathing two types of polarities to their daughter cells: magnetic and flagellar polarities. Indeed, these microorganisms align and move in the Earth’s magnetic field using an intracellular chain of nano-magnets that imparts a magnetic dipole to the cell. The paradox is that, after division occurs in cells, if the new flagellum is positioned opposite to the old pole devoid of a flagellum during cell division, the two daughter cells will have opposite magnetic polarities with respect to the positions of their flagella. Here we show that magnetotactic bacteria of the class Gammaproteobacteria pragmatically solve this problem by synthesizing a new flagellum at the division site. In addition, we model this particular structural inheritance during cell division. This finding opens up new questions regarding the molecular aspects of the new division mechanism, the way other polarly flagellated magnetotactic bacteria control the rotational direction of their flagella, and the positioning of organelles. PMID:25714711

  3. A divide-and-conquer strategy in tumor sampling enhances detection of intratumor heterogeneity in routine pathology: A modeling approach in clear cell renal cell carcinoma.

    PubMed

    Lopez, José I; Cortes, Jesús M

    2016-01-01

    Intratumor heterogeneity (ITH) is an inherent process in cancer development which follows for most of the cases a branched pattern of evolution, with different cell clones evolving independently in space and time across different areas of the same tumor. The determination of ITH (in both spatial and temporal domains) is nowadays critical to enhance patient treatment and prognosis. Clear cell renal cell carcinoma (CCRCC) provides a good example of ITH. Sometimes the tumor is too big to be totally analyzed for ITH detection and pathologists decide which parts must be sampled for the analysis. For such a purpose, pathologists follow internationally accepted protocols. In light of the latest findings, however, current sampling protocols seem to be insufficient for detecting ITH with significant reliability. The arrival of new targeted therapies, some of them providing promising alternatives to improve patient survival, pushes the pathologist to obtain a truly representative sampling of tumor diversity in routine practice. How large this sampling must be and how this must be performed are unanswered questions so far.  Here we present a very simple method for tumor sampling that enhances ITH detection without increasing costs. This method follows a divide-and-conquer (DAC) strategy, that is, rather than sampling a small number of large-size tumor-pieces as the routine protocol (RP) advises, we suggest sampling many small-size pieces along the tumor. We performed a computational modeling approach to show that the usefulness of the DAC strategy is twofold: first, we show that DAC outperforms RP with similar laboratory costs, and second, DAC is capable of performing similar to total tumor sampling (TTS) but, very remarkably, at a much lower cost. We thus provide new light to push forward a shift in the paradigm about how pathologists should sample tumors for achieving efficient ITH detection. PMID:27127618

  4. A divide-and-conquer strategy in tumor sampling enhances detection of intratumor heterogeneity in routine pathology: A modeling approach in clear cell renal cell carcinoma.

    PubMed

    Lopez, José I; Cortes, Jesús M

    2016-01-01

    Intratumor heterogeneity (ITH) is an inherent process in cancer development which follows for most of the cases a branched pattern of evolution, with different cell clones evolving independently in space and time across different areas of the same tumor. The determination of ITH (in both spatial and temporal domains) is nowadays critical to enhance patient treatment and prognosis. Clear cell renal cell carcinoma (CCRCC) provides a good example of ITH. Sometimes the tumor is too big to be totally analyzed for ITH detection and pathologists decide which parts must be sampled for the analysis. For such a purpose, pathologists follow internationally accepted protocols. In light of the latest findings, however, current sampling protocols seem to be insufficient for detecting ITH with significant reliability. The arrival of new targeted therapies, some of them providing promising alternatives to improve patient survival, pushes the pathologist to obtain a truly representative sampling of tumor diversity in routine practice. How large this sampling must be and how this must be performed are unanswered questions so far.  Here we present a very simple method for tumor sampling that enhances ITH detection without increasing costs. This method follows a divide-and-conquer (DAC) strategy, that is, rather than sampling a small number of large-size tumor-pieces as the routine protocol (RP) advises, we suggest sampling many small-size pieces along the tumor. We performed a computational modeling approach to show that the usefulness of the DAC strategy is twofold: first, we show that DAC outperforms RP with similar laboratory costs, and second, DAC is capable of performing similar to total tumor sampling (TTS) but, very remarkably, at a much lower cost. We thus provide new light to push forward a shift in the paradigm about how pathologists should sample tumors for achieving efficient ITH detection.

  5. Cloning of genes whose expression is correlated with mitosis and localized in dividing cells in root caps of Pisum sativum L.

    PubMed

    Woo, H H; Hawes, M C

    1997-12-01

    Removal of border cells from pea roots synchronizes and induces root cap cell division, wall biogenesis and differentiation. Three messages which are expressed differentially in such induced root caps have been cloned. Sequence analyses showed that the PsHRGP1-encoded protein has high homology with a homology with a hydroxyproline-rich glycoprotein. The PsCaP23-encoded protein has high homology with an alfalfa callus protein or translationally controlled human or mouse tumor protein P23. The PsRbL41-encoded protein has high homology with a highly basic 60S ribosomal protein L41. In situ hybridization showed that PsHRGP1. PsCaP23 and PsRbL41 messages are localized within dividing cells of the root cap. PsHRGP1 is highly expressed in uninduced root caps, but its message is repressed by 10-11 times as soon as cell division and differentiation begin. Expression of PsHRGP1 recovers to higher than (180%) its initial level in 30 min. PsHRGP1 is root-specific. PsCaP23 and PsRbL41 messages increase ca. 3-fold within 15 min after root cap induction. All three genes represent small families of 3-5 closely related genes in the pea genome.

  6. Digital Leadership Divide

    ERIC Educational Resources Information Center

    Consortium for School Networking (NJ1), 2004

    2004-01-01

    This 2004 survey from the Consortium for School Networking (CoSN) and Grunwald Associates includes data from 455 school district decision-makers for technology. The report points to large and growing disparities in funding for school technology and questions if these differences signal a growing digital divide. The survey reveals, schools are…

  7. Studies on process parameters for chlorine dioxide production using IrO2 anode in an un-divided electrochemical cell.

    PubMed

    Pillai, K Chandrasekara; Kwon, Tae Ouk; Park, Bo Bae; Moon, Il Shik

    2009-05-30

    Chlorine dioxide is potentially a powerful oxidant with environmentally compatible application in several strategic areas relating to pollution control typically for water disinfection, and its sustained production is a key factor for its successful application. Although increased attention has been paid for on-line chlorine dioxide generation by several chemical and electrochemical methods, the details are mostly confined as patents. We studied in this work the electrochemical generation of chlorine dioxide from an un-buffered solution of sodium chlorite and sodium chloride mixture in an un-divided electrochemical cell under constant current mode, with a view to optimize various process parameters, which have a direct bearing on the chlorine dioxide formation efficiency under laboratory conditions. The effect of feed flow rate (10-150 ml min(-1)), feed solution pH (2.3-5.0), concentration of sodium chloride (0-169.4mM), concentration of sodium chlorite (0-7.7 mM), and the applied current (100-1200 mA) on the formation of dissolved ClO(2) gas in solution and the pH of the product-containing solution was investigated by performing single pass experiments, with no circulation, in a cell set-up with Ti/IrO(2) anode and Ti/Pt cathode. The current efficiency and the power consumption were calculated for the optimized conditions. PMID:18838217

  8. Melting the Divide

    NASA Astrophysics Data System (ADS)

    Gibson, S. M.

    2014-12-01

    Presenting Quaternary Environmental Change to students who fall into Widening Participation criteria at the University of Cambridge, gives a unique opportunity to present academic debate in an approachable and entertaining way. Literally by discussing the melting of our ice caps, melts the divide Cambridge has between its reputation and the reality for the brightest, underprivileged, students. There is a balance between presenting cutting edge research with the need to come across as accessible (and importantly valuable to "learning"). Climate change over the Quaternary lends itself well to this aim. By lecturing groups of potential students through the entire Quaternary in an hour, stopping to discuss how our ancestors interacted with past Interglacials and what are the mechanisms driving change (in generalized terms), you are able to introduce cutting edge research (such as the latest NEEM ice core) to the students. This shows the evolution and importance of higher education and academic research. The lecture leads well onto group discussions (termed "supervisions" in Cambridge), to explore their opinions on the concern for present Anthropogenic Climate Change in relation to Past Climate Change after being presented with images that our ancestors "made it". Here discussion thrives off students saying obvious things (or sarcastic comments!) which quickly can lead into a deep technical discussion on their terms. Such discussions give the students a zest for higher education, simply throwing Ruddiman's (2003) "The Anthroprocene Started Several Thousand Years Ago" at them, questions in a second their concept of Anthropogenic Climate Change. Supervisions lend themselves well to bright, articulate, students and by offering these experiences to students of Widening Participation criteria we quickly melt the divide between the reputation of Cambridge ( and higher education as a whole) and the day to day practice. Higher education is not for the privileged, but a free and

  9. Monitoring the Digital Divide

    SciTech Connect

    Cottrell, Les

    2003-05-28

    It is increasingly important to support the large numbers of scientists working in remote areas and having low bandwidth access to the Internet. This will continue to be the case for years to come since there is evidence from PingER performance measurements that the, so-called, digital divide is not decreasing. In this work, we review the collaborative work of The Abdus Salam International Center for Theoretical Physics (ICTP) in Trieste, a leading organization promoting science dissemination in the developing world- and SLAC in Stanford, to monitor by PingER, Universities and Research Institutions all over the developing world following the recent ''Recommendations of Trieste'' to help bridge the digital divide. As a result, PingER's deployment now covers the real-time monitoring of worldwide Internet performance and, in particular, West and Central Africa for the first time. We report on the results from the ICTP sites and quantitatively identify regions with poor performance, identify trends, discuss experiences and future work.

  10. PULSE RATE DIVIDER

    DOEpatents

    McDonald, H.C. Jr.

    1962-12-18

    A compact pulse-rate divider circuit affording low impedance output and high input pulse repetition rates is described. The circuit features a single secondary emission tube having a capacitor interposed between its dynode and its control grid. An output pulse is produced at the anode of the tube each time an incoming pulse at the control grid drives the tube above cutoff and the duration of each output pulse corresponds to the charging time of the capacitor. Pulses incoming during the time the grid bias established by the discharging capacitor is sufficiently negative that the pulses are unable to drive the tube above cutoff do not produce output pulses at the anode; these pulses are lost and a dividing action is thus produced by the circuit. The time constant of the discharge path may be vanied to vary in turn the division ratio of the circuit; the time constant of the charging circuit may be varied to vary the width of the output pulses. (AEC)

  11. Cell sedimentation with gravity activation.

    PubMed

    Czerlinski, G; Goldman-Leikin, R; Reid, D

    1988-12-01

    Murine monoclonal antibody T101 has been coupled to thinly polymer-coated heavy alloy particles (LaMn2Ge2). These conjugates are coupled to cultured cells of the human T-cell leukemia line RPMI 8402 (T8402). The sedimentation velocities of cells, of particles, and of cells with particles attached are measured. After determining the mean radii of cells, of particles, and of cells with particles attached, one may compute a mean number of 33 particles attached to a cell. Independently one may compute a mean number of 144 particles/cell for surface saturation. The Appendix handles the underlying theory in three parts: number of particles/cell, saturation number of particles/cell, and resolution for gravity activation. Regarding the latter, cell radii from 4 to 10 microns and particle radii from 0.01 to 1 micron are considered.

  12. Fundamental Limitations Of Passive Power Dividers

    NASA Technical Reports Server (NTRS)

    Antsos, Dimitrios

    1995-01-01

    Report presents novel theoretical analysis of performance of passive, multiport power dividers like those used to distribute power to radiating elements of array antennas. Typically, unit cell of power-divider network is three- or four-port subnetwork with one port terminated, and subnetworks of network cascaded in multiple layers, so that insertion losses are of major concern.

  13. Divide and conquer: Blocking graft versus host but not graft versus leukemia T cells with agonist BTLA co-inhibitory signals.

    PubMed

    Thangavelu, Govindarajan; Anderson, Colin C

    2011-01-01

    One of the main objectives in allogeneic hematopoietic stem cell transplantation (aHSCT) research is the prevention of graft versus host disease (GVHD) while maintaining the graft versus leukemia/lymphoma (GVL) effect. Whether these two responses generated by donor T cells can be sufficiently separated and controlled remains controversial. While various approaches have been tested to achieve this goal, success has been relatively limited. Lymphocyte responses are negatively regulated by a series of receptors that function along with antigen receptors to deliver co-inhibitory signals. B and T lymphocyte associated (BTLA) is a novel co-inhibitory molecule expressed by activated T cells, B cells and other immune cells. A study by Albring et al. has now shown in a murine model that a single injection of agonistic anti-BTLA monoclonal antibody can inhibit GVHD long-term while maintaining GVL responses and immunity to infection. These studies suggest that future development of biologics to harness the function of co-inhibitory signals will be an important approach in the prevention of autoimmunity and GVHD and in protocols to achieve transplantation tolerance.

  14. The Digital Divide

    ERIC Educational Resources Information Center

    Hudson, Hannah Trierweiler

    2011-01-01

    Megan is a 14-year-old from Nebraska who just started ninth grade. She has her own digital camera, cell phone, Nintendo DS, and laptop, and one or more of these devices is usually by her side. Compared to the interactions and exploration she's engaged in at home, Megan finds the technology in her classroom falls a little flat. Most of the…

  15. The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle

    ERIC Educational Resources Information Center

    Scherer, Yvette D.

    2014-01-01

    In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

  16. Acoustofluidic Fluorescence Activated Cell Sorter.

    PubMed

    Nawaz, Ahmad Ahsan; Chen, Yuchao; Nama, Nitesh; Nissly, Ruth Helmus; Ren, Liqiang; Ozcelik, Adem; Wang, Lin; McCoy, J Philip; Levine, Stewart J; Huang, Tony Jun

    2015-12-15

    Selective isolation of cell subpopulations with defined biological characteristics is crucial for many biological studies and clinical applications. In this work, we present the development of an acoustofluidic fluorescence activated cell sorting (FACS) device that simultaneously performs on-demand, high-throughput, high-resolution cell detection and sorting, integrated onto a single chip. Our acoustofluidic FACS device uses the "microfluidic drifting" technique to precisely focus cells/particles three dimensionally and achieves a flow of single-file particles/cells as they pass through a laser interrogation region. We then utilize short bursts (150 μs) of standing surface acoustic waves (SSAW) triggered by an electronic feedback system to sort fluorescently labeled particles/cells with desired biological properties. We have demonstrated continuous isolation of fluorescently labeled HeLa cells from unlabeled cells at a throughput of ∼1200 events/s with a purity reaching 92.3 ± 3.39%. Furthermore, 99.18% postsort cell viability indicates that our acoustofluidic sorting technique maintains a high integrity of cells. Therefore, our integrated acoustofluidic FACS device is demonstrated to achieve two-way cell sorting with high purity, biocompatibility, and biosafety. We believe that our device has significant potential for use as a low-cost, high-performance, portable, and user-friendly FACS instrument.

  17. A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide

    PubMed Central

    Shokhirev, Maxim N; Almaden, Jonathan; Davis-Turak, Jeremy; Birnbaum, Harry A; Russell, Theresa M; Vargas, Jesse A D; Hoffmann, Alexander

    2015-01-01

    Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks. PMID:25680807

  18. Similar disturbances in B cell activity and regulatory T cell function in Henoch-Schonlein purpura and systemic lupus erythematosus

    SciTech Connect

    Beale, M.G.; Nash, G.S.; Bertovich, M.J.; MacDermott, R.P.

    1982-01-01

    The immunoglobulin synthesizing activities of peripheral mononuclear cells (MNC) from five patients with Henoch-Schonlein purpura (HSP) and eight patients with active systemic lupus erythematosus (SLE) were compared. Cumulative amounts of IgM, IgG, and IgA synthesized and secreted by unstimulated and PWM-stimulated patient cells over a 12-day period were determied in a solid-phase radioimmunoassay. In unstimulated control cultures mean rates of IgM, IgG, and IgA synthesis were less than 250 ng/ml. The synthetic activities of patient MNC were markedly increased. In HSP cultures IgA was the major immunoglobulin class produced (2810 x/divide 1.33 ng/ml) followed by IgG (1754 x/divide 1.32 ng/ml) and IgM (404 x/divide 1.16 ng/ml). In SLE cultures IgA and IgG syntheses were equally elevated (4427 x/divide 1.20 and 4438 x/divide 1.49 ng/ml, respectively) whereas IgM synthesis averaged 967 x/divide 1.66 ng/ml. PWM stimulation of pateient MNC caused a sharp decline in the synthesis of all three immunoglobulin classes. After T cell depletion B cell-enriched fractions from HSP and SLE patients maintained high levels of IgA and IgG synthesis that were inhibited by PWM and by normal allogeneic but not autologous T cells. In PWM-stimulted co-cultures, patient T cells nonspecifically suppressed the synthetic activities of autologous and control B cells. in contrast patient B cells achieved normal levels of immunoglobulin synthesis when cultured with control T cells plus PWM. In longitudinal studies patient B and T cell disturbances persisted despite clinical improvement.

  19. Divide and shape: an endosymbiont in action.

    PubMed

    Pyke, Kevin A

    2013-02-01

    The endosymbiotic evolution of the plastid within the host cell required development of a mechanism for efficient division of the plastid. Whilst a model for the mechanism of chloroplast division has been constructed, little is known of how other types of plastids divide, especially the proplastid, the progenitor of all plastid types in the cell. It has become clear that plastid shape is highly heterogeneous and dynamic, especially stromules. This article considers how such variation in morphology might be controlled and how such plastids might divide efficiently.

  20. M-cadherin-mediated intercellular interactions activate satellite cell division.

    PubMed

    Marti, Merce; Montserrat, Núria; Pardo, Cristina; Mulero, Lola; Miquel-Serra, Laia; Rodrigues, Alexandre Miguel Cavaco; Andrés Vaquero, José; Kuebler, Bernd; Morera, Cristina; Barrero, María José; Izpisua Belmonte, Juan Carlos

    2013-11-15

    Adult muscle stem cells and their committed myogenic precursors, commonly referred to as the satellite cell population, are involved in both muscle growth after birth and regeneration after damage. It has been previously proposed that, under these circumstances, satellite cells first become activated, divide and differentiate, and only later fuse to the existing myofiber through M-cadherin-mediated intercellular interactions. Our data show that satellite cells fuse with the myofiber concomitantly to cell division, and only when the nuclei of the daughter cells are inside the myofiber, do they complete the process of differentiation. Here we demonstrate that M-cadherin plays an important role in cell-to-cell recognition and fusion, and is crucial for cell division activation. Treatment of satellite cells with M-cadherin in vitro stimulates cell division, whereas addition of anti-M-cadherin antibodies reduces the cell division rate. Our results suggest an alternative model for the contribution of satellite cells to muscle development, which might be useful in understanding muscle regeneration, as well as muscle-related dystrophies.

  1. Essays on the Digital Divide

    ERIC Educational Resources Information Center

    Abdelfattah, Belal M. T.

    2013-01-01

    The digital divide is a phenomenon that is globally persistent, despite rapidly decreasing costs in technology. While much of the variance in the adoption and use of information communication technology (ICT) that defines the digital divide can be explained by socioeconomic and demographic variables, there is still significant unaccounted variance…

  2. Bridging the Macro- and Micro-Divide: Using an Activity Theory Model to Capture Sociocultural Complexity in Mathematics Teaching and Its Development

    ERIC Educational Resources Information Center

    Jaworski, Barbara; Potari, Despina

    2009-01-01

    This paper is methodologically based, addressing the study of mathematics teaching by linking micro- and macro-perspectives. Considering "teaching as activity", it uses "Activity Theory" and, in particular, the "Expanded Mediational Triangle" (EMT) to consider the role of the broader social frame in which classroom teaching is situated.…

  3. Cell-based flow cytometry assay to measure cytotoxic activity.

    PubMed

    Noto, Alessandra; Ngauv, Pearline; Trautmann, Lydie

    2013-12-17

    Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

  4. The serine 2481-autophosphorylated form of mammalian Target Of Rapamycin (mTOR) is localized to midzone and midbody in dividing cancer cells

    SciTech Connect

    Vazquez-Martin, Alejandro; Oliveras-Ferraros, Cristina; Bernado, Luis; Lopez-Bonet, Eugeni; Menendez, Javier A.

    2009-03-13

    Using a high-resolution, automated confocal high-content imaging system, we investigated the sub-cellular localization of the Serine 2481-autophosphorylated form of mTOR (PP-mTOR{sup Ser2481}) during mitosis and cytokinesis in human cancer cells. PP-mTOR{sup Ser2481} exhibited a punctate nuclear distribution in interphase cancer cells, with the number of PP-mTOR{sup Ser2481} nuclear speckles positively relating with the proliferative capacity of cancer cells. PP-mTOR{sup Ser2481} expression dynamically rearranged within the cytoplasm in a close association near and between separating chromosomes during early stages of mitosis. Towards the end of anaphase and in telophase, PP-mTOR{sup Ser2481} drastically focused on the midzone and ultimately in the centre of the midbody at the presumptive cleavage furrow. In cells at cytokinesis, PP-mTOR{sup Ser2481} appeared as a doublet facing each other at the apical ends of two daughter cells. Three-dimensional analysis confirmed that PP-mTOR{sup Ser2481} positioned at a ring structure wrapped round by microtubule bundles to connect daughter cells. These results reveal for the first time that PP-mTOR{sup Ser2481} may be unexpectedly involved in the terminal stages of cytokinesis.

  5. Conserved amino acids of the human immunodeficiency virus type 2 Vpx nuclear localization signal are critical for nuclear targeting of the viral preintegration complex in non-dividing cells

    SciTech Connect

    Belshan, Michael; Mahnke, Lisa A.; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-03-01

    The HIV-2 viral accessory protein Vpx is related to, but distinct from the Vpr protein of HIV-1. Vpx is packaged into virions and as a component of the viral preintegration complex (PIC) is required for efficient virus replication in non-dividing cells. We have previously reported that the minimal transferable region of Vpx that contained karyophilic properties was aa 65 to 72. Analysis of Vpx sequences from various HIV-2/SIV strains reveals that this region contains highly conserved amino acids, including two basic residues (K68, R70) and three tyrosines (Y66, Y69, Y71). Here, we demonstrate that mutation of the basic or tyrosine residues abolishes PIC nuclear import in arrested cells as assessed by PCR detection of viral integration. Examination of cell-free virus by Western blot indicated that all mutant proteins were incorporated into virions, suggesting that the lack of replication in arrested cells was not due to a loss of Vpx in target cells. Together, these studies map critical residues of the Vpx nuclear localization signal that are required for efficient infection of non-dividing cells.

  6. Sociology: The growing climate divide

    NASA Astrophysics Data System (ADS)

    Hoffman, Andrew J.

    2011-07-01

    Climate change has reached the level of a 'scientific consensus', but is not yet a 'social consensus'. New analysis highlights that a growing divide between liberals and conservatives in the American public is a major obstacle to achieving this end.

  7. Tracking and treating activated T cells

    PubMed Central

    Kim, N.H.; Nadithe, V.; Elsayed, M.; Merkel, O.M.

    2014-01-01

    Upon activation, T cells of various subsets are the most important mediators in cell-mediated immune responses. Activated T cells play an important role in immune system related diseases such as chronic inflammatory diseases, viral infections, autoimmune disease, transplant rejection, Crohn disease, diabetes, and many more. Therefore, efforts have been made to both visualize and treat activated T cells specifically. This review summarizes imaging approaches and selective therapeutics for activated T cells and gives an outlook on how tracking and treating can be combined into theragnositc agents for activated T cells. PMID:24660025

  8. Viral Evasion of Natural Killer Cell Activation

    PubMed Central

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-01-01

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections. PMID:27077876

  9. Viral Evasion of Natural Killer Cell Activation.

    PubMed

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-04-12

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

  10. Cell proliferation in vitro modulates fibroblast collagenase activity

    SciTech Connect

    Lindblad, W.J.; Flood, L.

    1986-05-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a /sup 14/C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/..mu..g DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of /sup 3/H-thymidine and /sup 3/H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion.

  11. [Chemosignals from isolated females have antimutagenic effect in dividing the cells of bone marrow from male mice of the CBA line].

    PubMed

    Daev, E V; Glinin, T S; Dukel'skaia, A V

    2014-01-01

    A level of X-ray induced mitotic disturbances in the cells of the bone marrow of male mice was studied under the modifying influence ofchemosignals from isolated adult female mice of the CBA line. It has been shown that the frequency of chromosomal aberrations in irradiated (4 Gr) males after exposing them for 24 hours on bedding soiled with female chemosignals is lower than in irradiated males in cages with clean bedding. The mechanisms and importance of the antimutagenic effect of female house mouse chemosignals are discussed.

  12. Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

    PubMed Central

    Jörnvall, H; Persson, M; Jeffery, J

    1981-01-01

    Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations. PMID:7027257

  13. CD27(-)CD45(+) γδ T cells can be divided into two populations, CD27(-)CD45(int) and CD27(-)CD45(hi) with little proliferation potential.

    PubMed

    Odaira, Kosuke; Kimura, Shin-Nosuke; Fujieda, Nao; Kobayashi, Yukari; Kambara, Kaori; Takahashi, Takuya; Izumi, Takamichi; Matsushita, Hirokazu; Kakimi, Kazuhiro

    2016-09-23

    In addition to the majority of T cells which carry the αβ T cell receptor (TCR) for antigen, a distinct subset of about 1-5% of human peripheral blood T cells expressing the γδ TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the Vδ2(+) TCR, usually paired with Vγ9, constitute the majority of these γδ T cells. Analogous to αβ T cells, they can be sorted into naive (CD27(+)CD45RA(+)), central memory (CD27(+)CD45RA(-)), effector memory (CD27(-)CD45RA(-)), and terminally-differentiated effector memory (CD27(-)CD45RA(+)) phenotypes. Here, we found that CD27(-)CD45RA(+) γδ T cells can be further divided into two populations based on the level of expression of CD45RA: CD27(-)CD45RA(int) and CD27(-)CD45RA(hi). Those with the CD27(-)CD45RA(hi) phenotype lack extensive proliferative capacity, while those with the CD27(-)CD45RA(int) phenotype can be easily expanded by culture with zoledronate and IL-2. These CD27(-)CD45RA(hi) potentially exhausted γδ T cells were found predominantly in cancer patients but also in healthy subjects. We conclude that γδ T cells can be divided into at least 5 subsets enabling discrimination of γδ T cells with poor proliferative capacity. It was one of our goals to predict the feasibility of γδ T cell expansion to sufficient amounts for adoptive immunotherapy without the necessity for conducting small-scale culture tests. Fulfilling the ≥1.5% criterion for γδ T cells with phenotypes other than CD27(-)CD45RA(hi), may help avoid small-scale culture testing and shorten the preparation period for adoptive γδ T cells by 10 days, which may be beneficial for patients with advanced cancer.

  14. CD27(-)CD45(+) γδ T cells can be divided into two populations, CD27(-)CD45(int) and CD27(-)CD45(hi) with little proliferation potential.

    PubMed

    Odaira, Kosuke; Kimura, Shin-Nosuke; Fujieda, Nao; Kobayashi, Yukari; Kambara, Kaori; Takahashi, Takuya; Izumi, Takamichi; Matsushita, Hirokazu; Kakimi, Kazuhiro

    2016-09-23

    In addition to the majority of T cells which carry the αβ T cell receptor (TCR) for antigen, a distinct subset of about 1-5% of human peripheral blood T cells expressing the γδ TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the Vδ2(+) TCR, usually paired with Vγ9, constitute the majority of these γδ T cells. Analogous to αβ T cells, they can be sorted into naive (CD27(+)CD45RA(+)), central memory (CD27(+)CD45RA(-)), effector memory (CD27(-)CD45RA(-)), and terminally-differentiated effector memory (CD27(-)CD45RA(+)) phenotypes. Here, we found that CD27(-)CD45RA(+) γδ T cells can be further divided into two populations based on the level of expression of CD45RA: CD27(-)CD45RA(int) and CD27(-)CD45RA(hi). Those with the CD27(-)CD45RA(hi) phenotype lack extensive proliferative capacity, while those with the CD27(-)CD45RA(int) phenotype can be easily expanded by culture with zoledronate and IL-2. These CD27(-)CD45RA(hi) potentially exhausted γδ T cells were found predominantly in cancer patients but also in healthy subjects. We conclude that γδ T cells can be divided into at least 5 subsets enabling discrimination of γδ T cells with poor proliferative capacity. It was one of our goals to predict the feasibility of γδ T cell expansion to sufficient amounts for adoptive immunotherapy without the necessity for conducting small-scale culture tests. Fulfilling the ≥1.5% criterion for γδ T cells with phenotypes other than CD27(-)CD45RA(hi), may help avoid small-scale culture testing and shorten the preparation period for adoptive γδ T cells by 10 days, which may be beneficial for patients with advanced cancer. PMID:27553282

  15. Dormancy activation mechanism of tracheal stem cells

    PubMed Central

    Li, Xin; Xu, Jing-xian; Jia, Xin-Shan; Li, Wen-ya; Han, Yi-chen; Wang, En-hua; Li, Fang

    2016-01-01

    Accurate markers and molecular mechanisms of stem cell dormancy and activation are poorly understood. In this study, the anti-cancer drug, 5-fluorouracil, was used to selectively kill proliferating cells of human bronchial epithelial (HBE) cell line. This method can enrich and purify stem cell population. The dormant versus active status of stem cells was determined by phosphorylation of RNAp II Ser2. The surviving stem cells were cultured to form stem cell spheres expressing stem cell markers and transplanted into nude mice to form a teratoma. The results demonstrated the properties of stem cells and potential for multi-directional differentiation. Bisulfite sequencing polymerase chain reaction showed that demethylation of the Sox2 promoter by 5-FU resulted in Sox2 expression in the dormant stem cells. This study shows that the dormancy and activation of HBE stem cells is closely related to epigenetic modification. PMID:27009861

  16. Dividing Fractions: A Pedagogical Technique

    ERIC Educational Resources Information Center

    Lewis, Robert

    2016-01-01

    When dividing one fraction by a second fraction, invert, that is, flip the second fraction, then multiply it by the first fraction. To multiply fractions, simply multiply across the denominators, and multiply across the numerators to get the resultant fraction. So by inverting the division of fractions it is turned into an easy multiplication of…

  17. Getting Past the "Digital Divide"

    ERIC Educational Resources Information Center

    McCollum, Sean

    2011-01-01

    As most educators know, there is a lot more to addressing the so-called "digital divide" than having enough working machines in classrooms. Effective information technology (IT) in schools requires useful software, reliable and speedy Internet access, effective teacher training, and well-considered goals with transformative outcomes. Educators who…

  18. Getting Past the "Digital Divide"

    ERIC Educational Resources Information Center

    McCollum, Sean

    2011-01-01

    In the last decade, "digital divide" has become a catchphrase for the stubborn disparity in IT resources between communities, especially in regard to education. Low-income, rural and minority populations have received special scrutiny as the technological "have-nots." This article presents success stories of educators who can work around obstacles…

  19. Celebrating Soft Matter's 10th Anniversary: Cell division: a source of active stress in cellular monolayers.

    PubMed

    Doostmohammadi, Amin; Thampi, Sumesh P; Saw, Thuan B; Lim, Chwee T; Ladoux, Benoit; Yeomans, Julia M

    2015-10-01

    We introduce the notion of cell division-induced activity and show that the cell division generates extensile forces and drives dynamical patterns in cell assemblies. Extending the hydrodynamic models of lyotropic active nematics we describe turbulent-like velocity fields that are generated by the cell division in a confluent monolayer of cells. We show that the experimentally measured flow field of dividing Madin-Darby Canine Kidney (MDCK) cells is reproduced by our modeling approach. Division-induced activity acts together with intrinsic activity of the cells in extensile and contractile cell assemblies to change the flow and director patterns and the density of topological defects. Finally we model the evolution of the boundary of a cellular colony and compare the fingering instabilities induced by cell division to experimental observations on the expansion of MDCK cell cultures.

  20. Cell culture density affects the proliferation activity of human adipose tissue stem cells.

    PubMed

    Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2016-01-01

    In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions.

  1. Antibacterial activity of human natural killer cells

    PubMed Central

    1989-01-01

    The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass- adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11- enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16- 24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing. PMID:2642532

  2. A transcriptional mechanism integrating inputs from extracellular signals to activate hippocampal stem cells.

    PubMed

    Andersen, Jimena; Urbán, Noelia; Achimastou, Angeliki; Ito, Ayako; Simic, Milesa; Ullom, Kristy; Martynoga, Ben; Lebel, Mélanie; Göritz, Christian; Frisén, Jonas; Nakafuku, Masato; Guillemot, François

    2014-09-01

    The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate of hippocampal stem cells, and inactivating Ascl1 blocks quiescence exit completely, rendering them unresponsive to activating stimuli. Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating the expression of cell-cycle regulatory genes. Ascl1 is similarly required for stem cell activation in the adult subventricular zone. Our results support a model whereby Ascl1 integrates inputs from both stimulatory and inhibitory signals and converts them into a transcriptional program activating adult neural stem cells.

  3. A Transcriptional Mechanism Integrating Inputs from Extracellular Signals to Activate Hippocampal Stem Cells

    PubMed Central

    Andersen, Jimena; Urbán, Noelia; Achimastou, Angeliki; Ito, Ayako; Simic, Milesa; Ullom, Kristy; Martynoga, Ben; Lebel, Mélanie; Göritz, Christian; Frisén, Jonas; Nakafuku, Masato; Guillemot, François

    2014-01-01

    Summary The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate of hippocampal stem cells, and inactivating Ascl1 blocks quiescence exit completely, rendering them unresponsive to activating stimuli. Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating the expression of cell-cycle regulatory genes. Ascl1 is similarly required for stem cell activation in the adult subventricular zone. Our results support a model whereby Ascl1 integrates inputs from both stimulatory and inhibitory signals and converts them into a transcriptional program activating adult neural stem cells. PMID:25189209

  4. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  5. Myosin II Activity Softens Cells in Suspension

    PubMed Central

    Chan, Chii J.; Ekpenyong, Andrew E.; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J.; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-01-01

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  6. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

  7. Active cell mechanics: Measurement and theory.

    PubMed

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  8. Wealth and the marital divide.

    PubMed

    Schneider, Daniel

    2011-09-01

    Marriage patterns differ dramatically in the United States by race and education. The author identifies a novel explanation for these marital divides, namely, the important role of personal wealth in marriage entry. Using event-history models and data from the National Longitudinal Survey of Youth 1979 cohort, the author shows that wealth is an important predictor of first marriage and that differences in asset ownership by race and education help to explain a significant portion of the race and education gaps in first marriage. The article also tests possible explanations for why wealth plays an important role in first marriage entry. PMID:22268248

  9. Cell death sensitization of leukemia cells by opioid receptor activation

    PubMed Central

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  10. Stem cell tracking with optically active nanoparticles

    PubMed Central

    Gao, Yu; Cui, Yan; Chan, Jerry KY; Xu, Chenjie

    2013-01-01

    Stem-cell-based therapies hold promise and potential to address many unmet clinical needs. Cell tracking with modern imaging modalities offers insight into the underlying biological process of the stem-cell-based therapies, with the goal to reveal cell survival, migration, homing, engraftment, differentiation, and functions. Adaptability, sensitivity, resolution, and non-invasiveness have contributed to the longstanding use of optical imaging for stem cell tracking and analysis. To identify transplanted stem cells from the host tissue, optically active probes are usually used to label stem cells before the administration. In comparison to the traditional fluorescent probes like fluorescent proteins and dyes, nanoparticle-based probes are advantageous in terms of the photo-stabilities and minimal changes to the cell phenotype. The main focus here is to overview the recent development of optically active nanoparticles for stem cells tracking. The related optical imaging modalities include fluorescence imaging, photoacoustic imaging, Raman and surface enhanced Raman spectroscopy imaging. PMID:23638335

  11. A house divided cannot stand

    SciTech Connect

    Gilbert, S.M. )

    1994-01-01

    When it comes to the relationships between electric utilities and public service commissions, utilities would do well to remember the words of Abraham Lincoln -- [open quotes]A house divided against itself cannot stand.[close quotes] For just as distrust, dissension, and division threatened the future of the United States during the Civil War, they threaten the future of utilities today.In an effort to lower their costs and increase their competitive advantage, utility companies are increasingly looking to reinvent cultures, reengineer work processes, and redefine corporate missions, values, and strategies. But unless utilities also rebuild regulatory relations, such efforts are doomed to fail. If this prognosis sounds overly simplistic or melodramatic -- especially as utilities appear to be moving toward an era of reduced regulation -- think again. History shows that regulatory relationships drive a utility's ability to successfully integrate demand-side management (DSM) programs that are often critical to business strategies and goals.

  12. Measurement of myeloid cell immune suppressive activity.

    PubMed

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  13. 40 CFR 1065.248 - Gas divider.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... testing. You may use critical-flow gas dividers, capillary-tube gas dividers, or thermal-mass-meter gas... PROCEDURES Measurement Instruments Flow-Related Measurements § 1065.248 Gas divider. (a) Application. You...

  14. Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis.

    PubMed

    Serrano-Mislata, Antonio; Schiessl, Katharina; Sablowski, Robert

    2015-11-16

    How cells regulate their dimensions is a long-standing question. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle, although there is evidence of cell-autonomous control in cell cultures. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures.

  15. The Southern Ocean biogeochemical divide

    NASA Astrophysics Data System (ADS)

    Marinov, I.; Gnanadesikan, A.; Toggweiler, J. R.; Sarmiento, J. L.

    2006-06-01

    Modelling studies have demonstrated that the nutrient and carbon cycles in the Southern Ocean play a central role in setting the air-sea balance of CO2 and global biological production. Box model studies first pointed out that an increase in nutrient utilization in the high latitudes results in a strong decrease in the atmospheric carbon dioxide partial pressure (pCO2). This early research led to two important ideas: high latitude regions are more important in determining atmospheric pCO2 than low latitudes, despite their much smaller area, and nutrient utilization and atmospheric pCO2 are tightly linked. Subsequent general circulation model simulations show that the Southern Ocean is the most important high latitude region in controlling pre-industrial atmospheric CO2 because it serves as a lid to a larger volume of the deep ocean. Other studies point out the crucial role of the Southern Ocean in the uptake and storage of anthropogenic carbon dioxide and in controlling global biological production. Here we probe the system to determine whether certain regions of the Southern Ocean are more critical than others for air-sea CO2 balance and the biological export production, by increasing surface nutrient drawdown in an ocean general circulation model. We demonstrate that atmospheric CO2 and global biological export production are controlled by different regions of the Southern Ocean. The air-sea balance of carbon dioxide is controlled mainly by the biological pump and circulation in the Antarctic deep-water formation region, whereas global export production is controlled mainly by the biological pump and circulation in the Subantarctic intermediate and mode water formation region. The existence of this biogeochemical divide separating the Antarctic from the Subantarctic suggests that it may be possible for climate change or human intervention to modify one of these without greatly altering the other.

  16. The Southern Ocean biogeochemical divide.

    PubMed

    Marinov, I; Gnanadesikan, A; Toggweiler, J R; Sarmiento, J L

    2006-06-22

    Modelling studies have demonstrated that the nutrient and carbon cycles in the Southern Ocean play a central role in setting the air-sea balance of CO(2) and global biological production. Box model studies first pointed out that an increase in nutrient utilization in the high latitudes results in a strong decrease in the atmospheric carbon dioxide partial pressure (pCO2). This early research led to two important ideas: high latitude regions are more important in determining atmospheric pCO2 than low latitudes, despite their much smaller area, and nutrient utilization and atmospheric pCO2 are tightly linked. Subsequent general circulation model simulations show that the Southern Ocean is the most important high latitude region in controlling pre-industrial atmospheric CO(2) because it serves as a lid to a larger volume of the deep ocean. Other studies point out the crucial role of the Southern Ocean in the uptake and storage of anthropogenic carbon dioxide and in controlling global biological production. Here we probe the system to determine whether certain regions of the Southern Ocean are more critical than others for air-sea CO(2) balance and the biological export production, by increasing surface nutrient drawdown in an ocean general circulation model. We demonstrate that atmospheric CO(2) and global biological export production are controlled by different regions of the Southern Ocean. The air-sea balance of carbon dioxide is controlled mainly by the biological pump and circulation in the Antarctic deep-water formation region, whereas global export production is controlled mainly by the biological pump and circulation in the Subantarctic intermediate and mode water formation region. The existence of this biogeochemical divide separating the Antarctic from the Subantarctic suggests that it may be possible for climate change or human intervention to modify one of these without greatly altering the other.

  17. Activity-driven fluctuations in living cells

    NASA Astrophysics Data System (ADS)

    Fodor, É.; Guo, M.; Gov, N. S.; Visco, P.; Weitz, D. A.; van Wijland, F.

    2015-05-01

    We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.

  18. Modeled Aeromagnetic Anomalies, Controlled By Radar Ice Sounding, As Evidence for Subglacial Volcanic Activity in the West Antarctic Rift System (WR) Beneath the Area of the Divide of the West Antarctic Ice Sheet (WAIS)

    NASA Astrophysics Data System (ADS)

    Behrendt, J. C.

    2014-12-01

    The Thwaites and Pine Island ice shelves, buttressing the WAIS, have passed the turning point as they are eaten away by warmer ocean waters (Joghin et al., 2014; Rignot et al., 2014). There is an increasing evidence (aeromagnetic, radar ice-sounding, high heat flow, subglacial volcanic seismicity, and several exposed and subglacial active volcanoes), for volcanic activity in the WR beneath the WAIS, which flows through it. The 5-km, orthogonally line spaced, central West Antarctica (CWA) aerogeophysical survey defined >400 high amplitude volcanic magnetic anomalies correlated with glacial bed topography. Modeled anomalies defined magnetic properties; interpreted volcanic edifices were mostly removed by the moving ice into which they were erupted. Very high apparent susceptibility contrasts (.001->.3 SI) are typical of measured properties from volcanic exposures in the WAIS area. About 90% of the magnetic sources have normal magnetization in the present field direction. Two explanations as to why the anomalies are not approximately 50% negative: (1) Volcanic activity resulting in these anomalies occurred in a predominantly normal field (unlikely). (2) Sources are a combination of induced and remanent magnetization resulting in anomalies of low amplitude (induced cancels remanent) and are not recognized because they are <100 nT (most probable). About 18 high relief, (~600-2000 m) "volcanic centers" beneath the WAIS surface, probably were erupted subaerially when the WAIS was absent; nine of these are in the general area beneath the divide of the WAIS. A 70-km wide, ring of interpreted subglacial volcanic rocks may define a volcanic caldera underlying thedivide (Behrendt et al., 1998). A 2 km-high subaerially erupted volcano (subglacial Mt Thiel, ~78o30'S, 111oW) ~ 100 km north of the WAISCORE, could be the source an ash layer observed in the core. Models by Tulaczyk and Hossainzadeh (2011) indicate >4mm/yr basal melting beneath the WAIS, supportive of high heat flow

  19. Single Cell Analysis of Transcriptional Activation Dynamics

    PubMed Central

    Rafalska-Metcalf, Ilona U.; Powers, Sara Lawrence; Joo, Lucy M.; LeRoy, Gary; Janicki, Susan M.

    2010-01-01

    Background Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells. Methodology/Principal Findings To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After α-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense. Conclusions/Significance This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after α-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation. PMID:20422051

  20. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-12-11

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

  1. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-01-01

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue. PMID:26658841

  2. 40 CFR 1065.248 - Gas divider.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 34 2013-07-01 2013-07-01 false Gas divider. 1065.248 Section 1065.248... PROCEDURES Measurement Instruments Flow-Related Measurements § 1065.248 Gas divider. (a) Application. You may use a gas divider to blend calibration gases. (b) Component requirements. Use a gas divider...

  3. 40 CFR 1065.248 - Gas divider.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 34 2012-07-01 2012-07-01 false Gas divider. 1065.248 Section 1065.248... PROCEDURES Measurement Instruments Flow-Related Measurements § 1065.248 Gas divider. (a) Application. You may use a gas divider to blend calibration gases. (b) Component requirements. Use a gas divider...

  4. 40 CFR 1065.248 - Gas divider.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Gas divider. 1065.248 Section 1065.248... PROCEDURES Measurement Instruments Flow-Related Measurements § 1065.248 Gas divider. (a) Application. You may use a gas divider to blend calibration gases. (b) Component requirements. Use a gas divider...

  5. 40 CFR 1065.248 - Gas divider.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Gas divider. 1065.248 Section 1065.248... PROCEDURES Measurement Instruments Flow-Related Measurements § 1065.248 Gas divider. (a) Application. You may use a gas divider to blend calibration gases. (b) Component requirements. Use a gas divider...

  6. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  7. Kinetic Discrimination in T-Cell Activation

    NASA Astrophysics Data System (ADS)

    Rabinowitz, Joshua D.; Beeson, Craig; Lyons, Daniel S.; Davis, Mark M.; McConnell, Harden M.

    1996-02-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

  8. Active oxygen and cell death in cereal aleurone cells.

    PubMed

    Fath, Angelika; Bethke, Paul; Beligni, Veronica; Jones, Russell

    2002-05-01

    The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

  9. Bursts of activity in collective cell migration

    PubMed Central

    Chepizhko, Oleksandr; Giampietro, Costanza; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stéphane; Alava, Mikko J.; Zapperi, Stefano; La Porta, Caterina A. M.

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. PMID:27681632

  10. Active lithium chloride cell for spacecraft power

    NASA Technical Reports Server (NTRS)

    Fleischmann, C. W.; Horning, R. J.

    1988-01-01

    An active thionyl chloride high rate battery is under development for spacecraft operations. It is a 540kC (150 Ah) battery capable of pulses up to 75A. This paper describes the design and initial test data on a 'state-of-the-art' cell that has been selected to be the baseline for the prototype cell for that battery. Initial data indicate that the specification can be met with fresh cells. Data for stored cells and additional environmental test data are in the process of being developed.

  11. E. adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat

    PubMed Central

    Hu, Yanchun; Luo, Biao; Wu, Lei; Qiao, Yan; Mo, Quan; Xu, Ruiguang; Zhou, Yancheng; Ren, Zhihua; Zuo, Zhicai; Deng, Junliang; Peng, Guangneng; He, Wei; Wei, Yahui

    2015-01-01

    The cytotoxicity effects of E. adenophorum on cell cycle and apoptosis of renal cells in Saanen goat was evaluated by TUNEL, DAPI, AO/EB staining, DNA fragmentation assay, Caspase activity, Western-blot, qRT-PCR and flow cytometry analysis. 16 saanen goats randomly divided into four groups were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The Results showed that E. adenophorum induced typical apoptotic features of renal cells. E. adenophorum significantly suppressed renal cells viability, caused cell cycle activity arrest and induced typical apoptotic features in a dose-dependent manner. However, the protein levels of Fas/FasL, Bid and caspase-8 did not appear significant changes in the process of E. adenophorum-induced apoptosis. Moreover, E. adenophorum administration slightly decreased Bcl-2 expression, promoted Bax translocation to mitochondria, triggered the release of Cyt c from mitochondria into cytosol and activated caspase-9, -3, and cleaved PARP. The mitochondrial p53 translocation was significantly activated, accompanied by a significant increase in the loss of ΔΨm, Cyt c release and caspase-9 activation. Above all, these data suggest that E. adenophorum induces renal cells apoptosis via the activation of mitochondria-mediated apoptosis pathway in renal cells. These findings may provide new insights to understand the mechanisms involved in E. adenophorum-caused cytotoxicity of renal cells. PMID:26382060

  12. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

    SciTech Connect

    Parekkadan, Biju; Poll, Daan van; Megeed, Zaki; Kobayashi, Naoya; Tilles, Arno W.; Berthiaume, Francois; Yarmush, Martin L.

    2007-11-16

    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-{alpha} abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

  13. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

    PubMed Central

    Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

  14. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

    PubMed Central

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A.; Kumar, Sheetal; Kalab, Petr

    2016-01-01

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  15. The Myth about the Digital Divide

    ERIC Educational Resources Information Center

    Hawkins, Brian L.; Oblinge, Diana G.

    2006-01-01

    Although computer ownership is not 100 percent, progress has been made on closing the digital divide. However, defining the digital divide according to the haves and have-nots of computer ownership is only a starting point. Beyond computer ownership, colleges and universities should explore the "second-level digital divide," which can be caused by…

  16. Tech, Teachers & Teens: Bridging the Divide

    ERIC Educational Resources Information Center

    Stuht, Amy Colcord; Colcord, Cean

    2011-01-01

    In past decades, the "digital divide" referred to the gap between those who could afford access to technology and those who could not. The divide has shifted in recent years to reflect the growing technological chasm between teachers and their students: today's schools and teenagers' worlds. The digital divide is widening and deepening…

  17. Comparing Internet and Mobile Phone Digital Divides.

    ERIC Educational Resources Information Center

    Rice, Ronald E.; Katz, James E.

    2002-01-01

    Discussion of the digital divide focuses on the Internet and mobile phone digital divide. Analyses of a telephone survey from 2000 considers similarities and differences in three kinds of digital dividers for both the Internet and the mobile phone: users and nonusers, users and dropouts, and recent and veteran users. (Author/LRW)

  18. Diversity, Disability, and Geographic Digital Divide

    ERIC Educational Resources Information Center

    Sumari, Melati; Carr, Erika; Ndebe-Ngovo, Manjerngie

    2006-01-01

    The phenomenon called digital divide was the focus of this paper. Diversity, disability, and geographical digital divide were relevant to this collaborative project. An extensive review of the literature was conducted for the completion of this project. The evidence for the digital divide in terms of race, level of education, and gender in the…

  19. Power Divider for Waveforms Rich in Harmonics

    NASA Technical Reports Server (NTRS)

    Sims, William Herbert, III

    2005-01-01

    A method for dividing the power of an electronic signal rich in harmonics involves the use of an improved divider topology. A divider designed with this topology could be used, for example, to propagate a square-wave signal in an amplifier designed with a push-pull configuration to enable the generation of more power than could be generated in another configuration.

  20. Entangled active matter: From cells to ants

    NASA Astrophysics Data System (ADS)

    Hu, D. L.; Phonekeo, S.; Altshuler, E.; Brochard-Wyart, F.

    2016-07-01

    Both cells and ants belong to the broad field of active matter, a novel class of non-equilibrium materials composed of many interacting units that individually consume energy and collectively generate motion or mechanical stresses. However cells and ants differ from fish and birds in that they can support static loads. This is because cells and ants can be entangled, so that individual units are bound by transient links. Entanglement gives cells and ants a set of remarkable properties usually not found together, such as the ability to flow like a fluid, spring back like an elastic solid, and self-heal. In this review, we present the biology, mechanics and dynamics of both entangled cells and ants. We apply concepts from soft matter physics and wetting to characterize these systems as well as to point out their differences, which arise from their differences in size. We hope that our viewpoints will spur further investigations into cells and ants as active materials, and inspire the fabrication of synthetic active matter.

  1. Critical telomerase activity for uncontrolled cell growth

    NASA Astrophysics Data System (ADS)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  2. Hippo Pathway Activity Influences Liver Cell Fate

    PubMed Central

    Yimlamai, Dean; Christodoulou, Constantina; Galli, Giorgio G.; Yanger, Kilangsungla; Pepe-Mooney, Brian; Gurung, Basanta; Shrestha, Kriti; Cahan, Patrick; Stanger, Ben Z.; Camargo, Fernando D.

    2014-01-01

    The Hippo signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo-pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo-pathway signaling in vivo is sufficient to de-differentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single cell level. We also identify the NOTCH signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate, and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration. PMID:24906150

  3. Critical telomerase activity for uncontrolled cell growth.

    PubMed

    Wesch, Neil L; Burlock, Laura J; Gooding, Robert J

    2016-01-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed. PMID:27500377

  4. Activated mast cells promote differentiation of B cells into effector cells

    PubMed Central

    Palm, Anna-Karin E.; Garcia-Faroldi, Gianni; Lundberg, Marcus; Pejler, Gunnar; Kleinau, Sandra

    2016-01-01

    Based on the known accumulation of mast cells (MCs) in B cell-dependent inflammatory diseases, including rheumatoid arthritis, we hypothesized that MCs directly modulate B cells. We show here that degranulated, and to a lesser extent naïve or IgE-sensitized, MCs activate both naïve and B cell receptor-activated B cells. This was shown by increased proliferation, blast formation, and expression of CD19, MHC class II and CD86 in the B cells. Further, MCs stimulated the secretion of IgM and IgG in IgM+ B cells, indicating that MCs can induce class-switch recombination in B cells. We also show that coculture of MCs with B cells promotes surface expression of L-selectin, a homing receptor, on the B cells. The effects of MCs on B cells were partly dependent on cell-cell contact and both follicular and marginal zone B cells could be activated by MCs. Our findings suggest that degranulated MCs support optimal activation of B cells, a finding that is in line with in vivo studies showing that MCs frequently degranulate in the context of B-cell driven pathologies such as arthritis. Together, our findings show that MCs have the capacity to differentiate B cells to effector cells. PMID:26847186

  5. Intracellular mechanisms of lymphoid cell activation.

    PubMed

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells. PMID:2642767

  6. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  7. Place cell activation predicts subsequent memory.

    PubMed

    Robitsek, R Jonathan; White, John A; Eichenbaum, Howard

    2013-10-01

    A major quandary in memory research is how hippocampal place cells, widely recognized as elements of a spatial map, contribute to episodic memory, our capacity to remember unique experiences that depends on hippocampal function. Here we recorded from hippocampal neurons as rats performed a T-maze alternation task in which they were required to remember a preceding experience over a delay in order to make a subsequent spatial choice. As it has been reported previously in other variations of this task, we observed differential firing that predicted correct subsequent choices, even as the animal traversed identical locations prior to the choice. Here we also observed that most place cells also fired differently on correct as compared to error trials. Among these cells, a large majority fired strongly before the delay or during the retrieval phase but were less active or failed to activate when the animal subsequently made an error. These findings join the place cell phenomenon with episodic memory performance dependent on the hippocampus, revealing that memory accuracy can be predicted by the activation of single place cells in the hippocampus.

  8. Mechanically activated artificial cell by using microfluidics

    PubMed Central

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  9. Mechanically activated artificial cell by using microfluidics.

    PubMed

    Ho, Kenneth K Y; Lee, Lap Man; Liu, Allen P

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  10. Mechanically activated artificial cell by using microfluidics.

    PubMed

    Ho, Kenneth K Y; Lee, Lap Man; Liu, Allen P

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

  11. Phenotypic models of T cell activation.

    PubMed

    Lever, Melissa; Maini, Philip K; van der Merwe, P Anton; Dushek, Omer

    2014-09-01

    T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters that govern the interactions between T cell receptors (TCRs) and peptide-MHC complexes (pMHC complexes). Despite extensive experimental studies, the relationship between the TCR-pMHC binding parameters and T cell activation remains controversial. To make sense of conflicting experimental data, a variety of verbal and mathematical models have been proposed. However, it is currently unclear which model or models are consistent or inconsistent with experimental data. A key problem is that a direct comparison between the models has not been carried out, in part because they have been formulated in different frameworks. For this Analysis article, we reformulated published models of T cell activation into phenotypic models, which allowed us to directly compare them. We find that a kinetic proofreading model that is modified to include limited signalling is consistent with the majority of published data. This model makes the intriguing prediction that the stimulation hierarchy of two different pMHC complexes (or two different TCRs that are specific for the same pMHC complex) may reverse at different pMHC concentrations.

  12. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  13. Shape memory polymers for active cell culture.

    PubMed

    Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

    2011-07-04

    Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date

  14. Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State

    PubMed Central

    Fisson, Sylvain; Darrasse-Jèze, Guillaume; Litvinova, Elena; Septier, Franck; Klatzmann, David; Liblau, Roland; Salomon, Benoît L.

    2003-01-01

    Despite a growing interest in CD4+ CD25+ regulatory T cells (Treg) that play a major role in self-tolerance and immunoregulation, fundamental parameters of the biology and homeostasis of these cells are poorly known. Here, we show that this population is composed of two Treg subsets that have distinct phenotypes and homeostasis in normal unmanipulated mice. In the steady state, some Treg remain quiescent and have a long lifespan, in the order of months, whereas the other Treg are dividing extensively and express multiple activation markers. After adoptive transfer, tissue-specific Treg rapidly divide and expand preferentially in lymph nodes draining their target self-antigens. These results reveal the existence of a cycling Treg subset composed of autoreactive Treg that are continuously activated by tissue self-antigens. PMID:12939344

  15. Transition metals activate TFEB in overexpressing cells.

    PubMed

    Peña, Karina A; Kiselyov, Kirill

    2015-08-15

    Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach. PMID:26251447

  16. Transition metals activate TFEB in overexpressing cells

    PubMed Central

    Peña, Karina A.; Kiselyov, Kirill

    2015-01-01

    Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach. PMID:26251447

  17. Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis

    PubMed Central

    Serrano-Mislata, Antonio; Schiessl, Katharina; Sablowski, Robert

    2015-01-01

    Summary How cells regulate their dimensions is a long-standing question [1, 2]. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed [3, 4, 5]. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle [1], although there is evidence of cell-autonomous control in cell cultures [6, 7, 8, 9]. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods [10]. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures. PMID:26526374

  18. The effects of Cyclosporine A and azathioprine on human T cells activated by different costimulatory signals

    PubMed Central

    Leitner, Judith; Drobits, Karin; Pickl, Winfried F.; Majdic, Otto; Zlabinger, Gerhard; Steinberger, Peter

    2011-01-01

    Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC50) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC50 for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation. PMID:21756939

  19. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-01-01

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  20. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  1. Insect cells respiratory activity in bioreactor

    PubMed Central

    Jorge, Soraia Athie Calil; Santos, Mariza Gerdulo; Yokomizo, Adriana Yurie; Pereira, Carlos Augusto; Tonso, Aldo

    2008-01-01

    Specific respiration rate ( \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}. Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 106 cells/mL and maximum specific respiration rate (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} \\max }} $$\\end{document}) of 7.3 × 10−17 molO2/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 106 cells/mL and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage

  2. Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

    PubMed

    Liang, Zhe; Brown, Roy C; Fletcher, Jennifer C; Opsahl-Sorteberg, Hilde-Gunn

    2015-09-01

    Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

  3. Social Welfare Implications of the Digital Divide

    ERIC Educational Resources Information Center

    Kim, Eunjin; Lee, Byungtae; Menon, Nirup M.

    2009-01-01

    The Internet plays a critical role in informing individuals about society, politics, business, and the environment. So much so that it has been said that the digital divide makes the segment of society on the ''right side'' of the divide (the digitally endowed group) better off and that on the ''wrong side'' (the digitally challenged group) worse…

  4. Bridge the Digital Divide for Educational Equity

    ERIC Educational Resources Information Center

    Mason, Christine Y.; Dodds, Richard

    2005-01-01

    Students' technological savvy has challenged schools to make greater use of computers and the Internet in their curricula, but unfortunately, not every student has the same access to it, and the inability to keep pace has created a digital divide that continues to widen. The digital divide particularly affects students who are black, Hispanic,…

  5. Parochial Geographies: Growing up in Divided Belfast

    ERIC Educational Resources Information Center

    Leonard, Madeleine

    2010-01-01

    This article explores the ways in which teenagers occupy and manage space in one divided community in Northern Ireland. Drawing on stories, maps and focus group discussions with 80 teenagers, from an interface area in Belfast, the article reveals their perceptions and experiences of divided cities, as risky landscapes. Teenagers respond to these…

  6. Resynchronization in neuronal network divided by femtosecond laser processing.

    PubMed

    Hosokawa, Chie; Kudoh, Suguru N; Kiyohara, Ai; Taguchi, Takahisa

    2008-05-01

    We demonstrated scission of a living neuronal network on multielectrode arrays (MEAs) using a focused femtosecond laser and evaluated the resynchronization of spontaneous electrical activity within the network. By an irradiation of femtosecond laser into hippocampal neurons cultured on a multielectrode array dish, neurites were cut at the focal point. After the irradiation, synchronization of neuronal activity within the network drastically decreased over the divided area, indicating diminished functional connections between neurons. Cross-correlation analysis revealed that spontaneous activity between the divided areas gradually resynchronized within 10 days. These findings indicate that hippocampal neurons have the potential to regenerate functional connections and to reconstruct a network by self-assembly. PMID:18418255

  7. Chronic variable stress activates hematopoietic stem cells

    PubMed Central

    Courties, Gabriel; Dutta, Partha; Iwamoto, Yoshiko; Zaltsman, Alex; von zur Muhlen, Constantin; Bode, Christoph; Fricchione, Gregory L.; Denninger, John; Lin, Charles P.; Vinegoni, Claudio; Libby, Peter; Swirski, Filip K.; Weissleder, Ralph; Nahrendorf, Matthias

    2014-01-01

    Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans. PMID:24952646

  8. An ultra wideband Wilkinson power divider

    NASA Astrophysics Data System (ADS)

    Hazeri, Ali Reza

    2012-04-01

    In this article, an ultra wideband power divider is proposed, analysed and designed. The design approach of the proposed power divider is derived from even/odd mode analysis. The design approach is validated, and the power divider is simulated by two full-wave electromagnetic simulators (ADS and Sonnet) and fabricated on a substrate with a thickness of 0.635 mm and relative constant of 10.2. Measured results of the proposed power divider show equal power split, excellent insertion loss and good return loss at all the three ports, and a good isolation between the two output ports is achieved over the specified 3.1-10.6 GHz ultra wideband range. In the input port return loss, there are two transmission poles around 5.2 and 9.5 GHz. The overall size of the proposed power divider is just ? .

  9. Persistent neural activity in head direction cells

    NASA Technical Reports Server (NTRS)

    Taube, Jeffrey S.; Bassett, Joshua P.; Oman, C. M. (Principal Investigator)

    2003-01-01

    Many neurons throughout the rat limbic system discharge in relation to the animal's directional heading with respect to its environment. These so-called head direction (HD) cells exhibit characteristics of persistent neural activity. This article summarizes where HD cells are found, their major properties, and some of the important experiments that have been conducted to elucidate how this signal is generated. The number of HD and angular head velocity cells was estimated for several brain areas involved in the generation of the HD signal, including the postsubiculum, anterior dorsal thalamus, lateral mammillary nuclei and dorsal tegmental nucleus. The HD cell signal has many features in common with what is known about how neural integration is accomplished in the oculomotor system. The nature of the HD cell signal makes it an attractive candidate for using neural network models to elucidate the signal's underlying mechanisms. The conditions that any network model must satisfy in order to accurately represent how the nervous system generates this signal are highlighted and areas where key information is missing are discussed.

  10. Cell Micromanipulation with an Active Handheld Micromanipulator

    PubMed Central

    Tabarés, Jaime Cuevas; MacLachlan, Robert A.; Ettensohn, Charles A.

    2012-01-01

    The paper describes the use of an active handheld micromanipulator, known as Micron, for micromanipulation of cells. The device enables users to manipulate objects on the order of tens of microns in size, with the natural ease of use of a fully handheld tool. Micron senses its own position using a purpose-built microscale optical tracker, estimates the erroneous or undesired component of hand motion, and actively corrects it by deflecting its own tool tip using piezoelectric actuators. Benchtop experiments in tip positioning show that active compensation can reduce positioning error by up to 51% compared to unaided performance. Preliminary experiments in bisection of sea urchin embryos exhibit an increased success rate when performed with the help of Micron. PMID:21096452

  11. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  12. Active stochastic stress fluctuations in the cell cytoskeleton stir the cell and activate primary cilia

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.; Fakhri, Nikta; Battle, Christopher; Ott, Carolyn M.; Wessel, Alok D.; Lippincott-Schwartz, Jennifer; Mackintosh, Frederick C.

    2015-03-01

    Cells are active systems with molecular force generation that drives complex dynamics at the supramolecular scale. Much of cellular dynamics is driven by myosin motors interacting with the actin cytoskeleton. We discovered active random ``stirring'' driven by cytoplasmic myosin as an intermediate mode of transport, different from both thermal diffusion and directed motor activity. We found a further manifestation of cytoskeletal dynamics in the active motion patterns of primary cilia generated by epithelial cells. These cilia were thought to be immotile due to the absence of dynein motors, but it turns out that their anchoring deeper inside the cell in combination with the strongly fluctuating cortex results in clearly measurable non-equilibrium fluctuations.

  13. Hymenoptera Allergy and Mast Cell Activation Syndromes.

    PubMed

    Bonadonna, Patrizia; Bonifacio, Massimiliano; Lombardo, Carla; Zanotti, Roberta

    2016-01-01

    Mast cell activation syndrome (MCAS) can be diagnosed in patients with recurrent, severe symptoms from mast cell (MC)-derived mediators, which are transiently increased in serum and are attenuated by mediator-targeting drugs. When KIT-mutated, clonal MC are detected in these patients, a diagnosis of primary MCAS can be made. Severe systemic reactions to hymenoptera venom (HV) represent the most common form of anaphylaxis in patients with mastocytosis. Patients with primary MCAS and HV anaphylaxis are predominantly males and do not have skin lesions in the majority of cases, and anaphylaxis is characterized by hypotension and syncope in the absence of urticaria and angioedema. A normal value of tryptase (≤11.4 ng/ml) in these patients does not exclude a diagnosis of mastocytosis. Patients with primary MCAS and HV anaphylaxis have to undergo lifelong venom immunotherapy, in order to prevent further potentially fatal severe reactions. PMID:26714690

  14. Hymenoptera Allergy and Mast Cell Activation Syndromes.

    PubMed

    Bonadonna, Patrizia; Bonifacio, Massimiliano; Lombardo, Carla; Zanotti, Roberta

    2016-01-01

    Mast cell activation syndrome (MCAS) can be diagnosed in patients with recurrent, severe symptoms from mast cell (MC)-derived mediators, which are transiently increased in serum and are attenuated by mediator-targeting drugs. When KIT-mutated, clonal MC are detected in these patients, a diagnosis of primary MCAS can be made. Severe systemic reactions to hymenoptera venom (HV) represent the most common form of anaphylaxis in patients with mastocytosis. Patients with primary MCAS and HV anaphylaxis are predominantly males and do not have skin lesions in the majority of cases, and anaphylaxis is characterized by hypotension and syncope in the absence of urticaria and angioedema. A normal value of tryptase (≤11.4 ng/ml) in these patients does not exclude a diagnosis of mastocytosis. Patients with primary MCAS and HV anaphylaxis have to undergo lifelong venom immunotherapy, in order to prevent further potentially fatal severe reactions.

  15. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  16. Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

    PubMed Central

    Chitikova, Zhanna V; Gordeev, Serguei A; Bykova, Tatiana V; Zubova, Svetlana G; Pospelov, Valery A; Pospelova, Tatiana V

    2014-01-01

    Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers. PMID:24626185

  17. T helper cell activation and human retroviral pathogenesis.

    PubMed Central

    Copeland, K F; Heeney, J L

    1996-01-01

    T helper (Th) cells are of central importance in regulating many critical immune effector mechanisms. The profile of cytokines produced by Th cells correlates with the type of effector cells induced during the immune response to foreign antigen. Th1 cells induce the cell-mediated immune response, while Th2 cells drive antibody production. Th cells are the preferential targets of human retroviruses. Infections with human T-cell leukemia virus (HTLV) or human immunodeficiency virus (HIV) result in the expansion of Th cells by the action of HTLV (adult T-cell leukemia) or the progressive loss of T cells by the action of HIV (AIDS). Both retrovirus infections impart a high-level activation state in the host immune cells as well as systemically. However, diverging responses to this activation state have contrasting effects on the Th-cell population. In HIV infection, Th-cell loss has been attributed to several mechanisms, including a selective elimination of cells by apoptosis. The induction of apoptosis in HIV infection is complex, with many different pathways able to induce cell death. In contrast, infection of Th cells with HTLV-1 affords the cell a protective advantage against apoptosis. This advantage may allow the cell to escape immune surveillance, providing the opportunity for the development of Th-cell cancer. In this review, we will discuss the impact of Th-cell activation and general immune activation on human retrovirus expression with a focus upon Th-cell function and the progression to disease. PMID:8987361

  18. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  19. Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

  20. Regulation of polymorphonuclear cell activation by thrombopoietin.

    PubMed Central

    Brizzi, M F; Battaglia, E; Rosso, A; Strippoli, P; Montrucchio, G; Camussi, G; Pegoraro, L

    1997-01-01

    Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation. PMID:9120001

  1. Web of Support: Families, Schools, and Communities: Bridging the Digital Divide. [Videotape].

    ERIC Educational Resources Information Center

    North Central Regional Educational Lab., Oak Brook, IL.

    Televisions, computers, cell phones, and pagers--technology seems to be everywhere. But for many, technology is not as commonplace as it seems. A vast divide, often called the "digital divide," exists in the availability of access for some individuals and communities. Addressing the issue of this digital divide means much more than getting boxes…

  2. Increased activity of chondroitin sulfate-synthesizing enzymes during proliferation of arterial smooth muscle cells

    SciTech Connect

    Hollmann, J.; Thiel, J.; Schmidt, A.; Buddecke, E.

    1986-12-01

    Cultured arterial smooth muscle cells incorporate (/sup 35/S)sulfate into the extracellular chondroitin sulfate/dermatan sulfate containing proteoglycans at a higher rate in the phase of logarithmic growth than do non-dividing cells. The cell growth-dependent decrease in /sup 35/S incorporation with increasing cell density is accompanied by a decrease in the activity of chondroitin sulfate-synthesizing enzymes. The specific activity of xylosyl transferase, N-acetylgalactosaminyl transferase I and chondroitin sulfotransferase declines as the cells proceed from low to high densities. The corresponding correlation coefficients are 0.86, 0.91 and 0.89. The ratio of C-60H/C-40H sulfation of chondroitin shows a cell proliferation-dependent decrease indicating an inverse correlation of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase activity. The observed changes in the expression of enzyme activities are thought to have some implications in the pathogenesis of arteriosclerosis, the initial stages of which are characterized by proliferation of arterial smooth muscle cells.

  3. Wideband unbalanced waveguide power dividers and combiners

    DOEpatents

    Halligan, Matthew; McDonald, Jacob Jeremiah; Strassner, II, Bernd H.

    2016-05-17

    The various technologies presented herein relate to waveguide dividers and waveguide combiners for application in radar systems, wireless communications, etc. Waveguide dividers-combiners can be manufactured in accordance with custom dimensions, as well as in accordance with waveguide standards such that the input and output ports are of a defined dimension and have a common impedance. Various embodiments are presented which can incorporate one or more septum(s), one or more pairs of septums, an iris, an input matching region, a notch located on the input waveguide arm, waveguide arms having stepped transformer regions, etc. The various divider configurations presented herein can be utilized in high fractional bandwidth applications, e.g., a fractional bandwidth of about 30%, and RF applications in the Ka frequency band (e.g., 26.5-40 GHz).

  4. WIDE BAND REGENERATIVE FREQUENCY DIVIDER AND MULTIPLIER

    DOEpatents

    Laine, E.F.

    1959-11-17

    A regenerative frequency divider and multiplier having wide band input characteristics is presented. The circuit produces output oscillations having frequencies related by a fixed ratio to input oscillations over a wide band of frequencies. In accomplishing this end, the divider-multiplier includes a wide band input circuit coupled by mixer means to a wide band output circuit having a pass band related by a fixed ratio to that of the input circuit. A regenerative feedback circuit derives a fixed frequency ratio feedback signal from the output circuit and applies same to the mixer means in proper phase relation to sustain fixed frequency ratio oscillations in the output circuit.

  5. MAIT cells are activated during human viral infections.

    PubMed

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Moore, Michael D; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M; Dustin, Lynn B; Ho, Ling-Pei; Thompson, Fiona M; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B; Screaton, Gavin R; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  6. Activated Muscle Satellite Cells Chase Ghosts.

    PubMed

    Mourikis, Philippos; Relaix, Frédéric

    2016-02-01

    The in vivo behaviors of skeletal muscle stem cells, i.e., satellite cells, during homeostasis and after injury are poorly understood. In this issue of Cell Stem Cell, Webster et al. (2016) now perform a tour de force intravital microscopic analysis of this population, showing that "ghost fiber" remnants act as scaffolds to guide satellite cell divisions after injury. PMID:26849298

  7. Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes.

    PubMed

    Sun, E; Zhang, L; Zeng, Y; Ge, Q; Zhao, M; Gao, W

    2000-03-01

    Although apoptosis is commonly viewed as a silent cell death without damage to adjacent tissues, the effect of apoptosis on immunity has been unclear. We have investigated the influence of apoptotic cells on T-cell activation. The K562 or HL-60 human leukemia cell lines that had been induced apoptosis by FTY720 or cycloheximide (CHX) were added into the culture of mouse spleen cells stimulated with Con A. Six to 20 h later, the expression of CD69, an early T-cell activation antigen, was detected using flowcytometry. Living cells and necrotic cells served as control groups. Apoptotic K562 or HL-60 cells induced by either FTY720 or CHX unanimously inhibited CD69 expression on the CD3+ mouse T cells while living and necrotic cells did not. The inhibition was proportional to the number of apoptotic cells and was different in the T-cell subsets, showing a rapid and transient inhibition on the CD3+CD8+ T-cell activation but with a slow and continuous inhibition on CD3+CD8- T-cell activation. In conclusion, the apoptotic cells actively inhibit a T-cell activation that is independent of the cell lines or the apoptotic inducers, indicating that the apoptotic cells dominantly regulate T-cell immunity. PMID:10736091

  8. Phagocytic cell function in active brucellosis.

    PubMed Central

    Ocon, P; Reguera, J M; Morata, P; Juarez, C; Alonso, A; Colmenero, J D

    1994-01-01

    In this study, we analyzed phagocytic cell function in 51 patients with active brucellosis and its relationship with different clinical, serological, and evolutionary variables. A control group was made up of 30 blood donors of similar geographic extraction, age, and sex, with no previous history of brucellosis or known exposure ot the infection or specific antibodies. The investigations were carried out at the time of diagnosis, at the conclusion of treatment, and after 6 months of follow-up. Polymorphonuclear leukocyte adherence and nitroblue tetrazolium reduction in response to Brucella antigen were significantly increased in the patients at the time of diagnosis with respect to the control group. In contrast, chemotaxis in response to Brucella antigen and phagocytosis were significantly reduced in the patients with respect to the control group. The alterations in phagocytic cell function were greater in patients with bacteremia, with focal forms of the disease, or with a longer diagnostic delay. Most of these initial alterations tended to normalize with treatment, indicating their transient character. PMID:8112863

  9. From Digital Divide to Digital Democracy.

    ERIC Educational Resources Information Center

    de los Santos, Gerardo E., Ed.; de los Santos, Alfredo G., Jr., Ed.; Milliron, Mark David, Ed.

    This publication is one of many efforts of the League for Innovation in the Community College to address the issue of societal technology access and learning needs. This work addresses the issue of the digital divide, which includes the often conflicting perspectives of information technology (IT) access and literacy needs held by government…

  10. Young People's Internet Use: Divided or Diversified?

    ERIC Educational Resources Information Center

    Boonaert, Tom; Vettenburg, Nicole

    2011-01-01

    This article critically analyses research on young people's internet use. Based on a literature analysis, it examines which young people do what on the internet. These results invite a reflection on the dominant discourse on the digital divide. Within this discourse, there is a strong focus on the use of the internet for information purposes only,…

  11. Project DIVIDE Instrument Development. Technical Report # 0810

    ERIC Educational Resources Information Center

    Ketterlin-Geller, Leanne; Jung, Eunju; Geller, Josh; Yovanoff, Paul

    2008-01-01

    In this technical report, we describe the development of cognitive diagnostic test items that form the basis of the diagnostic system for Project DIVIDE (Dynamic Instruction Via Individually Designed Environments). The construct underlying the diagnostic test is division of fractions. We include a description of the process we used to identify the…

  12. THE DEVELOPMENT OF THE TEACHING SPACE DIVIDER.

    ERIC Educational Resources Information Center

    BELLOMY, CLEON C.; CAUDILL, WILLIAM W.

    TYPES OF VERTICAL WORK SURFACES AND THE DEVELOPMENT OF A MODEL TEACHING SPACE DIVIDER ARE DISCUSSED IN THIS REPORT. THIS DESIGN IS BASED ON THE EXPRESSED NEED FOR MORE TACKBOARD AND SHELVING SPACE, AND FOR MOVABLE PARTITIONS. THE MODEL PANELS WHICH SERVE DIRECTLY AS PARTITIONS RATHER THAN BEING OVERLAID ON A PLASTERED SURFACE, INCLUDE THE…

  13. An automatic bridge for inductive voltage dividers

    SciTech Connect

    Chang, P.; Liang, C.P.; Hsiao, J.C.

    1994-12-31

    We describe an automatic, injection-type bridge for inductive voltage divider (IVD) applications at low audio frequencies. We used it to self-calibrate programmable IVDs fabricated in house, by an automated {open_quotes}boot-strap{close_quotes} procedure. It is the heart of our reference standard for ac voltage ratios as well as a calibration system for IVDs.

  14. Leveraging Resources to Close the Divide

    ERIC Educational Resources Information Center

    Rourke, James R.

    2007-01-01

    A host of real and perceived chasms divide students who achieve high academic standards and those who fail to meet them: varying expectations, economic status, resources, cultural and racial preconceptions, inappropriate standards, and so on. Yet as with all human endeavors throughout history, inventors, innovators, and risk takers rise to the…

  15. Dividing Fractions: What Is the Divisor's Role?

    ERIC Educational Resources Information Center

    Coughlin, Heather A.

    2010-01-01

    Dividing by fractions is considered by many to be one of the most complicated procedures in elementary mathematics. The computations are not only complicated but also challenging to explain in the context of a word problem. In this article, the author discusses the role of the divisor in the concept of division by fractions. She examines three…

  16. Hyperoxia Inhibits T Cell Activation in Mice

    NASA Astrophysics Data System (ADS)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  17. Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

    PubMed Central

    Xu, B.L.; Zhao, Q.Z.; Gao, X.Y.; Hou, G.J.

    2015-01-01

    Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway. PMID:26397976

  18. Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity.

    PubMed

    Xu, B L; Zhao, Q Z; Gao, X Y; Hou, G J

    2015-11-01

    Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

  19. Elasticity of adherent active cells on a compliant substrate

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya; Mertz, Aaron F.; Dufresne, Eric R.; Marchetti, M. Cristina

    2012-02-01

    We present a continuum mechanical model of rigidity sensing by livings cells adhering to a compliant substrate. The cell or cell colony is modeled as an elastic active gel, adapting recently developed continuum theories of active viscoelastic fluids. The coupling to the substrate enters as a boundary condition that relates the cell's deformation field to local stress gradients. In the presence of activity, the substrate induces spatially inhomogeneous contractile stresses and deformations, with a power law dependence of the total traction forces on cell or colony size. This is in agreement with recent experiments on keratinocyte colonies adhered to fibronectin coated surfaces. In the presence of acto-myosin activity, the substrate also enhances the cell polarization, breaking the cell's front-rear symmetry. Maximal polarization is observed when the substrate stiffness matches that of the cell, in agreement with experiments on stem cells.

  20. MAIT cells are activated during human viral infections

    PubMed Central

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C.; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Barnes, Eleanor; Ball, Jonathan; Burgess, Gary; Cooke, Graham; Dillon, John; Gore, Charles; Foster, Graham; Guha, Neil; Halford, Rachel; Herath, Cham; Holmes, Chris; Howe, Anita; Hudson, Emma; Irving, William; Khakoo, Salim; Koletzki, Diana; Martin, Natasha; Mbisa, Tamyo; McKeating, Jane; McLauchlan, John; Miners, Alec; Murray, Andrea; Shaw, Peter; Simmonds, Peter; Spencer, Chris; Targett-Adams, Paul; Thomson, Emma; Vickerman, Peter; Zitzmann, Nicole; Moore, Michael D.; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M.; Dustin, Lynn B.; Ho, Ling-Pei; Thompson, Fiona M.; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B.; Screaton, Gavin R.; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation—driving cytokine release and Granzyme B upregulation—is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  1. Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Levinskii, A B; Mel'nikov, P A; Cherepanov, S A; Chekhonin, V P

    2015-05-01

    The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.

  2. Activation of Complement by Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Smith, Thomas F.; Mcintosh, Kenneth; Fishaut, Mark; Henson, Peter M.

    1981-01-01

    The ability of respiratory syncytial virus (RSV)-infected HEp-2 cells in culture to activate complement was investigated. After incubation of cells with various complement sources and buffer, binding of C3b to surfaces of infected cells was demonstrated by immunofluorescence with a double-staining technique. Nonsyncytial and syncytial (i.e., fused, multinucleated) cells were separately enumerated. Also, lysis of RSV-infected cells was assessed by lactic dehydrogenase release. In this system only RSV-infected cells stained for C3b, and they did so only after incubation with functionally active complement. Blocking of classical pathway activation with ethylenediaminetetraacetic acid diminished the number of infected nonsyncytial cells positively stained for C3b, but had no effect on staining of syncytial cells. Blocking of alternative pathway activation with either zymosan incubation or heat treatment decreased the number of both syncytial and nonsyncytial cells stained for C3b. Decreasing immunoglobulin concentration of the serum used as the complement source also decreased numbers of both cell types stained for C3b. Eliminating specific anti-RSV antibody diminished numbers of both cell types stained for C3b, but staining was not eliminated. Lastly, incubation with functionally active complement markedly increased lactic dehydrogenase release from infected cells. This study demonstrated that RSV-infected nonsyncytial and syncytial cells are able to activate complement by both classical and alternative pathways. Activation of complement by syncytial cells appears to be less dependent on the classical pathway than is activation by nonsyncytial cells, and activation by syncytial cells may require immunoglobulin but not specific antibody. These experiments suggest the possibility of complement activation during respiratory tract infection by RSV. Implications of this are discussed. Images PMID:7263071

  3. Activation of anaphase-promoting complex by p53 induces a state of dormancy in cancer cells against chemotherapeutic stress

    PubMed Central

    Dai, Yafei; Wang, Lujuan; Tang, Jingqun; Cao, Pengfei; Luo, Zhaohui; Sun, Jun; Kiflu, Abraha; Sai, Buqing; Zhang, Meili; Wang, Fan; Li, Guiyuan; Xiang, Juanjuan

    2016-01-01

    Cancer dormancy is a stage in tumor progression in which residual disease remains occult and asymptomatic for a prolonged period. Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy. However, cancer dormancy is poorly characterized and the mechanisms of how cancer cells develop dormancy and relapse remain elusive. In this study, 5- fluorouracil (5-FU) was used to induce cancer cell dormancy. We found that cancer cells escape the cytotoxicity of 5-FU by becoming “dormant”. After exposure to 5-FU, residual non-small cell lung cancer (NSCLC) cells underwent epithelial-mesenchymal transition (EMT), followed by mesenchymal-epithelial transition (MET). These EMT-transformed NSCLC cells were in the state of cell quiescence where cells were not dividing and were arrested in the cell cycle in G0-G1. The dormant cells underwent an EMT showed characteristics of cancer stem cells. P53 is strongly accumulated in response to 5-FU-induced dormant cells through the activation of ubiquitin ligase anaphase-promoting complex (APC/C) and TGF-β/Smad signaling. In contrast to the EMT-transformed cells, MET-transformed cells showed an increased ability to proliferate, suggesting that dormant EMT cells were reactivated in the MET process. During the EMT-MET process, DNA repair including nonhomologous end joining (NHEJ) and homologous recombination (HR) is critical to dormant cell reactivation. Our findings provide a mechanism to unravel cancer cell dormancy and reactivation of the cancer cell population. PMID:27009858

  4. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  5. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    PubMed

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  6. The Digital Divide: A Global View

    NASA Astrophysics Data System (ADS)

    Ntoko, Alexander

    2011-04-01

    Huge progress was made in bridging the digital divide in first decade of 21^st century. This was largely due to the explosive growth of mobile, which saw numbers rise from under 500 million to over five billion mobile cellular subscriptions in just ten years. With household mobile penetration rates of over 50% even in rural areas of developing countries, we have achieved the dream of bringing all the world's people within reach of communications technology. We must now, however, replicate the mobile miracle for the Internet, and especially broadband, if we are to avoid creating a new broadband breach to replace the digital divide. Three things need to happen for this to be achieved: firstly, broadband needs to be brought to the top of the development agenda; secondly, broadband needs to become much more affordable and thirdly, security needs to be part of the strategy.

  7. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands

    PubMed Central

    Munich, Stephan; Sobo-Vujanovic, Andrea; Buchser, William J.; Beer-Stolz, Donna; Vujanovic, Nikola L.

    2012-01-01

    Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30–100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs. PMID:23170255

  8. Effects of valence and divided attention on cognitive reappraisal processes

    PubMed Central

    Leclerc, Christina M.; Kensinger, Elizabeth A.

    2014-01-01

    Numerous studies have investigated the neural substrates supporting cognitive reappraisal, identifying the importance of cognitive control processes implemented by prefrontal cortex (PFC). This study examined how valence and attention affect the processes used for cognitive reappraisal by asking participants to passively view or to cognitively reappraise positive and negative images with full or divided attention. When participants simply viewed these images, results revealed few effects of valence or attention. However, when participants engaged in reappraisal, there was a robust effect of valence, with the reappraisal of negative relative to positive images associated with more widespread activation, including within regions of medial and lateral PFC. There also was an effect of attention, with more lateral PFC recruitment when regulating with full attention and more medial PFC recruitment when regulating with divided attention. Within two regions of medial PFC and one region of ventrolateral PFC, there was an interaction between valence and attention: in these regions, divided attention reduced activity during reappraisal of positive but not negative images. Critically, participants continued to report reappraisal success even during the Divided Attention condition. These results suggest multiple routes to successful cognitive reappraisal, depending upon image valence and the availability of attentional resources. PMID:24493837

  9. Will the Nicaragua Canal connect or divide?

    PubMed

    Gross, Michael

    2014-11-01

    A century after the opening of the Panama Canal, a second inter-oceanic passage is set to be built in Central America, this time in Nicaragua. The ambitious and astronomically expensive project promises to bring economic opportunity to a poor country but it also carries risks to its tropical ecosystems. Will the new waterway ultimately link two oceans or divide a continent? Michael Gross investigates.

  10. Can Attention be Divided Between Perceptual Groups?

    NASA Technical Reports Server (NTRS)

    McCann, Robert S.; Foyle, David C.; Johnston, James C.; Hart, Sandra G. (Technical Monitor)

    1994-01-01

    Previous work using Head-Up Displays (HUDs) suggests that the visual system parses the HUD and the outside world into distinct perceptual groups, with attention deployed sequentially to first one group and then the other. New experiments show that both groups can be processed in parallel in a divided attention search task, even though subjects have just processed a stimulus in one perceptual group or the other. Implications for models of visual attention will be discussed.

  11. Lake Buchannan, Great Dividing Range, Queensland, Australia

    NASA Technical Reports Server (NTRS)

    1991-01-01

    Lake Buchannan, a small but blue and prominent in the center of the view, lies in the Great Dividing of Queensland, Australia (22.0S, 146.0E). The mountain range in this case is a low plateau of no more than 2,000 to 3,000 ft altitude. The interior is dry, mostly in pasture but the coastal zone in contrast, is wet tropical country where bananas and sugarcane are grown.

  12. Will the Nicaragua Canal connect or divide?

    PubMed

    Gross, Michael

    2014-11-01

    A century after the opening of the Panama Canal, a second inter-oceanic passage is set to be built in Central America, this time in Nicaragua. The ambitious and astronomically expensive project promises to bring economic opportunity to a poor country but it also carries risks to its tropical ecosystems. Will the new waterway ultimately link two oceans or divide a continent? Michael Gross investigates. PMID:25587585

  13. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  14. Firn Structure Evolution at WAIS Divide

    NASA Astrophysics Data System (ADS)

    Harwell, E. N.; Albert, M. R.; Gregory, S.; Keegan, K. M.

    2013-12-01

    The polar ice sheets serve as natural archives of past climate, as well as sensitive indicators of current climate change. The physical structure of snow and firn is sensitive to local environmental changes. The top 60-120 m of wide expanses of the Greenland and Antarctic ice sheets consists of firn, snow that is more than a year old. This porous structure serves as a natural archive of past atmospheric composition and plays an important role in the initiation of the ice core record of past atmospheres, and also plays a key role in remote sensing. This paper examines the physical nature of firn at the WAIS Divide ice core site in West Antarctica. Measurements of the density and permeability profiles are reported from the surface over the depth of the firn column profile. The WAIS Divide ice core site was chosen to be the Antarctic analog of the high-resolution GISP2 core from Summit, Greenland; both sites are cold sites with high accumulation rates. We describe similarities and differences in the structure of firn by comparing measurements from WAIS Divide and Summit, and we identify causes for differences.

  15. Splicing the Divide: A Review of Research on the Evolving Digital Divide among K-12 Students

    ERIC Educational Resources Information Center

    Dolan, Jennifer E.

    2016-01-01

    The digital divide has narrowed with regard to one definition of access to technology--the binary view of the "haves" and "have-nots." However, use of technology at home and in school is not equitable for all students. According to recent literature, a broader and more nuanced definition of the technological divide is necessary…

  16. Active unjamming of confluent cell layers

    NASA Astrophysics Data System (ADS)

    Marchetti, M. Cristina

    Cell motion inside dense tissues governs many biological processes, including embryonic development and cancer metastasis, and recent experiments suggest that these tissues exhibit collective glassy behavior. Motivated by these observations, we have studied a model of dense tissues that combines self-propelled particle models and vertex models of confluent cell layers. In this model, referred to as self-propelled Voronoi (SPV), cells are described as polygons in a Voronoi tessellation with directed noisy cell motility and interactions governed by a shape energy that incorporates the effects of cell volume incompressibility, contractility and cell-cell adhesion. Using this model, we have demonstrated a new density-independent solid-liquid transition in confluent tissues controlled by cell motility and a cell-shape parameter measuring the interplay of cortical tension and cell-cell adhesion. An important insight of this work is that the rigidity and dynamics of cell layers depends sensitively on cell shape. We have also used the SPV model to test a new method developed by our group to determine cellular forces and tissue stresses from experimentally accessible cell shapes and traction forces, hence providing the spatio-temporal distribution of stresses in motile dense tissues. This work was done with Dapeng Bi, Lisa Manning and Xingbo Yang. MCM was supported by NSF-DMR-1305184 and by the Simons Foundation.

  17. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    PubMed

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  18. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    SciTech Connect

    Kist, M.; Koester, H.; Bredt, W.

    1985-06-01

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic /sup 75/selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by /sup 51/Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances.

  19. Mycoplasma arthritidis mitogen up-regulates human NK cell activity.

    PubMed Central

    D'Orazio, J A; Cole, B C; Stein-Streilein, J

    1996-01-01

    While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms. PMID

  20. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    PubMed Central

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  1. Shape control and compartmentalization in active colloidal cells

    PubMed Central

    Spellings, Matthew; Engel, Michael; Klotsa, Daphne; Sabrina, Syeda; Drews, Aaron M.; Nguyen, Nguyen H. P.; Bishop, Kyle J. M.; Glotzer, Sharon C.

    2015-01-01

    Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core–shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble–crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non–momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier–Stokes equation. PMID:26253763

  2. The DNA methylation profile of activated human natural killer cells.

    PubMed

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  3. Remote Control of T Cell Activation Using Magnetic Janus Particles.

    PubMed

    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  4. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins. PMID:14514663

  5. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

  6. Electrodes and electrochemical storage cells utilizing tin-modified active materials

    DOEpatents

    Anani, Anaba; Johnson, John; Lim, Hong S.; Reilly, James; Schwarz, Ricardo; Srinivasan, Supramaniam

    1995-01-01

    An electrode has a substrate and a finely divided active material on the substrate. The active material is ANi.sub.x-y-z Co.sub.y Sn.sub.z, wherein A is a mischmetal or La.sub.1-w M.sub.w, M is Ce, Nd, or Zr, w is from about 0.05 to about 1.0, x is from about 4.5 to about 5.5, y is from 0 to about 3.0, and z is from about 0.05 to about 0.5. An electrochemical storage cell utilizes such an electrode as the anode. The storage cell further has a cathode, a separator between the cathode and the anode, and an electrolyte.

  7. Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells.

    PubMed

    Di Giorgi-Gerevini, V; Melchiorri, D; Battaglia, G; Ricci-Vitiani, L; Ciceroni, C; Busceti, C L; Biagioni, F; Iacovelli, L; Canudas, A M; Parati, E; De Maria, R; Nicoletti, F

    2005-08-01

    The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders. PMID:15947794

  8. Lactoperoxidase activity in milk is correlated with somatic cell count in dairy cows.

    PubMed

    Isobe, N; Kubota, H; Yamasaki, A; Yoshimura, Y

    2011-08-01

    Lactoperoxidase (LPO) is a milk protein with antimicrobial function. The present study was undertaken to examine the correlation between LPO activity and somatic cell count (SCC) in milk to use LPO activity as an indicator of mastitis. Composite milk of 36 cows and quarter milk of 3 cows were collected once per week from 0 to 300 d postpartum and twice per day for 1 wk, respectively. For the measurement of LPO activity, milk was mixed with tetramethylbenzidine solution and incubated at 37°C for 30 min, followed by the measurement of optical density. When only milk with low SCC (132±12×10(3) cells/mL) was used, a significant decrease in LPO activity was detected in primiparous cows from 0 to 4 mo postpartum. Lactoperoxidase activities of primiparous cows in mo 1, 2, and 3 postpartum were significantly higher than those in multiparous cows. When composite milk was divided based on LPO activity, the SCC was significantly higher in the groups with LPO activity >5 and from 3 to 3.9 U/mL in the second- and fourth-parity cows, respectively, compared with the group with LPO activity <2U/mL. Extremely high SCC were found in the ≥fifth-parity cows, even in low-LPO activity groups. In the case of quarter milk, higher LPO activity was associated with increased SCC in all 3 cows. The percentage of quarter milk samples with high SCC (4,062±415×10(3) cells/mL) increased with an increase in the LPO activity. The percentage of quarter milk samples with high SCC was 50.0 to 100% in the milk with LPO activity ≥5 U/mL. These results indicate that the correlation of LPO activity to the SCC in bovine milk may point to the potential use of the former as an indicator of SCC.

  9. Senescence of activated stellate cells limits liver fibrosis

    PubMed Central

    Krizhanovsky, Valery; Yon, Monica; Dickins, Ross A.; Hearn, Stephen; Simon, Janelle; Miething, Cornelius; Yee, Herman; Zender, Lars; Lowe, Scott W.

    2011-01-01

    Summary Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to non-cancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage. PMID:18724938

  10. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  11. Heteronomous rhythmic activity of neurosecretory cells in the silkmoth.

    PubMed

    Ichikawa, Toshio; Kamimoto, Satoshi

    2003-08-21

    Electrical action potentials of neurosecretory cells producing pheromone biosynthesis-activating neuropeptide (PBAN) and electrocardiograms were recorded from female pupae of Bombyx mori and the correlation between firing activity of the cells and cardiac activity was analyzed. PBAN producing cells localized in the suboesophageal ganglion (SOG) generated clusters of action potentials at an interval of 30-60 min. The firing activity rhythm at a middle pupal period was closely related to heartbeat reversal rhythm: an active phase of the cells was usually apparent during anterograde pulse phases. Electrocardiograms at a late pupal period often revealed brief oscillatory potentials (15-25 Hz in frequency) of unknown origin. The firing activity rhythm of PBAN cells closely correlated with the rhythmic appearance of clustered oscillatory potentials. Transection of connectives between the brain and SOG abolished rhythmic activity of the cells. These results suggest that a rhythmic firing activity of the PBAN cell system is heteronomously generated by a cerebral neuronal mechanism and the cerebral mechanism relates the cell system to other neuronal mechanisms controlling cardiac activity and oscillatory potential rhythms. PMID:12873731

  12. Preferential solvation: dividing surface vs excess numbers.

    PubMed

    Shimizu, Seishi; Matubayasi, Nobuyuki

    2014-04-10

    How do osmolytes affect the conformation and configuration of supramolecular assembly, such as ion channel opening and actin polymerization? The key to the answer lies in the excess solvation numbers of water and osmolyte molecules; these numbers are determinable solely from experimental data, as guaranteed by the phase rule, as we show through the exact solution theory of Kirkwood and Buff (KB). The osmotic stress technique (OST), in contrast, purposes to yield alternative hydration numbers through the use of the dividing surface borrowed from the adsorption theory. However, we show (i) OST is equivalent, when it becomes exact, to the crowding effect in which the osmolyte exclusion dominates over hydration; (ii) crowding is not the universal driving force of the osmolyte effect (e.g., actin polymerization); (iii) the dividing surface for solvation is useful only for crowding, unlike in the adsorption theory which necessitates its use due to the phase rule. KB thus clarifies the true meaning and limitations of the older perspectives on preferential solvation (such as solvent binding models, crowding, and OST), and enables excess number determination without any further assumptions. PMID:24689966

  13. Preferential solvation: dividing surface vs excess numbers.

    PubMed

    Shimizu, Seishi; Matubayasi, Nobuyuki

    2014-04-10

    How do osmolytes affect the conformation and configuration of supramolecular assembly, such as ion channel opening and actin polymerization? The key to the answer lies in the excess solvation numbers of water and osmolyte molecules; these numbers are determinable solely from experimental data, as guaranteed by the phase rule, as we show through the exact solution theory of Kirkwood and Buff (KB). The osmotic stress technique (OST), in contrast, purposes to yield alternative hydration numbers through the use of the dividing surface borrowed from the adsorption theory. However, we show (i) OST is equivalent, when it becomes exact, to the crowding effect in which the osmolyte exclusion dominates over hydration; (ii) crowding is not the universal driving force of the osmolyte effect (e.g., actin polymerization); (iii) the dividing surface for solvation is useful only for crowding, unlike in the adsorption theory which necessitates its use due to the phase rule. KB thus clarifies the true meaning and limitations of the older perspectives on preferential solvation (such as solvent binding models, crowding, and OST), and enables excess number determination without any further assumptions.

  14. Dividing phase-dependent cytotoxicity profiling of human embryonic lung fibroblast identifies candidate anticancer reagents.

    PubMed

    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Tang, Wei; Sekimizu, Kazuhisa

    2016-01-01

    Human Embryonic Lung fibroblasts (HEL cells) are widely used as a normal cell in studies of cell biology and can be easily maintained in the resting phase. Here we aimed to discover compounds that exhibit cytotoxicity against HEL cells in the dividing phase, but not in the resting phase. The cytotoxicity of each compound against HEL cells either in the resting phase or in the dividing phase was determined by MTT assay. Ratios of the IC50 of cells in the resting phase and that of cells in the dividing phase (RRD) for these compounds were compared. We selected 44 compounds that exhibited toxic effects on HEL cells in the dividing phase from a chemical library containing 325 anticancer drugs and enzyme inhibitors. The RRD values of those compounds were widely distributed. Paclitaxel and docetaxel, which are clinically used as anticancer drugs, had RRD values larger than 2000. On the other hand, the RRD value of dimethyl sulfoxide, an organic solvent, was 1. The cytotoxic effect of paclitaxel on HEL cells in the dividing phase was attenuated by aphidicolin, hydroxyurea, and nocodazole, confirming that the cytotoxic effects of paclitaxel are dependent on cells being in the dividing phase. Thapsigargin, whose RRD value was 800, the third highest RRD value in the library, exhibited therapeutic effects in a mouse model of FM3A ascites carcinoma. We suggest that compounds with high RRD values for HEL cells are candidate anticancer chemotherapy seeds. PMID:27594296

  15. Activated leukocyte cell adhesion molecule regulates the interaction between pancreatic cancer cells and stellate cells

    PubMed Central

    Zhang, Wei-Wei; Zhan, Shu-Hui; Geng, Chang-Xin; Sun, Xin; Erkan, Mert; Kleeff, Jörg; Xie, Xiang-Jun

    2016-01-01

    Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein that is involved in tumor progression and metastasis. In the present study, the expression and functional role of ALCAM in pancreatic cancer cells and pancreatic stellate cells (PSCs) was investigated. Tissue specimens were obtained from patients with pancreatic ductal adenocarcinoma (n=56) or chronic pancreatitis (CP; n=10), who underwent pancreatic resection, and from normal pancreatic tissue samples (n=10). Immunohistochemistry was used to analyze the localization and expression of ALCAM in pancreatic tissues. Subsequently, reverse transcription-quantitative polymerase chain reaction and immunoblotting were applied to assess the expression of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, as well as in PSCs. An enzyme-linked immunosorbent assay was used to measure ALCAM levels in cell culture medium stimulated by hypoxia, tumor necrosis factor (TNF)-α and transforming growth factor-β. Silencing of ALCAM was performed using ALCAM small interfering (si)RNA and immunocytochemistry was used to analyze the inhibition efficiency. An invasion assay and a cell interaction assay were performed to assess the invasive ability and co-cultured adhesive potential of Panc-1 and T3M4 cells, as well as PSCs. Histologically, ALCAM expression was generally weak or absent in pancreatic cancer cells, but was markedly upregulated in PSCs in pancreatic cancer tissues. ALCAM was highly expressed in PSCs from CP tissues and PSCs surrounding pancreatic intraepithelial neoplasias, as well as in pancreatic cancer cells. ALCAM mRNA was highly expressed in PSCs, with a low to moderate expression in T3M4 and Panc-1 cells. Similar to the mRNA expression, immunoblotting demonstrated that ALCAM protein levels were high in PSCs and T3M4 cells, but low in Panc-1 cells. The expression of TNF-α increased, while hypoxia decreased the secretion of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, and also in

  16. Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model

    PubMed Central

    Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

    2016-01-01

    Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

  17. Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model

    PubMed Central

    Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

    2016-01-01

    Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression. PMID:27602116

  18. Borrelia burgdorferi Spirochetes Induce Mast Cell Activation and Cytokine Release

    PubMed Central

    Talkington, Jeffrey; Nickell, Steven P.

    1999-01-01

    The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. PMID:10024550

  19. Automatically activated, 300 ampere-hour silver-zinc cell

    NASA Technical Reports Server (NTRS)

    Hennigan, T. J.

    1972-01-01

    A prototype silver zinc cell is reported for which the electrolyte is being stored in a separate tank; the cell is being activated when additional power is required by collapsing the neoprene bellows container and thus forcing the electrolyte into cell through a plastic connection. A solar array is proposed as main power source for the flow actuator.

  20. Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores*

    PubMed Central

    Patel, Vimal A.; Massenburg, Donald; Vujicic, Snezana; Feng, Lanfei; Tang, Meiyi; Litbarg, Natalia; Antoni, Angelika; Rauch, Joyce; Lieberthal, Wilfred; Levine, Jerrold S.

    2015-01-01

    Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. PMID:26183782

  1. NF-κB Is Activated in CD4+ iNKT Cells by Sickle Cell Disease and Mediates Rapid Induction of Adenosine A2A Receptors

    PubMed Central

    Yu, Jennifer C.; Ken, Ruey; Neuberg, Donna; Nathan, David G.; Linden, Joel

    2013-01-01

    Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11–7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists. PMID:24124453

  2. Experimental evaluation of decrease in bacterial activity due to cell death and activity decay in activated sludge.

    PubMed

    Hao, Xiaodi; Wang, Qilin; Zhang, Xiangping; Cao, Yali; van Mark Loosdrecht, C M

    2009-08-01

    Decrease in bacterial activity (cell decay) in activated sludge can be attributed to cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The aim of this study was to experimentally differentiate between cell death and activity decay as a source of decrease in microbial activity. By means of measuring maximal oxygen uptake rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in-situ hybridization, the decay rates and the death rates of ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and ordinary heterotrophic organisms (OHOs) were determined respectively in a nitrifying sequencing batch reactor (SBR) and a heterotrophic SBR. The experiments revealed that in the nitrifying system activity decay contributed 47% and 82% to the decreased activities of AOB and NOB and that cell death was responsible for 53% and 18% of decreases in their respective activities. In the heterotrophic system, activity decay took a share of 78% in the decreased activity of OHOs, and cell death was only responsible for 22% of decrease in their activity. The difference between the importance of cell death on the decreased activities of AOB and OHOs might be caused by the mechanisms of substrate storage and/or cryptic growth/death-regeneration of OHOs. The different nutrient sources for AOB and NOB might be the reason for a relatively smaller fraction of cell death in NOB.

  3. GATA3 inhibits GCM1 activity and trophoblast cell invasion

    PubMed Central

    Chiu, Yueh Ho; Chen, Hungwen

    2016-01-01

    Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion. PMID:26899996

  4. Library outreach: addressing Utah's "Digital Divide".

    PubMed

    McCloskey, K M

    2000-10-01

    A "Digital Divide" in information and technological literacy exists in Utah between small hospitals and clinics in rural areas and the larger health care institutions in the major urban area of the state. The goals of the outreach program of the Spencer S. Eccles Health Sciences Library at the University of Utah address solutions to this disparity in partnership with the National Network of Libraries of Medicine-- Midcontinental Region, the Utah Department of Health, and the Utah Area Health Education Centers. In a circuit-rider approach, an outreach librarian offers classes and demonstrations throughout the state that teach information-access skills to health professionals. Provision of traditional library services to unaffiliated health professionals is integrated into the library's daily workload as a component of the outreach program. The paper describes the history, methodology, administration, funding, impact, and results of the program.

  5. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells.

    PubMed

    Maher, Katarina; Konjar, Spela; Watts, Colin; Turk, Boris; Kopitar-Jerala, Natasa

    2014-01-01

    Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

  6. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    PubMed

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  7. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  8. Calcium alloy as active material in secondary electrochemical cell

    DOEpatents

    Roche, Michael F.; Preto, Sandra K.; Martin, Allan E.

    1976-01-01

    Calcium alloys such as calcium-aluminum and calcium-silicon, are employed as active material within a rechargeable negative electrode of an electrochemical cell. Such cells can use a molten salt electrolyte including calcium ions and a positive electrode having sulfur, sulfides, or oxides as active material. The calcium alloy is selected to prevent formation of molten calcium alloys resulting from reaction with the selected molten electrolytic salt at the cell operating temperatures.

  9. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells

    PubMed Central

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  10. Neural progenitor cells regulate microglia functions and activity.

    PubMed

    Mosher, Kira I; Andres, Robert H; Fukuhara, Takeshi; Bieri, Gregor; Hasegawa-Moriyama, Maiko; He, Yingbo; Guzman, Raphael; Wyss-Coray, Tony

    2012-11-01

    We found mouse neural progenitor cells (NPCs) to have a secretory protein profile distinct from other brain cells and to modulate microglial activation, proliferation and phagocytosis. NPC-derived vascular endothelial growth factor was necessary and sufficient to exert at least some of these effects in mice. Thus, neural precursor cells may not only be shaped by microglia, but also regulate microglia functions and activity.

  11. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated

  12. Light activated cell migration in synthetic extracellular matrices.

    PubMed

    Guo, Qiongyu; Wang, Xiaobo; Tibbitt, Mark W; Anseth, Kristi S; Montell, Denise J; Elisseeff, Jennifer H

    2012-11-01

    Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.

  13. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Ray, E.R.; Veyo, S.E.

    1995-12-31

    This reports on a solid oxide fuel cell demonstration program in which utilities are provided fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units serve to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  14. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    SciTech Connect

    Bulleit, R.F.; Zimmerman, E.F.

    1984-09-15

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with (3H)arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.

  15. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  16. Activation of peripheral blood mononuclear cells in bronchoalveolar lavage fluid from patients with sarcoidosis: visualisation of single cell activation products.

    PubMed Central

    Pantelidis, P.; Southcott, A. M.; Cambrey, A. D.; Laurent, G. J.; du Bois, R. M.

    1994-01-01

    BACKGROUND--Interstitial lung diseases are characterised by the recruitment of mononuclear cells to disease sites where maturation occurs and activation products, including lysozyme (LZM), are released. Analysis of in vitro cell culture supernatants for activation products masks the functional heterogeneity of cell populations. It is therefore necessary to examine the secretion of activation products by single cells to assess whether the activation of newly recruited mononuclear phagocytes at the sites of disease in the lung is uniform and controlled by the local microenvironment. METHODS--The reverse haemolytic plaque assay was used to evaluate, at a single cell level, the ability of bronchoalveolar lavage (BAL) fluid from seven patients with sarcoidosis to activate Ficoll-Hypaque-separated peripheral blood mononuclear cells by comparison with BAL fluid from six normal volunteers and nine patients with systemic sclerosis. Monolayers of peripheral blood mononuclear cells and sheep red blood cells were cultured either alone or in the presence of 20% (v/v) BAL fluid with a polyclonal anti-LZM antibody. LZM/anti-LZM complexes bound to red blood cells surrounding the secreting cells were disclosed following complement lysis of red blood cells and quantification of plaque dimensions using microscopy and image analysis. RESULTS--Bronchoalveolar lavage fluid from all the patients with sarcoidosis increased LZM secretion by peripheral blood mononuclear cells compared with unstimulated mononuclear cells. By contrast, BAL fluid from the other individuals had no effect on LZM secretion. CONCLUSIONS--Single cells activated by BAL fluid can be evaluated by the reverse haemolytic plaque assay. BAL fluid from patients with sarcoidosis, but not from patients with systemic sclerosis or normal individuals, contains components capable of activating mononuclear phagocytes to secrete lysozyme. Images PMID:7831632

  17. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

    PubMed Central

    Carroll-Portillo, Amanda; Cannon, Judy L.; te Riet, Joost; Holmes, Anna; Kawakami, Yuko; Kawakami, Toshiaki; Cambi, Alessandra

    2015-01-01

    Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell–cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell–cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC–DC synapse suggest a new role for intercellular crosstalk in defining the immune response. PMID:26304724

  18. Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus

    PubMed Central

    Fan, H; Liu, F; Dong, G; Ren, D; Xu, Y; Dou, J; Wang, T; Sun, L; Hou, Y

    2014-01-01

    B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19+CD27- and CD19+IgM+ B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19+ B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients. PMID:25210799

  19. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Veyo, S.E.

    1995-08-01

    The development of a viable fuel cell driven electrical power generation system involves not only the development of cell and stack technology, but also the development of the overall system concept, the strategy for control, and the ancillary subsystems. The design requirements used to guide system development must reflect a customer focus in order to evolve a commercial product. In order to obtain useful customer feedback, Westinghouse has practiced the deployment with customers of fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units have served to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  20. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  1. [The presence of an endogenous peroxidase activity in hairy cell leukemia cells].

    PubMed

    Reyes, F; Gourdin, M F; Farcet, J P; Dreyfus, B; Breton-Gorius, J

    1977-02-01

    Mononuclear cells from hairy cell leukemia have been studied in three cases by ultrastructural immunocytochemistry. Cells have fairly detectable surface immunoglobulins, without monoclonal distribution however. In addition these cells have a peroxidatic activity which is revealed in the perinuclear space and strands of endoplasmic reticulum. PMID:404081

  2. Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

    PubMed Central

    Bikker, Angela; Kruize, Aike A.; van der Wurff-Jacobs, Kim M. G.; Peters, Rogier P.; Kleinjan, Marije; Redegeld, Frank; de Jager, Wilco; Lafeber, Floris P. J. G.; van Roon, Joël A. G.

    2014-01-01

    Objectives To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation. PMID:24740301

  3. T Cell Activation Thresholds are Affected by Gravitational

    NASA Technical Reports Server (NTRS)

    Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

    1999-01-01

    T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

  4. CD27-CD70 interactions regulate B-cell activation by T cells.

    PubMed Central

    Kobata, T; Jacquot, S; Kozlowski, S; Agematsu, K; Schlossman, S F; Morimoto, C

    1995-01-01

    CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis. PMID:7479974

  5. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  6. Bacterial lipopolysaccharide activates CD57-negative human NK cells.

    PubMed

    Kanevskiy, L M; Erokhina, S A; Streltsova, M A; Telford, W G; Sapozhnikov, A M; Kovalenko, E I

    2014-12-01

    NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.

  7. Quantitative Measurement of the Digital Divide

    NASA Astrophysics Data System (ADS)

    Cottrell, Roger

    2007-04-01

    Bandwidth and the Internet infrastructure are the life-blood of the world's knowledge economy, but they are often scarcest where most needed. Measuring the numbers of users of the Internet infrastructure is not easy in developing countries because many people share accounts, use corporate and academic networks, or visit the rapidly growing number of cyber cafes, telecentres and business services. Also measuring the number of users does not take into account the level of use. One valuable indicator for measuring the Internet infrastructure is the international Internet performance of a country or region. One of the major aims of the PingER project is to provide an historical archive of extensive, publicly accessible, up-to-date, measurements, analyses and reports of multiple Internet performance indicators (such as delay, loss, throughput, reachability, and jitter) between sites, countries and regions of the world. This talk will briefly describe the PingER project and then compare and contrast the Internet performance and its trends within and between countries and regions of the world. By means of extensive case studies it will also identify which regions need the greatest attention, together with their major issues and possible approaches to reducing the divide.

  8. Bridging the divide between science and journalism.

    PubMed

    Van Eperen, Laura; Marincola, Francesco M; Strohm, Jennifer

    2010-03-10

    There are countless reasons nearly every scientist should learn how to communicate effectively with the media, including increased understanding of critical research findings to attract or sustain funding and build new professional partnerships that will further propel forward research. But where do scientists begin? Bridging the Divide between Science and Journalism offers practical tips for any scientist looking to work with the media.Given the traditional and internet-based sources for medical research and healthcare-related news now available, it is imperative that scientists know how to communicate their latest findings through the appropriate channels. The credible media channels are managed by working journalists, so learning how to package vast, technical research in a form that is appetizing and "bite-sized" in order to get their attention, is an art. Reducing years of research into a headline can be extremely difficult and certainly doesn't come naturally to every scientist, so this article provides suggestions on how to work with the media to communicate your findings.

  9. Twin Knudsen Cell Configuration for Activity Measurements by Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Jacobson, N. S.

    1996-01-01

    A twin Knudsen cell apparatus for alloy activity measurements by mass spectrometry is described. Two Knudsen cells - one containing an alloy and one containing a pure component - are mounted on a single flange and translated into the sampling region via a motorized x-y table. Mixing of the molecular beams from the cells is minimized by a novel system of shutters. Activity measurements were taken on two well-characterized alloys to verify the operation of the system. Silver activity measurements are reported for Ag-Cu alloys and aluminum activity measurements are reported for Fe-Al alloys. The temperature dependence of activity for a 0.474 mol fraction Al-Fe alloy gives a partial molar heat of aluminum. Measurements taken with the twin cell show good agreement with literature values for these alloys.

  10. T cell-activating protein on murine lymphocytes.

    PubMed

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-12-01

    A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.

  11. Active Cellular Mechanics and Information Processing in the Living Cell

    NASA Astrophysics Data System (ADS)

    Rao, M.

    2014-07-01

    I will present our recent work on the organization of signaling molecules on the surface of living cells. Using novel experimental and theoretical approaches we have found that many cell surface receptors are organized as dynamic clusters driven by active currents and stresses generated by the cortical cytoskeleton adjoining the cell surface. We have shown that this organization is optimal for both information processing and computation. In connecting active mechanics in the cell with information processing and computation, we bring together two of the seminal works of Alan Turing.

  12. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    PubMed

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.

  13. L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

    PubMed

    Yi, Young-Su; Baek, Kwang-Soo; Cho, Jae Youl

    2014-06-01

    L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility. PMID:24974583

  14. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  15. Knowledge Activism: Bridging the Research/Policy Divide

    ERIC Educational Resources Information Center

    Gillies, Donald

    2014-01-01

    How research can better inform policy and how policy can have a better research base are longstanding issues both in educational research and across public policy generally. Drawing on the work of Hannah Arendt, this article argues that progress in increasing the impact of research can be made through a clearer understanding of the nature of…

  16. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  17. CD4 T cell activation by B cells in human Leishmania (Viannia) infection

    PubMed Central

    2014-01-01

    Background An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. Methods CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. Results Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. Conclusions The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to

  18. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    PubMed Central

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  19. A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation

    PubMed Central

    Love, Nicola K.; Keshavan, Nandaki; Lewis, Rebecca; Harris, William A.; Agathocleous, Michalis

    2014-01-01

    In many growing tissues, slowly dividing stem cells give rise to rapidly proliferating progenitors that eventually exit the cell cycle and differentiate. Growth rates are limited by nutrient availability, but it is unclear which steps of the proliferation-differentiation programme are particularly sensitive to fuel supplies. We examined how nutrient deprivation (ND) affects stem and progenitor cells in the ciliary marginal zone (CMZ) of the amphibian retina, a well-characterised neurogenic niche. We show that ND specifically blocks the proliferation and differentiation of progenitor cells through an mTOR-mediated mechanism. By contrast, the identity and proliferation of retinal stem cells are insensitive to ND and mTOR inhibition. Re-feeding starved retinas in vitro rescues both proliferation and differentiation, and activation of mTOR is sufficient to stimulate differentiation even in ND retinas. These results suggest that an mTOR-mediated restriction point operates in vivo to couple nutrient abundance to the proliferation and differentiation programme in retinal progenitor cells. PMID:24449845

  20. Monocytic Cells Become Less Compressible but More Deformable upon Activation

    PubMed Central

    Ravetto, Agnese; Wyss, Hans M.; Anderson, Patrick D.; den Toonder, Jaap M. J.; Bouten, Carlijn V. C.

    2014-01-01

    Aims Monocytes play a significant role in the development of atherosclerosis. During the process of inflammation, circulating monocytes become activated in the blood stream. The consequent interactions of the activated monocytes with the blood flow and endothelial cells result in reorganization of cytoskeletal proteins, in particular of the microfilament structure, and concomitant changes in cell shape and mechanical behavior. Here we investigate the full elastic behavior of activated monocytes in relation to their cytoskeletal structure to obtain a better understanding of cell behavior during the progression of inflammatory diseases such as atherosclerosis. Methods and Results The recently developed Capillary Micromechanics technique, based on exposing a cell to a pressure difference in a tapered glass microcapillary, was used to measure the deformation of activated and non-activated monocytic cells. Monitoring the elastic response of individual cells up to large deformations allowed us to obtain both the compressive and the shear modulus of a cell from a single experiment. Activation by inflammatory chemokines affected the cytoskeletal organization and increased the elastic compressive modulus of monocytes with 73–340%, while their resistance to shape deformation decreased, as indicated by a 25–88% drop in the cell’s shear modulus. This decrease in deformability is particularly pronounced at high strains, such as those that occur during diapedesis through the vascular wall. Conclusion Overall, monocytic cells become less compressible but more deformable upon activation. This change in mechanical response under different modes of deformation could be important in understanding the interplay between the mechanics and function of these cells. In addition, our data are of direct relevance for computational modeling and analysis of the distinct monocytic behavior in the circulation and the extravascular space. Lastly, an understanding of the changes of monocyte

  1. Cytolytic activity of Naegleria fowleri cell-free extract.

    PubMed

    Fulford, D E; Marciano-Cabral, F

    1986-11-01

    The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.

  2. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles. PMID:25184988

  3. Patterns of plasminogen activator production in cultured normal embryonic cells

    PubMed Central

    1977-01-01

    Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor. PMID:21193

  4. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

  5. Real-time transposable element activity in individual live cells.

    PubMed

    Kim, Neil H; Lee, Gloria; Sherer, Nicholas A; Martini, K Michael; Goldenfeld, Nigel; Kuhlman, Thomas E

    2016-06-28

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE's orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  6. Real-time transposable element activity in individual live cells

    PubMed Central

    Lee, Gloria; Martini, K. Michael

    2016-01-01

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE’s orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  7. Autoimmunity, polyclonal B-cell activation and infection.

    PubMed

    Granholm, N A; Cavallo, T

    1992-02-01

    It is widely believed that autoimmunity is an integral part of the immune system, and that genetic, immunologic, hormonal, environmental and other factors contribute to the pathogenesis of autoimmune disease. Thus, autoimmune disease may represent an abnormal expression of immune functions instead of loss of tolerance to self, and it can be organ specific or systemic in its manifestations. We review the various factors that contribute to the development of autoimmune disease; we also review the mechanisms of polyclonal B-cell activation, with emphasis on the role of infectious agents. We consider systemic lupus erythematosus in humans and in experimental animals as prototypic autoimmune disease, and we summarize data to indicate that polyclonal B-cell activation is central to the pathogenesis of systemic autoimmune disease. The effect of polyclonal B-cell activation, brought about by injections of a B-cell activator-lipopolysaccharide from Gram-negative bacteria-is sufficient to cause autoimmune disease in an immunologically normal host. In fact, autoimmune disease can be arrested if excessive polyclonal B-cell activation is suppressed; alternatively, autoimmune disease can be exacerbated if polyclonal B-cell activation is enhanced. We explore the mechanism of tissue injury when autoimmune disease is induced or exacerbated, and we consider the pathogenic roles of autoantibodies, immune complexes, complement, the blood cell carrier system, and the mononuclear phagocyte system. Although polyclonal B-cell activation may be the mechanism whereby various factors can cause or exacerbate systemic autoimmune disease, polyclonal B-cell activation may cause autoimmune disease on its own.

  8. Laser activated nanothermolysis of leukemia cells monitored by photothermal microscopy

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri; Lukianova, Ekaterina; Shnip, Alexander; Zheltov, George; Potapnev, Michail; Savitsky, Valeriy; Klimovich, Olga; Oraevsky, Alexander

    2005-04-01

    We are developing new diagnostic and therapeutic technologies for leukemia based on selective targeting of leukemia cells with gold nanoparticles and thermomechanical destruction of the tumor cells with laser-induced microbubbles. Clusters of spherical gold nanoparticles that have strong optical absorption of laser pulses at 532 nm served as nucleation sites of vapor microbubbles. The nanoparticles were targeted selectively to leukemia cells using leukemia-specific surface receptors and a set of two monoclonal antibodies. Application of a primary myeloid-specific antibody to tumor cells followed by targeting the cells with 30-nm nanoparticles conjugated with a secondary antibody (IgG) resulted in formation of nanoparticulate clusters due to aggregation of IgGs. Formation of clusters resulted in substantial decrease of the damage threshold for target cells. The results encourage development of Laser Activated Nanothermolysis as a Cell Elimination Therapy (LANCET) for leukemia. The proposed technology can be applied separately or in combination with chemotherapy for killing leukemia cells without damage to other blood cells. Potential applications include initial reduction of concentration of leukemia cells in blood prior to chemotherapy and treatment of residual tumor cells after the chemotherapy. Laser-induced bubbles in individual cells and cell damage were monitored by analyzing profile of photothermal response signals over the entire cell after irradiation with a single 10-ns long laser pulse. Photothermal microscopy was utilized for imaging formation of microbubbles around nanoparticulate clusters.

  9. Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

    PubMed Central

    Posadas, Inmaculada; Santos, Pablo; Ceña, Valentín

    2012-01-01

    Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β. PMID:23166834

  10. Functional activity of mitochondria in cultured neural precursor cells.

    PubMed

    Plotnikov, E Yu; Marei, M V; Podgornyi, O V; Aleksandrova, M A; Zorov, D B; Sukhikh, G T

    2006-01-01

    We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity. PMID:16929986

  11. The regulation and activation of lupus-associated B cells.

    PubMed

    Fields, Michele L; Hondowicz, Brian D; Wharton, Gina N; Adair, Brigette S; Metzgar, Michele H; Alexander, Shawn T; Caton, Andrew J; Erikson, Jan

    2005-04-01

    Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

  12. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

    PubMed

    Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

    1999-08-01

    We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. PMID:10411512

  13. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  14. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    PubMed

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

  15. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    PubMed

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk. PMID:27569912

  16. Divide Et Impera--cellular auxin compartmentalization.

    PubMed

    Barbez, Elke; Kleine-Vehn, Jürgen

    2013-02-01

    The phytohormone auxin is an essential regulator for plant growth and development. Decades of intensive research revealed the mutual importance of auxin metabolism and intercellular cell-to-cell transport for the regulation of spatiotemporal auxin distribution. Just recently, intracellular putative auxin carriers, such as the PIN-FORMED (PIN)5/PIN8 and the PIN-LIKES (PILS)2/PILS5 were discovered at the endoplasmic reticulum (ER) and seem to limit nuclear auxin signaling via an auxin sequestration mechanism. Moreover, these auxin carriers at the ER might provide a link between auxin compartmentalization and auxin conjugation-based metabolism. Here we review the recent findings on auxin compartmentalization at the ER and discuss its potential contribution to cellular auxin homeostasis and its importance for plant development. PMID:23200033

  17. Natural killer cell activity in cigarette smokers and asbestos workers

    SciTech Connect

    Ginns, L.C.; Ryu, J.H.; Rogol, P.R.; Sprince, N.L.; Oliver, L.C.; Larsson, C.J.

    1985-06-01

    In order to evaluate the effects of cigarette smoking and asbestos exposure on cellular immunity, the authors tested a group of cigarette smokers and asbestos workers for natural killer (NK) activity in the peripheral blood. The mean NK activity in cigarette smokers was lower than in normal subjects (13.7 +/- 1.6 versus 29.0 +/- 3%; p less than 0.05). As a group, the mean NK activity for the asbestos-exposed group was also reduced compared with that of the nonsmoking control group (22.6 +/- 3.2%; p less than 0.05). When divided according to the smoking status, the asbestos workers who were nonsmokers or ex-smokers showed similar decreases in NK activity compared with normal subjects (19.5 +/- 6.2 and 21.2 +/- 4.5%, respectively; p less than 0.05). A subgroup of asbestos-exposed subjects who currently smoked showed no decrease in NK activity. The data show that NK activity is reduced in the peripheral blood of cigarette smokers and asbestos workers. The relatively normal NK activity found in asbestos workers who also smoked is unexplained. Impairment of NK activity is a potential mechanism for the increased incidence of infection and cancer in smokers and neoplasia in asbestos workers.

  18. Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.

    PubMed Central

    Park, J; Cartwright, C A

    1995-01-01

    Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher throughout all phases of the HT-29 colon carcinoma cell cycle than in corresponding phases of the fibroblast cycle. In addition, during mitosis of HT-29 cells, Src specific activity increased two- to threefold more, while Yes activity and abundance decreased threefold. The decreased steady-state protein levels of Yes during mitosis appeared to be due to both decreased synthesis and increased degradation of the protein. Inhibition of tyrosine but not serine/threonine phosphatases abolished the mitotic activation of Src. Mitotic Src was phosphorylated at novel serine and threonine sites and dephosphorylated at Tyr-527. Two cellular proteins (p160 and p180) were phosphorylated on tyrosine only during mitosis. Tyrosine phosphorylation of several other proteins decreased during mitosis. Thus, Src in HT-29 colon carcinoma cells, similar to Src complexed to polyomavirus middle T antigen or activated by mutation at Tyr-527, is highly active in all phases of the cell cycle. Moreover, Src activity further increases during mitosis, whereas Yes activity and abundance decrease. Thus, Src and Yes appear to be regulated differently during mitosis of HT-29 colon carcinoma cells. PMID:7739521

  19. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    SciTech Connect

    Beebe, D.P.; Wood, L.L.; Moos, M. )

    1990-07-15

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between {sup 125}I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.

  20. A Model Approach to the Electrochemical Cell: An Inquiry Activity

    ERIC Educational Resources Information Center

    Cullen, Deanna M.; Pentecost, Thomas C.

    2011-01-01

    In an attempt to address some student misconceptions in electrochemistry, this guided-inquiry laboratory was devised to give students an opportunity to use a manipulative that simulates the particulate-level activity within an electrochemical cell, in addition to using an actual electrochemical cell. Students are led through a review of expected…

  1. Increased Mitochondrial Activity in Anthrax-Induced Cell Death

    PubMed Central

    Li, Chi

    2009-01-01

    Pathogenesis of anthrax lethal toxin (LT) is attributed to its ability to cause death of infected cells. New work has demonstrated that increase of mitochondrial F1F0 ATPase activity and subsequent depletion of cellular ATP level are critical early events during LT-induced cell death. PMID:26124679

  2. Comparison of the proliferation, cytotoxic activity and cytokine secretion function of cascade primed immune cells and cytokine-induced killer cells in vitro.

    PubMed

    Li, Gui-Xin; Zhao, Shu-Shu; Zhang, Xu-Guang; Wang, Wen-Hao; Liu, Jin; Xue, Ke-Wei; Li, Xiao-Yan; Guo, Ying-Xue; Wang, Li-Hua

    2015-08-01

    The present study aimed to compare the antitumor effects of cascade primed immune (CAPRI) cells and cytokine-induced killer (CIK) cells in vitro, through investigating cell morphology, proliferation, cytotoxic activity to tumor cells and the ability of these cells to secrete cytokines. Peripheral blood samples (50 ml) were obtained from three healthy volunteers and peripheral blood mononuclear cells (PBMCs) were obtained from each via Ficoll-Conray density gradient centrifugation. Each suspension of PBMCs (1 x 10(6)/ml) was divided into two parts; CAPRI cells were obtained from one part through a series of induction, amplification and cytokine cultures, while CIK cells were obtained from the other part through induction with different cytokines. During the culture process, the proliferation and morphological changes were observed for the two cell types using Trypan blue staining. At day 14, the cytotoxic activity of the two cell types was examined through determining lactate dehydrogenase release in the presence of K562 leukemia cells and MCF-7 breast cancer cells. In addition, secretory levels of interferon (IFN)-γ and interleukin (IL)-2 were detected using enzyme-linked immunospot (ELISPOT) technology. The results revealed that at day 5 and 14 of culture, there were significantly fewer CAPRI cells compared with CIK cells (P<0.001), although the survival rate of each cell type was >95%. The cytotoxic activity of CAPRI cells towards the K562 cell line was effector-target ratio-dependent (40:1 and 20:1) with values of 55.1 ± 3.25 and 35.0 ± 2.65%, respectively, which were significantly reduced compared with the corresponding data in CIK cells, 60.0 ± 3.03 and 39.7 ± 3.42% (P=0.004 and 0.005, respectively). Furthermore, the cytotoxic activity of CAPRI cells towards MCF-7 cells were 71.5 ± 3.06, 56.0 ± 3.76 and 40.2 ± 2.90% at effector-target ratios 40:1, 20:1 and 10:1, respectively. These data were significantly higher than the corresponding values in CIK

  3. Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles

    PubMed Central

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted

  4. Beyond the Academic-Corporate Divide

    ERIC Educational Resources Information Center

    Siegel, David J.

    2012-01-01

    Academics often view intercourse with business as a dirty, unchaste affair. Yet in some realms of activity, academic institutions practice greater virtue not by rebuffing corporate interests but by being in bed with them. Cross-sector social partnership, one of the terms of art applied to this sort of interbreeding, offers a potent means of…

  5. Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity.

    PubMed

    Gao, Chunyan; Xie, Rui; Li, Wen; Zhou, Jin; Liu, Shuchuan; Cao, Fenglin; Liu, Yue; Ma, Ruishuang; Si, Yu; Liu, Yan; Bi, Yayan; Gilbert, Gary E; Shi, Jialan

    2013-06-01

    Abundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches includingflow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

  6. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  7. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    PubMed Central

    Shih, Tsung-Chieh; Hsieh, Sen-Yung; Hsieh, Yi-Yueh; Chen, Tse-Chin; Yeh, Chien-Yu; Lin, Chun-Jung; Lin, Deng-Yn; Chiu, Cheng-Tang

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn’s disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (τ = 0.2145, P = 0.0281) and histologic grade (τ = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis. PMID:18350607

  8. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    PubMed Central

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  9. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

    PubMed

    Pinton, Laura; Solito, Samantha; Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-12

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

  10. The activation of protease-activated receptor 1 mediates proliferation and invasion of nasopharyngeal carcinoma cells.

    PubMed

    Zhu, Qingyao; Luo, Jianchao; Wang, Tao; Ren, Jinghua; Hu, Kai; Wu, Gang

    2012-07-01

    Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein, which is involved in physiological and malignant invasion processes. It is activated by serine proteases such as thrombin through a unique form or by specific synthetic peptides. In this study, we determined the expression of PAR-1 in five nasopharyngeal carcinoma (NPC) cell lines with different characteristics of invasiveness and metastasis, and found that the levels of PAR-1 expression were higher in invasive or metastatic cell lines than those in low invasive or metastatic ones. Of the five NPC cell lines, CNE1-LMP1 cells had the highest expression levels of PAR-1, which was mainly distributed at the membrane and in the cytoplasm of tumor cells. Further study showed that the thrombin receptor synthetic activating peptide SFLLRN could stimulate the growth of CNE1-LMP1 cells in a dose-dependent manner. However, thrombin itself had a dual effect on the proliferation of NPC cells. Concentrations of thrombin in the range of 0.1-0.5 U/ml promoted cell growth, but concentrations higher than 0.5 U/ml impaired cell growth. Moreover, thrombin and SFLLRN also enhanced the invasive capabilities of CNE1-LMP1 cells in vitro, and this was partly due to enhancing the activities of MMP-2 and MMP-9. Our findings suggest that PAR-1 may contribute to the growth and invasive potential of NPC cells. PMID:22562397

  11. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    NASA Technical Reports Server (NTRS)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  12. Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

    SciTech Connect

    Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene; Raptis, Leda

    2010-03-10

    Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

  13. Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

    PubMed

    Wang, Han Chin; Lin, Chun-Chieh; Cheung, Rocky; Zhang-Hooks, YingXin; Agarwal, Amit; Ellis-Davies, Graham; Rock, Jason; Bergles, Dwight E

    2015-12-01

    Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells. PMID:26627734

  14. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  15. Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation

    PubMed Central

    Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

    2011-01-01

    Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

  16. Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

    PubMed Central

    Lee, Jee Youn

    2014-01-01

    Background Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. Methods Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. Results Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. Conclusion Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone. PMID:25309797

  17. Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

    PubMed Central

    Marentette, John O.; Hauser, Paul J.; Hurst, Robert E.; Klumpp, David J.; Rickard, Alice; McHowat, Jane

    2013-01-01

    The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS. PMID:23922867

  18. Autoinflammation and autoimmunity: bridging the divide.

    PubMed

    Doria, A; Zen, M; Bettio, S; Gatto, M; Bassi, N; Nalotto, L; Ghirardello, A; Iaccarino, L; Punzi, L

    2012-11-01

    As soon as autoinflammatory diseases (AIDs) emerged as new entities, they have been linked to the well known world of autoimmunity. In fact, AIDs and systemic autoimmune diseases (ADs), share some characteristics: they start with the prefix "auto" to define a pathological process directed against self; they are systemic diseases, frequently involving musculoskeletal system; both include monogenic and polygenic diseases. From the pathogenetic point of view, they are characterized by a chronic activation of immune system, which eventually leads to tissue inflammation in genetically predisposed individuals. Nevertheless, the specific effectors of the damage are different in the two groups of diseases: in AIDs the innate immune system directly causes tissue inflammation, whereas in ADs the innate immune system activates the adaptive immune system which, in turn, is responsible for the inflammatory process. Mutations in inflammasome-related proteins, particularly in NOD-like receptor (NLR) genes, have been strongly associated to the occurrence of AIDs, whereas the link between inflammasome and ADs is less clear. However, a role for this multiprotein-complex in some ADs can be postulated, since a wide spectrum of endogenous danger signals can activate NLRs and inflammasome products, including IL-1ß, can activate adaptive immunity. An association between single nucleotide polymorphisms (SNPs) localized in the inflammasome gene NLRP1 and systemic lupus erythematosus has recently been reported. AIDs and ADs are currently subdivided into two different groups, but looking at their similarities they might be considered as a single group of diseases with a large immune pathological and clinical spectrum which includes at one end pure ADs and at the other end pure AIDs. PMID:22878274

  19. Minimal model for spontaneous cell polarization and edge activity in oscillating, rotating and migrating cells

    NASA Astrophysics Data System (ADS)

    Raynaud, Franck; Ambühl, Mark E.; Gabella, Chiara; Bornert, Alicia; Sbalzarini, Ivo F.; Meister, Jean-Jacques; Verkhovsky, Alexander B.

    2016-04-01

    How cells break symmetry and organize activity at their edges to move directionally is a fundamental question in cell biology. Physical models of cell motility commonly incorporate gradients of regulatory proteins and/or feedback from the motion itself to describe the polarization of this edge activity. These approaches, however, fail to explain cell behaviour before the onset of polarization. We use polarizing and moving fish epidermal cells as a model system to bridge the gap between cell behaviours before and after polarization. Our analysis suggests a novel and simple principle of self-organizing cell activity, in which local cell-edge dynamics depends on the distance from the cell centre, but not on the orientation with respect to the front-back axis. We validate this principle with a stochastic model that faithfully reproduces a range of cell-migration behaviours. Our findings indicate that spontaneous polarization, persistent motion and cell shape are emergent properties of the local cell-edge dynamics controlled by the distance from the cell centre.

  20. The TACI receptor regulates T-cell-independent marginal zone B cell responses through innate activation-induced cell death.

    PubMed

    Figgett, William A; Fairfax, Kirsten; Vincent, Fabien B; Le Page, Mélanie A; Katik, Indzi; Deliyanti, Devy; Quah, Pin Shie; Verma, Pali; Grumont, Raelene; Gerondakis, Steve; Hertzog, Paul; O'Reilly, Lorraine A; Strasser, Andreas; Mackay, Fabienne

    2013-09-19

    Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independent B cell responses.

  1. Volume Changes During Active Shape Fluctuations in Cells

    NASA Astrophysics Data System (ADS)

    Taloni, Alessandro; Kardash, Elena; Salman, Oguz Umut; Truskinovsky, Lev; Zapperi, Stefano; La Porta, Caterina A. M.

    2015-05-01

    Cells modify their volume in response to changes in osmotic pressure but it is usually assumed that other active shape variations do not involve significant volume fluctuations. Here we report experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. We develop and simulate a model of fluid-mediated membrane-cortex deformations and show that a permeable membrane is necessary for bleb formation which is otherwise impaired. Taken together, our experimental and theoretical results emphasize the subtle balance between hydrodynamics and elasticity in actively driven cell morphological changes.

  2. Volume Changes During Active Shape Fluctuations in Cells

    NASA Astrophysics Data System (ADS)

    La Porta, Caterina A. M.; Taloni, Alessandro; Kardash, Elena; Salman, Oguz Umut; Truskinovsky, Lev; Zapperi, Stefano

    Cells modify their volume in response to changes in osmotic pressure but it is usually assumed that other active shape variations do not involve significant volume fluctuations. Here we report experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. We develop and simulate a model of fluid-mediated membrane-cortex deformations and show that a permeable membrane is necessary for bleb formation which is otherwise impaired. Taken together, our experimental and theoretical results emphasize the subtle balance between hydrodynamics and elasticity in actively driven cell morphological changes.

  3. Synergistic activation of cells by Epstein-Barr virus and B-cell growth factor.

    PubMed Central

    Hutt-Fletcher, L M

    1987-01-01

    Infection with Epstein-Barr virus (EBV) is initiated by virus binding to the C3dg-C3d receptor CR2. Several workers have implicated this receptor in the control of B-cell activation by examining the effects of antibodies to CR2 and isolated C3d on B-cell proliferation and differentiation. We report here on the activating effects of irradiated EBV, which retains its capacity to bind to CR2 but loses its ability to function as a T-independent B-cell activator. EBV synergized with B-cell growth factor in the induction of uptake of tritiated thymidine by T cell-depleted leukocytes from seronegative donors but did not induce secretion of immunoglobulin. Synergism could be inhibited with an anti-viral antibody that inhibited binding of EBV to CR2. No similar synergism was found between EBV and recombinant interleukin 2, interleukin 1 alpha, or gamma interferon or with the lipid A fraction of bacterial lipopolysaccharide. EBV may thus initiate B-cell activation as it binds to CR2. Infectious virus may, under normal circumstances, induce the cell to make those growth factors necessary to support B-cell proliferation; the difficulty of transforming cells with transfected EBV DNA may in part reflect the absence of an activation event provided by intact virus as it attaches to CR2. The synergism of EBV and B-cell growth factor more clearly distinguishes the effects of B-cell growth factor from those of interleukin 1 and interleukin 2 in other models of B-cell activation. Thus, this may be a useful model for further delineation of unique effects of B-cell growth factor on B-cell function. PMID:3027404

  4. Computer and Video Games in Family Life: The Digital Divide as a Resource in Intergenerational Interactions

    ERIC Educational Resources Information Center

    Aarsand, Pal Andre

    2007-01-01

    In this ethnographic study of family life, intergenerational video and computer game activities were videotaped and analysed. Both children and adults invoked the notion of a digital divide, i.e. a generation gap between those who master and do not master digital technology. It is argued that the digital divide was exploited by the children to…

  5. Activation of Human T-Helper/Inducer Cell, T-Cytotoxic Cell, B-Cell, and Natural Killer (NK)-Cells and induction of Natural Killer Cell Activity against K562 Chronic Myeloid Leukemia Cells with Modified Citrus Pectin

    PubMed Central

    2011-01-01

    Background Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells. Methods MCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD). Results MCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends. Conclusions MCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP. PMID:21816083

  6. Decrease in T Cell Activation and Calcium Flux during Clinorotation

    NASA Technical Reports Server (NTRS)

    Sams, Clarence; Holtzclaw, J. David

    2006-01-01

    We investigated the effect of altered gravitational environments on T cell activation. We isolated human, naive T cells (CD3+CD14-CD19-CD16-CD56-CD25-CD69-CD45RA-) following IRB approved protocols. These purified T cells were then incubated with 6 mm polystyrene beads coated with OKT3 (Ortho Biotech, Raritan, NJ) and antiCD28 (Becton Dickinson (BD), San Jose, CA) at 37 C for 24 hours. Antibodies were at a 1:1 ratio and the bead-to-cell ratio was 2:1. Four incubation conditions existed: 1) static or "1g"; 2) centrifugation at 10 relative centrifugal force (RCF) or "10g"; 3) clinorotation at 25 RPM (functional weightlessness or "0g"); and 4) clinorotation at 80 RPM ("1g" plus net shear force approx.30 dynes/sq cm). Following incubation, T cells were stained for CD25 expression (BD) and intracellular calcium (ratio of Fluo4 to Fura Red, Molecular Probes, Eugene, OR) and analyzed by flow cytometry (Coulter EPICS XL, Miami, FL). Results: Static or "1g" T cells had the highest level of CD25 expression and intracellular calcium. T cells centrifuged at 10 RCF ("10g") had lower CD25 expression and calcium levels compared to the static control. However, cells centrifuged at 10 RCF had higher CD25 expression and calcium levels than those exposed to 24 RPM clinorotation ("0g"). T cells exposed to 24 RPM clinorotation had lower CD25 expression, but the approximately the same calcium levels than T cells exposed to 80 RPM clinorotation. These data suggest that stress-activated calcium channel exist in T cells and may play a role during T cell activation.

  7. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    SciTech Connect

    Nakajima, Hideaki . E-mail: hnakajim@ims.u-tokyo.ac.jp; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-02-03

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

  8. Nattokinase-promoted tissue plasminogen activator release from human cells.

    PubMed

    Yatagai, Chieko; Maruyama, Masugi; Kawahara, Tomoko; Sumi, Hiroyuki

    2008-01-01

    When heated to a temperature of 70 degrees C or higher, the strong fibrinolytic activity of nattokinase in a solution was deactivated. Similar results were observed in the case of using Suc-Ala-Ala-Pro-Phe-pNA and H-D-Val-Leu-Lys-pNA, which are synthetic substrates of nattokinase. In the current study, tests were conducted on the indirect fibrinolytic effects of the substances containing nattokinase that had been deactivated through heating at 121 degrees C for 15 min. Bacillus subtilis natto culture solutions made from three types of bacteria strain were heat-treated and deactivated, and it was found that these culture solutions had the ability to generate tissue plasminogen activators (tPA) from vascular endothelial cells and HeLa cells at certain concentration levels. For example, it was found that the addition of heat-treated culture solution of the Naruse strain (undiluted solution) raises the tPA activity of HeLa cells to about 20 times that of the control. Under the same conditions, tPA activity was raised to a level about 5 times higher for human vascular endothelial cells (HUVEC), and to a level about 24 times higher for nattokinase sold on the market. No change in cell count was observed for HeLa cells and HUVEC in the culture solution at these concentrations, and the level of activity was found to vary with concentration.

  9. Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

    PubMed Central

    Kolodecik, Thomas R.; Shugrue, Christine A.; Thrower, Edwin C.; Levin, Lonny R.; Buck, Jochen; Gorelick, Fred S.

    2012-01-01

    An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis. PMID:22844459

  10. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes. PMID:25785438

  11. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  12. Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart

    PubMed Central

    Singla, Dinender; Wang, Jing

    2016-01-01

    We hypothesized that fibroblast growth factor-9 (FGF-9) would enhance angiogenesis via activating c-kit positive stem cells in the infarcted nondiabetic and diabetic heart. In brief, animals were divided into three groups: Sham, MI, and MI+FGF-9. Two weeks following MI or sham surgery, our data suggest that treatment with FGF-9 significantly diminished vascular apoptosis compared to the MI group in both C57BL/6 and db/db mice (p < 0.05). Additionally, the number of c-kit+ve/SM α-actin+ve cells and c-kit+ve/CD31+ve cells were greatly enhanced in the MI+FGF-9 groups relative to the MI suggesting FGF-9 enhances c-Kit cell activation and their differentiation into vascular smooth muscle cells and endothelial cells, respectively (p < 0.05). Histology shows that the total number of vessels were quantified for all groups and our data suggest that the FGF-9 treated groups had significantly more vessels than their MI counterparts (p < 0.05). Finally, echocardiographic data suggests a significant improvement in left ventricular output, as indicated by fractional shortening and ejection fraction in both nondiabetic and diabetic animals treated with FGF-9 (p < 0.05). Overall, our data suggests FGF-9 has the potential to attenuate vascular cell apoptosis, activate c-Kit progenitor cells, and enhance angiogenesis and neovascularization in C57BL/6 and db/db mice leading to improved cardiac function. PMID:26682010

  13. Plasmin Activation of Glial Cells through Protease-Activated Receptor 1.

    PubMed

    Greenidge, André R; Hall, Kiana R; Hambleton, Ian R; Thomas, Richelle; Monroe, Dougald M; Landis, R Clive

    2013-01-01

    The objective of this study was to determine whether plasmin could induce morphological changes in human glial cells via PAR1. Human glioblastoma A172 cells were cultured in the presence of plasmin or the PAR1 specific activating hexapeptide, SFLLRN. Cells were monitored by flow cytometry to detect proteolytic activation of PAR1 receptor. Morphological changes were recorded by photomicroscopy and apoptosis was measured by annexinV staining. Plasmin cleaved the PAR1 receptor on glial cells at 5 minutes (P = 0.02). After 30 minutes, cellular processes had begun to retract from the basal substratum and by 4 hours glial cells had become detached. Similar results were obtained by generating plasmin de novo from plasminogen. Morphological transformation was blocked by plasmin inhibitors aprotinin or epsilon-aminocaproic acid (P = 0.03). Cell viability was unimpaired during early morphological changes, but by 24 hours following plasmin treatment 22% of glial cells were apoptotic. PAR1 activating peptide SFLLRN (but not inactive isomer FSLLRN) promoted analogous glial cell detachment (P = 0.03), proving the role for PAR1 in this process. This study has identified a plasmin/PAR1 axis of glial cell activation, linked to changes in glial cell morophology. This adds to our understanding of pathophysiological disease mechanisms of plasmin and the plasminogen system in neuroinjury. PMID:23431500

  14. Angiotensin I converting enzyme activity in rabbit corneal endothelial cells.

    PubMed

    Neels, H M; Vanden Berghe, D A; Neetens, A J; Delgadillo, R A; Scharpe, S L

    1983-01-01

    Angiotensin I converting enzyme (ACE) was studied in Vero cells, rabbit corneal fibroblasts, and rabbit corneal endothelial cells. The enzyme activity was determined by means of an assay employing hippuryl-glycyl-glycine as a substrate. The hippuric acid end product was separated from the substrate by reversed phase liquid chromatography and measured spectrophotometrically at 228 nm. The enzyme was further characterized by a captopril inhibition study. Significant ACE activity was found in rabbit corneal endothelial cells but not in other types of cells tested. This is the first report of the presence of this enzyme in a specific ocular cell type and suggests that angiotensin II may play a role in normal ocular physiology.

  15. Isolation of osteogenic cells from the trauma-activated periosteum

    NASA Astrophysics Data System (ADS)

    Wu, Chang-Hsiao; Bullock, John

    1987-12-01

    Closed, greenstick type fractures were created in adult male white New Zealand rabbits. After a waiting period of 5 days the developing callous and bone approximately 1 cm to each side of the callous was harvested and cell cultures established. Biochemical assays for total protein, alkaline phosphatase activity and glycosamino-glycan content were performed on spent media collected at each change and upon the cells after their termination, in an attempt to more fully characterize the osteoblast population. Since little is known about bone forming cells isolated from this source it is important to establish baseline data so as to be able to relate reactions of these cells to altered environmental conditions.

  16. Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

    PubMed

    Bitencourt, Shanna; Mesquita, Fernanda; Basso, Bruno; Schmid, Júlia; Ferreira, Gabriela; Rizzo, Lucas; Bauer, Moises; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis; Mannaerts, Inge; van Grunsven, Leo Adrianus; Oliveira, Jarbas

    2014-03-01

    Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro. PMID:23955514

  17. Substrate elasticity affects bovine satellite cell activation kinetics in vitro.

    PubMed

    Lapin, M R; Gonzalez, J M; Johnson, S E

    2013-05-01

    Satellite cells support efficient postnatal skeletal muscle hypertrophy through fusion into the adjacent muscle fiber. Nuclear contribution allows for maintenance of the fiber myonuclear domain and proficient transcription of myogenic genes. Niche growth factors affect satellite cell biology; however, the interplay between fiber elasticity and microenvironment proteins remains largely unknown. The objective of the experiment was to examine the effects of hepatocyte growth factor (HGF) and surface elasticity on bovine satellite cell (BSC) activation kinetics in vitro. Young's elastic modulus was calculated for the semimembranosus (SM) and LM muscles of young bulls (5 d; n = 8) and adult cows (27 mo; n = 4) cattle. Results indicate that LM elasticity decreased (P < 0.05) with age; no difference in Young's modulus for the SM was noted. Bovine satellite cells were seeded atop polyacrylamide bioscaffolds with surface elasticities that mimic young bull and adult cow LM or traditional cultureware. Cells were maintained in low-serum media supplemented with 5 ng/mL HGF or vehicle only for 24 or 48 h. Activation was evaluated by proliferating cell nuclear antigen (PCNA) immunocytochemistry. Results indicate that BSC maintained on rigid surfaces were activated at 24 h and refractive to HGF supplementation. By contrast, fewer (P < 0.05) BSC had exited quiescence after 24 h of culture on surfaces reflective of either young bull (8.1 ± 1.7 kPa) or adult cow (14.6 ± 1.6 kPa) LM. Supplementation with HGF promoted activation of BSC cultured on bioscaffolds as measured by an increase (P < 0.05) in PCNA immunopositive cells. Culture on pliant surfaces affected neither activation kinetics nor numbers of Paired box 7 (Pax7) immunopositive muscle stem cells (P > 0.05). However, with increasing surface elasticity, an increase (P < 0.05) in the numbers of muscle progenitors was observed. These results confirm that biophysical and biochemical signals regulate BSC activation.

  18. Fate of retinoic acid-activated embryonic cell lineages.

    PubMed

    Dollé, Pascal; Fraulob, Valérie; Gallego-Llamas, Jabier; Vermot, Julien; Niederreither, Karen

    2010-12-01

    Retinoic acid (RA), a vitamin A derivative, is synthesized by specific cell populations and acts as a diffusible embryonic signal activating ligand-inducible transcription factors, the RA receptors (RARs). RA-activatable transgenic systems have revealed many discrete, transient sites of RA action during development. However, there has been no attempt to permanently label the RA-activated cell lineages during mouse ontogenesis. We describe the characterization of a RA-activatable Cre transgene, which through crosses with a conditional reporter strain (the ROSA26R lacZ reporter), leads to a stable labeling of the cell populations experiencing RA signaling during embryogenesis. RA response-element (RARE)-driven Cre activity mimics at early stages the known activity of the corresponding RARE-lacZ transgene (Rossant et al.,1991). Stable labeling of the Cre-excised cell populations allows to trace the distribution of the RA-activated cell lineages at later stages. These are described in relationship with current models of RA activity in various developmental systems, including the embryonic caudal region, limb buds, hindbrain, sensory organs, and heart. PMID:21046629

  19. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  20. Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells

    PubMed Central

    Cordeau, Martine; Belounis, Assila; Lelaidier, Martin; Cordeiro, Paulo; Sartelet, Hervé; Duval, Michel

    2016-01-01

    High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB. PMID:27716850

  1. Passive versus active local microrheology in mammalian cells and amoebae

    NASA Astrophysics Data System (ADS)

    Riviere, C.; Gazeau, F.; Marion, S.; Bacri, J.-C.; Wilhelm, C.

    2004-12-01

    We compare in this paper the rotational magnetic microrheology detailed by Marion et al [18] and Wilhelm et al [19] to the passive tracking microrheology. The rotational microrheology has been designed to explore, using magnetic rotating probes, the local intracellular microenvironment of living cells in terms of viscoelasticity. Passive microrheology techniques is based on the analysis of spontaneous diffusive motions of Brownian probes. The dependence of mean square displacement (MSD) with the time then directly reflects the type of movement (sub-, hyper- or diffusive motions). Using the same intracellular probes, we performed two types of measurements (active and passive). Based on the fluctuation-dissipation theorem, one should obtain the same information from the both techniques in a thermally equilibrium system. Interestingly, our measurements differ, and the discordances directly inform on active biological processes, which add to thermally activated fluctuations in our out-of equilibrium systems. In both cell models used, mammalian Hela cells and amoebae Entamoeba Histolytica, a hyper-diffusive regime at a short time is observed, which highlights the presence of an active non-thermal driving force, acting on the probe. However, the nature of this active force in mammalian cells and amoebae is different, according to their different phenotypes. In mammalian cells active processes are governed by the transport, via molecular motors, on the microtubule network. In amoebae, which are highly motile cells free of microtubule network, the active processes are dominated by strong fluxes of cytoplasm driven by extension of pseudopodia, in random directions, leading to an amplitude of motion one order of magnitude higher than for mammalian cells. Figs 7, Refs 32.

  2. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  3. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  4. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    PubMed

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-01

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  5. Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

    PubMed Central

    Yang, Yi; Nguyen, Thanh Thi; Jeong, Min-Hye; Crişan, Florin; Yu, Young Hyun; Ha, Hyung-Ho; Choi, Kyung Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl; Kim, Kyung Keun; Hur, Jae-Seoun; Kim, Hangun

    2016-01-01

    Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action. PMID:26751081

  6. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

    PubMed

    Kim, Dong Eun; Kim, Yunha; Cho, Dong-Hyung; Jeong, Seong-Yun; Kim, Sung-Bae; Suh, Nayoung; Lee, Jung Shin; Choi, Eun Kyung; Koh, Jae-Young; Hwang, Jung Jin; Kim, Choung-Soo

    2015-01-01

    Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

  7. Binding of tissue plasminogen activator to cultured human endothelial cells.

    PubMed Central

    Hajjar, K A; Hamel, N M; Harpel, P C; Nachman, R L

    1987-01-01

    Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen. PMID:3119664

  8. Janus particles as artificial antigen-presenting cells for T cell activation.

    PubMed

    Chen, Bo; Jia, Yilong; Gao, Yuan; Sanchez, Lucero; Anthony, Stephen M; Yu, Yan

    2014-01-01

    Here we show that the multifunctionality of Janus particles can be exploited for in vitro T cell activation. We engineer bifunctional Janus particles on which the spatial distribution of two ligands, anti-CD3 and fibronectin, mimics the "bull's eye" protein pattern formed in the membrane junction between a T cell and an antigen-presenting cell. Different levels of T cell activation can be achieved by simply switching the spatial distribution of the two ligands on the surfaces of the "bull's eye" particles. We find that the ligand pattern also affects clustering of intracellular proteins. This study demonstrates that anisotropic particles, such as Janus particles, can be developed as artificial antigen-presenting cells for modulating T cell activation. PMID:25343426

  9. Active Biochemical Regulation of Cell Volume and a Simple Model of Cell Tension Response.

    PubMed

    Tao, Jiaxiang; Sun, Sean X

    2015-10-20

    Active contractile forces exerted by eukaryotic cells play significant roles during embryonic development, tissue formation, and cell motility. At the molecular level, small GTPases in signaling pathways can regulate active cell contraction. Here, starting with mechanical force balance at the cell cortex, and the recent discovery that tension-sensitive membrane channels can catalyze the conversion of the inactive form of Rho to the active form, we show mathematically that this active regulation of cellular contractility together with osmotic regulation can robustly control the cell size and membrane tension against external mechanical or osmotic shocks. We find that the magnitude of active contraction depends on the rate of mechanical pulling, but the cell tension can recover. The model also predicts that the cell exerts stronger contractile forces against a stiffer external environment, and therefore exhibits features of mechanosensation. These results suggest that a simple system for maintaining homeostatic values of cell volume and membrane tension could explain cell tension response and mechanosensation in different environments. PMID:26488645

  10. Streptococcus induces circulating CLA(+) memory T-cell-dependent epidermal cell activation in psoriasis.

    PubMed

    Ferran, Marta; Galván, Ana B; Rincón, Catalina; Romeu, Ester R; Sacrista, Marc; Barboza, Erika; Giménez-Arnau, Ana; Celada, Antonio; Pujol, Ramon M; Santamaria-Babí, Luis F

    2013-04-01

    Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

  11. Radiosensitivity of human natural killer cells: Binding and cytotoxic activities of natural killer cell subsets

    SciTech Connect

    Rana, R.; Vitale, M.; Mazzotti, G.; Manzoli, L.; Papa, S. )

    1990-10-01

    The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.

  12. Plasma-activated medium induced apoptosis on tumor cells

    NASA Astrophysics Data System (ADS)

    Hori, Masaru; Tanaka, Hiromasa; Mizuno, Masaaki; Nakamura, Kae; Kajiyama, Hiroaki; Takeda, Keigo; Ishikawa, Kenji; Kano, Hiroyuki; Kikkawa, Fumitaka

    2013-09-01

    The non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. In this study, the fresh medium was treated with our developed NEAPP, ultra-high electron density (approximately 2 × 1016 cm-3). The medium called the plasma-activated medium (PAM) killed not normal cells but tumor cells through induction of apoptosis. Cell proliferation assays showed that the tumor cells were selectively killed by the PAM. Those cells induced apoptosis using an apoptotic molecular marker, cleaved Caspase3/7. The molecular mechanisms of PAM-mediated apoptosis in the tumor cells were also found that the PAM downregulated the expression of AKT kinase, a marker molecule in a survival signal transduction pathway. These results suggest that PAM may be a promising tool for tumor therapy by downregulating the survival signals in cancers.

  13. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  14. Shed syndecan-2 enhances tumorigenic activities of colon cancer cells

    PubMed Central

    Choi, Sojoong; Choi, Youngsil; Jun, Eunsung; Kim, In-San; Kim, Seong-Eun; Jung, Sung-Ae; Oh, Eok-Soo

    2015-01-01

    Because earlier studies showed the cell surface heparan sulfate proteoglycan, syndecan-2, sheds from colon cancer cells in culture, the functional roles of shed syndecan-2 were assessed. A non-cleavable mutant of syndecan-2 in which the Asn148-Leu149 residues were replaced with Asn148-Ile149, had decreased shedding, less cancer-associated activities of syndecan-2 in vitro, and less syndecan-2-mediated metastasis of mouse melanoma cells in vivo, suggesting the importance of shedding on syndecan-2-mediated pro-tumorigenic functions. Indeed, shed syndecan-2 from cancer-conditioned media and recombinant shed syndecan-2 enhanced cancer-associated activities, and depletion of shed syndecan-2 abolished these effects. Similarly, shed syndecan-2 was detected from sera of patients from advanced carcinoma (625.9 ng/ml) and promoted cancer-associated activities. Furthermore, a series of syndecan-2 deletion mutants showed that the tumorigenic activity of shed syndecan-2 resided in the C-terminus of the extracellular domain and a shed syndecan-2 synthetic peptide (16 residues) was sufficient to establish subcutaneous primary growth of HT29 colon cancer cells, pulmonary metastases (B16F10 cells), and primary intrasplenic tumor growth and liver metastases (4T1 cells). Taken together, these results demonstrate that shed syndecan-2 directly enhances colon cancer progression and may be a promising therapeutic target for controlling colon cancer development. PMID:25686828

  15. Anti-tumor activity of safranal against neuroblastoma cells

    PubMed Central

    Samarghandian, Saeed; Shoshtari, Mohammad Ebrahim; Sargolzaei, Javad; Hossinimoghadam, Hosna; Farahzad, Jabbari Azad

    2014-01-01

    Objective: Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, C10H14O) is an active ingredient in the saffron, which is used in traditional medicine, and also, the biological activity of saffron in anti-cancer is in development. It has been reported to have anti-oxidant effects, but its anti-tumor effects remain uncertain. The aim of this study was to evaluate effects of safranal on anti-tumor on neuroblastoma cells. Materials and Methods: Neuroblastoma cells were cultured and exposed to safranal (0, 10, 15, 20, 50 μg/ml). Cell proliferation was examined using the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic cells, cell cycle distribution, and sub-G1 fraction were analyzed using flow cytometric analysis after propidium iodide staining. Results: Safranal inhibited the growth of malignant cells in a dose-and time-dependent manner. The IC (50) values against the neuroblastoma cell line were determined as 11.1 and 23.3 μg/ml after 24 and 48 h, respectively. Safranal induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in safranal toxicity. Conclusions: Our pre-clinical study demonstrated a neuroblastoma cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not yet clearly understood, it appears to have potential as a therapeutic agent. PMID:24991121

  16. Melatonin modulates aromatase activity and expression in endothelial cells.

    PubMed

    Alvarez-García, Virginia; González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

    2013-05-01

    Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens. PMID:23450505

  17. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

  18. Bovine viral diarrhea virus 2 infection activates the unfolded protein response in MDBK cells, leading to apoptosis.

    PubMed

    Maeda, Kouji; Fujihara, Masatoshi; Harasawa, Ryô

    2009-06-01

    Bovine viral diarrhea virus 2 (BVDV-2) strains are divided into cytopathic and non-cytopathic biotypes based on the ablity to induce cytopathic effects in cultured cells. The mechanism of cytopathogenicity of BVDV-2 is not well understood. We examined cytopathogenesis in MDBK cells resulting from BVDV-2 infections by microscopic examinations and microarray analysis. We found that BVDV-2 activates endoplasmic reticulum (ER) stress signaling pathways that contribute to apoptosis of infected cells. We also monitored the expression of ER stress marker gene by RT-PCR during BVDV-2 infection and demonstrated that infection of MDBK cells with a cytopathic strain of BVDV-2 induces glucose-regulated protein 78 expression. Infection with BVDV-2 also induces DNA-damage-inducible transcript 3 expression and downregulates the lectin-galactoside-binding soluble 1 level. These results show that cytopathic strains of BVDV-2 induce an ER stress response resulting in apoptosis.

  19. Vinculin contributes to Cell Invasion by Regulating Contractile Activation

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2008-07-01

    Vinculin is a component of the focal adhesion complex and is described as a mechano-coupling protein connecting the integrin receptor and the actin cytoskeleton. Vinculin knock-out (k.o.) cells (vin-/-) displayed increased migration on a 2-D collagen- or fibronectin-coated substrate compared to wildtype cells, but the role of vinculin in cell migration through a 3-D connective tissue is unknown. We determined the invasiveness of established tumor cell lines using a 3-D collagen invasion assay. Gene expression analysis of 4 invasive and 4 non-invasive tumor cell lines revealed that vinculin expression was significantly increased in invasive tumor cell lines. To analyze the mechanisms by which vinculin increased cell invasion in a 3-D gel, we studied mouse embryonic fibroblasts wildtype and vin-/- cells. Wildtype cells were 3-fold more invasive compared vin-/- cells. We hypothesized that the ability to generate sufficient traction forces is a prerequisite for tumor cell migration in a 3-D connective tissue matrix. Using traction microscopy, we found that wildtype exerted 3-fold higher tractions on fibronectin-coated polyacrylamide gels compared to vin-/- cells. These results show that vinculin controls two fundamental functions that lead to opposite effects on cell migration in a 2-D vs. a 3-D environment: On the one hand, vinculin stabilizes the focal adhesions (mechano-coupling function) and thereby reduces motility in 2-D. On the other hand, vinculin is also a potent activator of traction generation (mechano-regulating function) that is important for cell invasion in a 3-D environment.

  20. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations. PMID:23299308

  1. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  2. Plasminogen activator inhibitor-1 enhances radioresistance and aggressiveness of non-small cell lung cancer cells

    PubMed Central

    Youn, HyeSook; Kim, Joong Sun; Youn, BuHyun

    2016-01-01

    Acquired resistance of tumor cells during treatment limits the clinical efficacy of radiotherapy. Recent studies to investigate acquired resistance under treatment have focused on intercellular communication because it promotes survival and aggressiveness of tumor cells, causing therapy failure and tumor relapse. Accordingly, a better understanding of the functional communication between subpopulations of cells within a tumor is essential to development of effective cancer treatment strategies. Here, we found that conditioned media (CM) from radioresistant non-small cell lung cancer (NSCLC) cells increased survival of radiosensitive cells. Comparative proteomics analysis revealed plasminogen activator inhibitor-1 (PAI-1) as a key molecule in the secretome that acts as an extracellular signaling trigger to strengthen resistance to radiation. Our results revealed that expression and secretion of PAI-1 in radioresistant cells was increased by radiation-induced transcription factors, including p53, HIF-1α, and Smad3. When CM from radioresistant cells was applied to radiosensitive cells, extracellular PAI-1 activated the AKT and ERK1/2 signaling pathway and inhibited caspase-3 activity. Our study also proposed that PAI-1 activates the signaling pathway in radiosensitive cells via extracellular interaction with its binding partners, not clathrin-mediated endocytosis. Furthermore, secreted PAI-1 increased cell migration capacity and expression of EMT markers in vitro and in vivo. Taken together, our findings demonstrate that PAI-1 secreted from radioresistant NSCLC cells reduced radiosensitivity of nearby cells in a paracrine manner, indicating that functional inhibition of PAI-1 signaling has therapeutic potential because it prevents sensitive cells from acquiring radioresistance. PMID:27004408

  3. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  4. Evidence that a critical threshold of DNA polymerase-alpha activity may be required for the initiation of DNA synthesis in mammalian cell heterokaryons.

    PubMed

    Pendergrass, W R; Saulewicz, A C; Burmer, G C; Rabinovitch, P S; Norwood, T H; Martin, G M

    1982-10-01

    The specific activity of DNA polymerase (90% alpha) was determined in nine "neoplastoid" cell lines (Martin and Sprague, 1973) and in three different strains of HDF (human diploid fibroblast-like cells), all examined in logarithmic phases of growth. This was compared to the ability of each cell type to "rescue" (reinitiate DNA synthesis in) senescent HDF cells subsequent to polyethylene glycol-mediated cell fusions. A sharp "threshold" value of DNA polymerase activity was observed below which reinitiation of DNA synthesis in heterokaryons with senescent HDF does not occur. This threshold was especially obvious when the specific activity of DNA polymerase (p moles dTTP incorporated per mg protein or per cell) was divided by the percent of S-phase cells present in each culture as determined by flow microfluorometry. Our results indicate that the specific activity of DNA polymerase-alpha (or some other factor tightly coregulated with it) in "recessive" cell types (those unable to rescue senescent cells) is only about two times this theoretical "threshold" value, and that fusion of recessive cell types to senescent HDF cells reduces the specific activity in the heterokaryon to below this minimum, thus preventing the cells from entering S phase.

  5. Apoptotic Cells Activate NKT Cells through T Cell Ig-Like Mucin-Like–1 Resulting in Airway Hyperreactivity

    PubMed Central

    Lee, Hyun-Hee; Meyer, Everett H.; Goya, Sho; Pichavant, Muriel; Kim, Hye Young; Bu, Xia; Umetsu, Sarah E.; Jones, Jennifer C.; Savage, Paul B.; Iwakura, Yoichiro; Casasnovas, Jose M.; Kaplan, Gerardo; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Umetsu, Dale T.

    2011-01-01

    T cell Ig-like mucin-like–1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1–dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma. PMID:20889552

  6. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  7. Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

    PubMed

    Czuczman, Natalie M; Barth, Matthew J; Gu, Juan; Neppalli, Vishala; Mavis, Cory; Frys, Sarah E; Hu, Qiang; Liu, Song; Klener, Pavel; Vockova, Petra; Czuczman, Myron S; Hernandez-Ilizaliturri, Francisco J

    2016-03-01

    Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL. PMID:26675347

  8. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly. PMID:26524645

  9. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly.

  10. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  11. [An electrochemical method for measuring metabolic activity and counting cells].

    PubMed

    Kuznetsov, B a; Khlupova, M e; Shleev, S V; Kaprel'iants, A S; Iaropolov, A I

    2006-01-01

    An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 10-5 cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed. PMID:17066962

  12. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  13. Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis.

    PubMed

    Suryavanshi, Shweta; Sharma, Deepak; Checker, Rahul; Thoh, Maikho; Gota, Vikram; Sandur, Santosh K; Sainis, Krishna B

    2015-08-01

    Hematopoietic stem cells and progenitor cells (HSPC) are low in abundance and exhibit high radiosensitivity and their ability to divide dramatically decreases following exposure to ionizing radiation. Our earlier studies have shown antiapoptotic, immune-stimulatory, and antioxidant effects of chlorophyllin, a constituent of the over the counter drug derifil. Here we describe the beneficial effects of chlorophyllin against radiation-induced hematopoietic syndrome. Chlorophyllin administration significantly enhanced the abundance of HSPC in vivo. It induced a transient cell cycle arrest in lineage-negative cells in the bone marrow. However, the chlorophyllin-treated mice exposed to whole body irradiation (WBI) had a significantly higher proportion of actively dividing HSPC in the bone marrow as compared to only WBI-exposed mice. It significantly increased the number of colony forming units (CFUs) by bone marrow cells in vitro and spleen CFUs in irradiated mice in vivo. Pharmacokinetic study showed that chlorophyllin had a serum half-life of 141.8 min in mice. Chlorophyllin upregulated antiapoptotic genes and antioxidant machinery via activation of prosurvival transcription factors Nrf-2 and NF-κB and increased the survival and recovery of bone marrow cells in mice exposed to WBI. Chlorophyllin stimulated granulocyte production in bone marrow and increased the abundance of peripheral blood neutrophils by enhancing serum levels of granulocyte-colony stimulation factor (GCSF). Most importantly, prophylactic treatment of mice with chlorophyllin significantly abrogated radiation-induced mortality. Chlorophyllin mitigates radiation-induced hematopoietic syndrome by increasing the abundance of hematopoietic stem cells, enhancing granulopoiesis, and stimulating prosurvival pathways in bone marrow cells and lymphocytes.

  14. The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

    PubMed

    Barbosa-Lorenzi, Valéria Cintra; Buranello, Patrícia Andressa de Almeida; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance; Pereira-da-Silva, Gabriela

    2011-12-16

    Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

  15. Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

    PubMed

    Cui, X; Zhang, L; Magli, A R; Catera, R; Yan, X-J; Griffin, D O; Rothstein, T L; Barrientos, J; Kolitz, J E; Allen, S L; Rai, K R; Chiorazzi, N; Chu, C C

    2016-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  16. Premalignant Oral Lesion Cells Elicit Increased Cytokine Production and Activation of T-cells

    PubMed Central

    JOHNSON, SARA D.; LEVINGSTON, CORINNE; YOUNG, M. RITA I.

    2016-01-01

    Background Head and neck squamous cell carcinomas (HNSCC) are known to evade the host immune response. How premalignant oral lesions modulate the immune response, however, has yet to be elucidated. Materials and Methods A mouse model of oral carcinogenesis was used to determine how mediators from premalignant oral lesion cells vs. HNSCC cells impact on immune cytokine production and activation. Results Media conditioned by premalignant lesion cells elicited an increased production of T cell-associated cytokines and proinflammatory mediators from cervical lymph node cells compared to media conditioned by HNSCC cells or media alone. In the presence of premalignant lesion cell-conditioned media, CD4+ T cell expression of the IL-2 receptor CD25 and CD8+ T cell expression of the activation marker CD69 was greater, compared to what was induced in HNSCC cell-conditioned media or media alone. Conclusion Premalignant lesion cells promote a proinflammatory environment and induce immune changes before HNSCC tumors are established. PMID:27354582

  17. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells.

    PubMed

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N

    2016-06-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5-CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion.

  18. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells

    PubMed Central

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N.

    2016-01-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5–CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion. PMID:27335323

  19. Mucosal Regulatory T Cells and T Helper 17 Cells in HIV-Associated Immune Activation

    PubMed Central

    Pandiyan, Pushpa; Younes, Souheil-Antoine; Ribeiro, Susan Pereira; Talla, Aarthi; McDonald, David; Bhaskaran, Natarajan; Levine, Alan D.; Weinberg, Aaron; Sekaly, Rafick P.

    2016-01-01

    Residual mucosal inflammation along with chronic systemic immune activation is an important feature in individuals infected with human immunodeficiency virus (HIV), and has been linked to a wide range of co-morbidities, including malignancy, opportunistic infections, immunopathology, and cardiovascular complications. Although combined antiretroviral therapy (cART) can reduce plasma viral loads to undetectable levels, reservoirs of virus persist, and increased mortality is associated with immune dysbiosis in mucosal lymphoid tissues. Immune-based therapies are pursued with the goal of improving CD4+ T-cell restoration, as well as reducing chronic immune activation in cART-treated patients. However, the majority of research on immune activation has been derived from analysis of circulating T cells. How immune cell alterations in mucosal tissues contribute to HIV immune dysregulation and the associated risk of non-infectious chronic complications is less studied. Given the significant differences between mucosal T cells and circulating T cells, and the immediate interactions of mucosal T cells with the microbiome, more attention should be devoted to mucosal immune cells and their contribution to systemic immune activation in HIV-infected individuals. Here, we will focus on mucosal immune cells with a specific emphasis on CD4+ T lymphocytes, such as T helper 17 cells and CD4+Foxp3+ regulatory T cells (Tregs), which play crucial roles in maintaining mucosal barrier integrity and preventing inflammation, respectively. We hypothesize that pro-inflammatory milieu in cART-treated patients with immune activation significantly contributes to enhanced loss of Th17 cells and increased frequency of dysregulated Tregs in the mucosa, which in turn may exacerbate immune dysfunction in HIV-infected patients. We also present initial evidence to support this hypothesis. A better comprehension of how pro-inflammatory milieu impacts these two types of cells in the mucosa will shed light

  20. Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2

    PubMed Central

    Rong, Yuan; Yuan, Chun-Hui; Qu, Zhen; Zhou, Hu; Guan, Qing; Yang, Na; Leng, Xiao-Hua; Bu, Lang; Wu, Ke; Wang, Fu-Bing

    2016-01-01

    Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4+CD25− T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors. PMID:27032536

  1. Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2.

    PubMed

    Rong, Yuan; Yuan, Chun-Hui; Qu, Zhen; Zhou, Hu; Guan, Qing; Yang, Na; Leng, Xiao-Hua; Bu, Lang; Wu, Ke; Wang, Fu-Bing

    2016-01-01

    Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4(+)CD25(-) T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors. PMID:27032536

  2. Dectin-1-activated dendritic cells trigger potent antitumour immunity through the induction of Th9 cells

    PubMed Central

    Zhao, Yinghua; Chu, Xiao; Chen, Jintong; Wang, Ying; Gao, Sujun; Jiang, Yuxue; Zhu, Xiaoqing; Tan, Guangyun; Zhao, Wenjie; Yi, Huanfa; Xu, Honglin; Ma, Xingzhe; Lu, Yong; Yi, Qing; Wang, Siqing

    2016-01-01

    Dectin-1 signalling in dendritic cells (DCs) has an important role in triggering protective antifungal Th17 responses. However, whether dectin-1 directs DCs to prime antitumour Th9 cells remains unclear. Here, we show that DCs activated by dectin-1 agonists potently promote naive CD4+ T cells to differentiate into Th9 cells. Abrogation of dectin-1 in DCs completely abolishes their Th9-polarizing capability in response to dectin-1 agonist curdlan. Notably, dectin-1 stimulation of DCs upregulates TNFSF15 and OX40L, which are essential for dectin-1-activated DC-induced Th9 cell priming. Mechanistically, dectin-1 activates Syk, Raf1 and NF-κB signalling pathways, resulting in increased p50 and RelB nuclear translocation and TNFSF15 and OX40L expression. Furthermore, immunization of tumour-bearing mice with dectin-1-activated DCs induces potent antitumour response that depends on Th9 cells and IL-9 induced by dectin-1-activated DCs in vivo. Our results identify dectin-1-activated DCs as a powerful inducer of Th9 cells and antitumour immunity and may have important clinical implications. PMID:27492902

  3. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  4. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  5. Spectral perspective on the electromagnetic activity of cells.

    PubMed

    Kučera, Ondrej; Červinková, Kateřina; Nerudová, Michaela; Cifra, Michal

    2015-01-01

    In this mini-review, we summarize the current hypotheses, theories and experimental evidence concerning the electromagnetic activity of living cells. We systematically classify the bio-electromagnetic phenomena in terms of frequency and we assess their general acceptance in scientific community. We show that the electromagnetic activity of cells is well established in the low frequency range below 1 kHz and on optical wavelengths, while there is only limited evidence for bio-electromagnetic processes in radio- frequency and millimeter-wave ranges. This lack of generally accepted theory or trustful experimental results is the cause for controversy which accompanies this topic. We conclude our review with the discussion of the relevance of the electromagnetic activity of cells to human medicine.

  6. Structured variability in Purkinje cell activity during locomotion

    PubMed Central

    Sauerbrei, Britton A.; Lubenov, Evgueniy V.; Siapas, Athanassios G.

    2015-01-01

    Summary The cerebellum is a prominent vertebrate brain structure that is critically involved in sensorimotor function. During locomotion, cerebellar Purkinje cells are rhythmically active, shaping descending signals and coordinating commands from higher brain areas with the step cycle. However, the variation in this activity across steps has not been studied, and its statistical structure, afferent mechanisms, and relationship to behavior remain unknown. Here, using multi-electrode recordings in freely moving rats, we show that behavioral variables systematically influence the shape of the step-locked firing rate. This effect depends strongly on the phase of the step cycle and reveals a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. PMID:26291165

  7. Optochemical Activation of Kinase Function in Live Cells

    PubMed Central

    Karginov, Andrei V.; Hahn, Klaus M.; Deiters, Alexander

    2015-01-01

    Summary Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases, and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases (RapR-kinases) in conjunction with a photocaged analog of rapamycin. PMID:24718793

  8. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    NASA Astrophysics Data System (ADS)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  9. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  10. Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

    PubMed

    Watson, Amanda L; Chiu, Norman H L

    2016-09-01

    Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

  11. Biologically active substances-enriched diet regulates gonadotrope cell activation pathway in liver of adult and old rats.

    PubMed

    Oszkiel, Hanna; Wilczak, Jacek; Jank, Michał

    2014-09-01

    According to the Hippocrates' theorem "Let food be your medicine and medicine be your food", dietary interventions may induce changes in the metabolic and inflammatory state by modulating the expression of important genes involved in the chronic disorders. The aim of the present study was to evaluate the influence of long-term (14 months) use of biologically active substances-enriched diet (BASE-diet) on transcriptomic profile of rats' liver. The experiment was conducted on 36 Sprague-Dawley rats divided into two experimental groups (fed with control or BASE-diet, both n = 18). Control diet was a semi-synthetic diet formulated according to the nutritional requirements for laboratory animals. The BASE-diet was enriched with a mixture of polyphenolic compounds, β-carotene, probiotics, and n-3 and n-6 polyunsaturated fatty acids. In total, n = 3,017 differentially expressed (DE) genes were identified, including n = 218 DE genes between control and BASE groups after 3 months of feeding and n = 1,262 after 14 months. BASE-diet influenced the expression of genes involved particularly in the gonadotrope cell activation pathway and guanylate cyclase pathway, as well as in mast cell activation, gap junction regulation, melanogenesis and apoptosis. Especially genes involved in regulation of GnRH were strongly affected by BASE-diet. This effect was stronger with the age of animals and the length of diet use. It may suggest a link between the diet, reproductive system function and aging. PMID:25156242

  12. Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity.

    PubMed

    Wu, Keren; Li, Ning; Sun, Huaqin; Xu, Tao; Jin, Fa; Nie, Jifeng

    2015-10-23

    In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. PMID:26361144

  13. Cytotoxic Activity from Curcuma zedoaria Through Mitochondrial Activation on Ovarian Cancer Cells.

    PubMed

    Shin, Yujin; Lee, Yongkyu

    2013-12-31

    α-Curcumene is one of the physiologically active components of Curcuma zedoaria, which is believed to perform anti-tumor activities, the mechanisms of which are poorly understood. In the present study, we investigated the mechanism of the apoptotic effect of α-curcumene on the growth of human overian cancer, SiHa cells. Upon treatment with α-curcumene, cell viability of SiHa cells was inhibited > 73% for 48 h incubation. α-Curcumene treatment showed a characteristic nucleosomal DNA fragmentation pattern and the percentage of sub-diploid cells was increased in a concentration-dependent manner, hallmark features of apoptosis. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of α-curcumene, which mediates cell death. These results suggest that the apoptotic effect of α-curcumene on SiHa cells may converge caspase-3 activation through the release of mitochondrial cytochrome c.

  14. Investigation of MEK activity in COS7 cells entering mitosis.

    PubMed

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Luo, Jun

    2014-12-01

    Although the mitogen-activated protein kinase (MAPK) pathway has been extensively investigated, numerous events remain unclear. In the present study, we examined mitogen-activated protein kinase kinase (MEK) expression from interphase to mitosis. Following nocodazole treatment, COS7 cells gradually became round as early as 4 h after treatment. Cyclin B1 expression gradually increased from 4 to 24 h in the presence of nocodazole. When cells were treated with nocodazole for 4 h, the level of epidermal growth factor (EGF)-mediated MEK phosphorylation did not significantly change between nocodazole-untreated and -treated (4 h) cells (P>0.05). However, EGF-mediated MEK phosphorylation was significantly inhibited upon treatment with nocodazole for 8 and 24 h compared to nocodazole-untreated cells (P<0.05). MEK phosphorylation levels were comparable between 1, 5, 10 and 50 ng/ml EGF treatments. Phorbol 12-myristic 13-acetate (PMA) did not activate MEK in mitotic cells. Following treatment of COS7 cells at the interphase with AG1478 or U0126, MEK phosphorylation was blocked. In addition, the investigation of the expression of proteins downstream of MEK demonstrated that EGF does not significantly affect the phosphorylation level of extracellular-signal-regulated kinase (ERK), ribosomal protein S6 kinase (RSK) and Elk in mitotic cells (P>0.05). The results showed that MEK expression is gradually inhibited from cell interphase to mitosis, and that MEK downstream signaling is affected by this inhibition, which probably reflects the requirements of cell physiology during mitosis.

  15. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    PubMed

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics. PMID:26068799

  16. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-12-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  17. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed Central

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-01-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  18. Biphasic activity of chloroquine in human colorectal cancer cells.

    PubMed

    Park, Deokbae; Lee, Youngki

    2014-12-01

    Autophagy is a homeostatic degradation process that is involved in tumor development and normal development. Autophagy is induced in cancer cells in response to chemotherapeutic agents, and inhibition of autophagy results in enhanced cancer cell death or survival. Chloroquine (CQ), an anti-malarial devrepug, is a lysosomotropic agent and is currently used as a potential anticancer agent as well as an autophagy inhibitor. Here, we evaluate the characteristics of these dual activities of CQ using human colorectal cancer cell line HCT15. The results show that CQ inhibited cell viability in dose-and time-dependent manner in the range between 20 to 80 uM, while CQ did not show any antiproliferative activity at 5 and 10 uM. Cotreatment of CQ with antitumor agent NVP-BEZ235, a dual inhibitor of PI3K/mTOR, rescued the cell viability at low concentrations meaning that CQ acted as an autophagy inhibitor, but CQ induced the lethal effect at high concentrations. Acridine orange staining revealed that CQ at high doses induced lysosomal membrane permeabilization (LMP). High doses of CQ produced cellular reactive oxygen species (ROS) and cotreatment of antioxidants, such as NAC and trolox, with high doses of CQ rescued the cell viability. These results suggest that CQ may exert its dual activities, as autophagy inhibitor or LMP inducer, in concentration-dependent manner. PMID:25949192

  19. Biphasic Activity of Chloroquine in Human Colorectal Cancer Cells

    PubMed Central

    Park, Deokbae; Lee, Youngki

    2014-01-01

    Autophagy is a homeostatic degradation process that is involved in tumor development and normal development. Autophagy is induced in cancer cells in response to chemotherapeutic agents, and inhibition of autophagy results in enhanced cancer cell death or survival. Chloroquine (CQ), an anti-malarial devrepug, is a lysosomotropic agent and is currently used as a potential anticancer agent as well as an autophagy inhibitor. Here, we evaluate the characteristics of these dual activities of CQ using human colorectal cancer cell line HCT15. The results show that CQ inhibited cell viability in dose-and time-dependent manner in the range between 20 to 80 uM, while CQ did not show any antiproliferative activity at 5 and 10 uM. Cotreatment of CQ with antitumor agent NVP-BEZ235, a dual inhibitor of PI3K/mTOR, rescued the cell viability at low concentrations meaning that CQ acted as an autophagy inhibitor, but CQ induced the lethal effect at high concentrations. Acridine orange staining revealed that CQ at high doses induced lysosomal membrane permeabilization (LMP). High doses of CQ produced cellular reactive oxygen species (ROS) and cotreatment of antioxidants, such as NAC and trolox, with high doses of CQ rescued the cell viability. These results suggest that CQ may exert its dual activities, as autophagy inhibitor or LMP inducer, in concentration-dependent manner. PMID:25949192

  20. Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis

    PubMed Central

    Saini, Manoj; Pearson, Claire

    2009-01-01

    Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo. PMID:19357399

  1. Antihelminthic niclosamide modulates dendritic cells activation and function.

    PubMed

    Wu, Chieh-Shan; Li, Yi-Rong; Chen, Jeremy J W; Chen, Ying-Che; Chu, Chiang-Liang; Pan, I-Hong; Wu, Yu-Shan; Lin, Chi-Chen

    2014-01-01

    Dendritic cells (DCs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. Accordingly, DCs are considered to be a major target in the development of immunomodulating compounds. In this study, the effect of niclosamide, a Food and Drug Administration-approved antihelminthic drug, on the activation of lipopolysaccharide (LPS)-stimulated murine bone marrow-derived DCs was examined. Our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of LPS-activated DCs. In addition, niclosamide also affected the expression of MHC and costimulatory molecules and influenced the ability of the cells to take up antigens. Therefore, in mixed cell cultures composed of syngeneic OVA-specific T cells and DCs, niclosamide-treated DCs showed a decreased ability to stimulate T cell proliferation and IFN-γ production. Furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (CHS) in mice during sensitization with 2,4-dinitro-1-fluorobenzene. Blocking the LPS-induced activation of MAPK-ERK, JNK and NF-κB may contribute to the inhibitory effect of niclosamide on DC activation. Collectively, our findings suggest that niclosamide can manipulate the function of DCs. These results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or DC-mediated autoimmune diseases. PMID:24561310

  2. Activation of cells using femtosecond laser beam (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Batabyal, Subrata; Satpathy, Sarmishtha; Kim, Young-tae; Mohanty, Samarendra K.

    2016-03-01

    Study of communication in cellular systems requires precise activation of targeted cell(s) in the network. In contrast to chemical, electrical, thermal, mechanical stimulation, optical stimulation is non-invasive and is better suited for stimulation of targeted cells. As compared to visible lasers, the near infrared (NIR) microsecond/nanosecond pulsed laser beams are being used as preferred stimulation tool as they provide higher penetration depth in tissues. Femotosecond (FS) laser beams in NIR are also being used for direct and indirect (i.e. via two-photon optogenetics) stimulation of cells. Here, we present a comparative evaluation of efficacy of NIR FS laser beam for direct (no optogenetic sensitization) and 2ph optogenetic stimulation of cells. Further, for the first time, we demonstrate the use of blue (~450 nm, obtained by second harmonic generation) FS laser beam for stimulation of cells with and without Channelrhodopisn-2 (ChR2) expression. Comparative analysis of photocurrent generated by blue FS laser beam and continuous wave blue light for optogenetics stimulation of ChR2 transfected HEK cells will be presented. The use of ultrafast laser micro-beam for focal, non-contact, and repeated stimulation of single cells in a cellular circuitry allowed us to study the communication between different cell types.

  3. AEA Cell-Bypass-Switch Activation: An Update

    NASA Technical Reports Server (NTRS)

    Keys, Denney; Rao, Gopalakrishna M.; Wannemacher, Harry

    2002-01-01

    The objectives of this project included the following: (1) verify the performance of AEA cell bypass protection device (CBPD) under simulated EOS-Aqua/Aura flight hardware configuration; (2) assess the safety of the hardware under an inadvertent firing of CBPD switch, as well as the closing of CBPD; and (3) confirm that the mode of operation of CBPD switch is the formation of a continuous low impedance path (a homogeneous low melting point alloy). The nominal performance of AEA CBPD under flight operating conditions (vacuum except zero-G, and high impedance cell) has been demonstrated. There is no evidence of cell rupture or excessive heat production during or after CBPD switch activation under simulated high cell impedance (open-circuit cell failure mode). The formation of a continuous low impedance path (a homogeneous low melting point alloy) has been confirmed.

  4. Rare earth elements activate endocytosis in plant cells

    PubMed Central

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-01-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms. PMID:25114214

  5. Rare earth elements activate endocytosis in plant cells.

    PubMed

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-09-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms.

  6. Pattern matching based active optical sorting of colloids/cells

    NASA Astrophysics Data System (ADS)

    Verma, R. S.; Dasgupta, R.; Ahlawat, S.; Kumar, N.; Uppal, A.; Gupta, P. K.

    2013-08-01

    We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.

  7. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  8. Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells

    PubMed Central

    Bozic, Iva; Savic, Danijela; Stevanovic, Ivana; Pekovic, Sanja; Nedeljkovic, Nadezda; Lavrnja, Irena

    2015-01-01

    Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes—catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and ·O−2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells. PMID:26388737

  9. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  10. KIPase activity is a novel caspase-like activity associated with cell proliferation.

    PubMed

    Medina-Palazon, Cahora; Bernard, Emmanuelle; Frost, Victoria; Morley, Simon; Sinclair, Alison J

    2004-07-01

    A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.

  11. Analyzing electrical activities of pancreatic β cells using mathematical models.

    PubMed

    Cha, Chae Young; Powell, Trevor; Noma, Akinori

    2011-11-01

    Bursts of repetitive action potentials are closely related to the regulation of glucose-induced insulin secretion in pancreatic β cells. Mathematical studies with simple β-cell models have established the central principle that the burst-interburst events are generated by the interaction between fast membrane excitation and slow cytosolic components. Recently, a number of detailed models have been developed to simulate more realistic β cell activity based on expanded findings on biophysical characteristics of cellular components. However, their complex structures hinder our intuitive understanding of the underlying mechanisms, and it is becoming more difficult to dissect the role of a specific component out of the complex network. We have recently developed a new detailed model by incorporating most of ion channels and transporters recorded experimentally (the Cha-Noma model), yet the model satisfies the charge conservation law and reversible responses to physiological stimuli. Here, we review the mechanisms underlying bursting activity by applying mathematical analysis tools to representative simple and detailed models. These analyses include time-based simulation, bifurcation analysis and lead potential analysis. In addition, we introduce a new steady-state I-V (ssI-V) curve analysis. We also discuss differences in electrical signals recorded from isolated single cells or from cells maintaining electrical connections within multi-cell preparations. Towards this end, we perform simulations with our detailed pancreatic β-cell model.

  12. Long noncoding RNAs in B-cell development and activation

    PubMed Central

    Brazão, Tiago F.; Johnson, Jethro S.; Müller, Jennifer; Heger, Andreas; Ponting, Chris P.

    2016-01-01

    Long noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia. PMID:27381906

  13. Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter.

    PubMed

    Schmid, Lothar; Weitz, David A; Franke, Thomas

    2014-10-01

    We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications.

  14. Tomato waste: Carotenoids content, antioxidant and cell growth activities.

    PubMed

    Stajčić, Sladjana; Ćetković, Gordana; Čanadanović-Brunet, Jasna; Djilas, Sonja; Mandić, Anamarija; Četojević-Simin, Dragana

    2015-04-01

    The carotenoid content, antioxidant and cell growth activities of tomato waste extracts, obtained from five different tomato genotypes, was investigated. High performance liquid chromatography was used to identify and quantify the main carotenoids present in tomato waste extracts. The antioxidant activity of tomato waste extracts was tested using spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power assay. The highest DPPH scavenging activity (IC50 = 0.057 mg/ml) was obtained for Bačka extract. The Knjaz extract showed the best reducing power (IC50 = 2.12 mg/ml). Cell growth effects were determined in HeLa, MCF7 and MRC-5 cell lines by sulforhodamine B test. Anti-proliferative effects were observed in all cell lines at higher concentrations (⩾ 0.125 mg/ml). The carotenoid contents exhibited a strong correlation with antioxidant and anti-proliferation activity. The results obtained indicated that tomato waste should be regarded as potential nutraceutic resource and may be used as a functional food ingredient.

  15. Tomato waste: Carotenoids content, antioxidant and cell growth activities.

    PubMed

    Stajčić, Sladjana; Ćetković, Gordana; Čanadanović-Brunet, Jasna; Djilas, Sonja; Mandić, Anamarija; Četojević-Simin, Dragana

    2015-04-01

    The carotenoid content, antioxidant and cell growth activities of tomato waste extracts, obtained from five different tomato genotypes, was investigated. High performance liquid chromatography was used to identify and quantify the main carotenoids present in tomato waste extracts. The antioxidant activity of tomato waste extracts was tested using spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power assay. The highest DPPH scavenging activity (IC50 = 0.057 mg/ml) was obtained for Bačka extract. The Knjaz extract showed the best reducing power (IC50 = 2.12 mg/ml). Cell growth effects were determined in HeLa, MCF7 and MRC-5 cell lines by sulforhodamine B test. Anti-proliferative effects were observed in all cell lines at higher concentrations (⩾ 0.125 mg/ml). The carotenoid contents exhibited a strong correlation with antioxidant and anti-proliferation activity. The results obtained indicated that tomato waste should be regarded as potential nutraceutic resource and may be used as a functional food ingredient. PMID:25442547

  16. CTNNB1 Signaling in Sertoli Cells Downregulates Spermatogonial Stem Cell Activity via WNT4

    PubMed Central

    Boyer, Alexandre; Yeh, Jonathan R.; Zhang, Xiangfan; Paquet, Marilène; Gaudin, Aurore; Nagano, Makoto C.; Boerboom, Derek

    2012-01-01

    Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development. PMID:22253774

  17. Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

    PubMed

    Seo, Yo-Seob; Yim, Min-Ji; Kim, Bok-Hee; Kang, Kyung-Rok; Lee, Sook-Young; Oh, Ji-Su; You, Jae-Seek; Kim, Su-Gwan; Yu, Sang-Joun; Lee, Gyeong-Je; Kim, Do Kyung; Kim, Chun Sung; Kim, Jin-Soo; Kim, Jae-Sung

    2015-12-01

    In the present study, we investigated berberine‑induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria‑dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck squamous carcinoma.

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

    PubMed

    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.

  19. PARP activation promotes nuclear AID accumulation in lymphoma cells

    PubMed Central

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

    2016-01-01

    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. PMID:26921193

  20. Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

    PubMed

    Collins, Cheryl C; Bashant, Kathleen; Erikson, Cuixia; Thwe, Phyu Myat; Fortner, Karen A; Wang, Hong; Morita, Craig T; Budd, Ralph C

    2016-01-01

    γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress. PMID:27431410

  1. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

    SciTech Connect

    Wang, Lei; Nishihara, Hiroshi; Kimura, Taichi; Kato, Yasutaka; Tanino, Mishie; Nishio, Mitsufumi; Obara, Masato; Endo, Tomoyuki; Koike, Takao; Tanaka, Shinya

    2010-04-23

    DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.

  2. Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

    PubMed

    Martino, Nicola; Bossio, Caterina; Vaquero Morata, Susana; Lanzani, Guglielmo; Antognazza, Maria Rosa

    2016-01-28

    Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported

  3. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  4. Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

    PubMed

    Martino, Nicola; Bossio, Caterina; Vaquero Morata, Susana; Lanzani, Guglielmo; Antognazza, Maria Rosa

    2016-01-01

    Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported

  5. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    PubMed

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  6. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  7. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

    PubMed

    Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

    1996-02-15

    Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

  8. Oxytocin-stimulated NFAT transcriptional activation in human myometrial cells.

    PubMed

    Pont, Jason N A; McArdle, Craig A; López Bernal, Andrés

    2012-10-01

    Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca(2+) stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1-C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.

  9. Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1986-05-15

    The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4..beta..-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Results of cell cycle analysis support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen stimulated T cells through the first round of the cell cycle.

  10. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  11. Blockade of mast cell activation reduces cutaneous scar formation.

    PubMed

    Chen, Lin; Schrementi, Megan E; Ranzer, Matthew J; Wilgus, Traci A; DiPietro, Luisa A

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound.

  12. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

    PubMed

    Liang, H; Reich, C F; Pisetsky, D S; Lipsky, P E

    2000-08-01

    Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

  13. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  14. Immunomodulation of phloretin by impairing dendritic cell activation and function.

    PubMed

    Lin, Chi-Chen; Chu, Ching-Liang; Ng, Chin-Sheng; Lin, Ching-Yen; Chen, Der-Yuan; Pan, I-Hong; Huang, Kao-Jean

    2014-05-01

    Dietary compounds in fruits and vegetables have been shown to exert many biological activities. In addition to antioxidant effects, a number of flavonoids are able to modulate inflammatory responses. Here, we demonstrated that phloretin (PT), a natural dihydrochalcone found in many fruits, suppressed the activation and function of mouse dendritic cells (DCs). Phloretin disturbed the multiple intracellular signaling pathways in DCs induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), including ROS, MAPKs (ERK, JNK, p38 MAPK), and NF-κB, and thereby reducing the production of inflammatory cytokines and chemokines. Phloretin also effectively suppressed the activation of DCs treated with different dosages of LPS or various TLR agonists. The LPS-induced DC maturation was attenuated by phloretin because the expression levels of the MHC class II and the co-stimulatory molecules were down-regulated, which then inhibited the LPS-stimulating DCs and the subsequent naïve T cell activation in a mixed lymphocyte reaction. Moreover, in vivo administration of phloretin suppressed the phenotypic maturation of the LPS-challenged splenic DCs and decreased the IFN-γ production from the activated CD4 T cells. Thus, we suggest that phloretin may potentially be an immunomodulator by impairing the activation and function of DCs and phloretin-contained fruits may be helpful in the improvement of inflammation and autoimmune diseases. PMID:24651121

  15. Cloud Activation Characteristics of Airborne Erwinia carotovora Cells.

    NASA Astrophysics Data System (ADS)

    Franc, Gary D.; Demott, Paul J.

    1998-10-01

    Several strains of plant pathogenic bacteria, Erwinia carotovora carotovora and E. carotovora atroseptica, were observed to be active as cloud condensation nuclei (CCN). The CCN supersaturation spectra of bacterial aerosols were measured in the laboratory and compared to the activity of ammonium sulfate. Approximately 25%-30% of the aerosolized bacterial cells activated droplets at 1% water supersaturation compared to 80% activation of the ammonium sulfate aerosol. Physical and numerical simulations of cloud droplet activation and growth on bacteria were also performed. Both simulations predict that aerosolized bacteria will be incorporated into cloud droplets during cloud formation. Results strongly support the hypothesis that significant numbers of the tested bacterial strains are actively involved in atmospheric cloud formation and precipitation processes following natural aerosolization and vertical transport to cloud levels.

  16. Inhibition of peroxisome proliferator-activated receptor gamma prevents the melanogenesis in murine B16/F10 melanoma cells.

    PubMed

    Chen, Jiun-Han; Chang, Junn-Liang; Chen, Pei-Ru; Chuang, Yun-Ju; Tang, Shih-Tsang; Pan, Shwu-Fen; Lin, Tzer-Bin; Chen, Kang-Hua; Chen, Mei-Jung

    2014-01-01

    The purpose of this study was to investigate if PPARγ plays a role in the melanogenesis. B16/F10 cells were divided into five groups: control, melanin stimulating hormone (α-MSH), α-MSH+retinol, α-MSH+GW9662 (PPARγ antagonist), and GW9662. Cells in the control group were cultured in the Dulbecco's modified Eagle's medium (DMEM) for 48 hrs. To initiate the melanogenesis, cells in all α-MSH groups were cultured in medium containing α-MSH (10 nM) for 48 hrs. Cells were treated simultaneously with retinol (5 μM) in the α-MSH+retinol group. Instead of retinol, GW9662 (10 μM) was cocultured in the α-MSH+GW9662 group. Cells in the final group were cultured in the DMEM with GW9662. All the analyses were carried out 48 hours after treatments. The α-MSH was able to increase cell number, melanin production, and the activity of tyrosinase, the limiting enzyme in melanogenesis. These α-MSH-induced changes were prevented either by retinol or by GW9662. Further analyses of the activities of antioxidant enzymes including glutathione, catalase, and the superoxide dismutase (SOD) showed that α-MSH treatment raised the activity of SOD which was dependent on PPARγ level. According to our results, the α-MSH-induced melanogenesis was PPARγ dependent, which also modulated the expression of SOD. PMID:25250328

  17. The predominance of alternatively activated macrophages following challenge with cell wall peptide-polysaccharide after prior infection with Sporothrix schenckii.

    PubMed

    Alegranci, Pamela; de Abreu Ribeiro, Livia Carolina; Ferreira, Lucas Souza; Negrini, Thais de Cássia; Maia, Danielle Cardoso Geraldo; Tansini, Aline; Gonçalves, Amanda Costa; Placeres, Marisa Campos Polesi; Carlos, Iracilda Zeppone

    2013-08-01

    Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into "classic" or "alternatively" activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-β, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-β production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.

  18. A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

    NASA Technical Reports Server (NTRS)

    Platt, Donald W.; Hoover, Richard B.

    2009-01-01

    A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

  19. Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

    PubMed Central

    Diu, A; Gougeon, M L; Moreau, J L; Reinherz, E L; Thèze, J

    1987-01-01

    The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle. PMID:2962196

  20. Active p21-activated kinase 1 rescues MCF10A breast epithelial cells from undergoing anoikis.

    PubMed

    Menard, Raymond E; Jovanovski, Andrew P; Mattingly, Raymond R

    2005-07-01

    The protein kinase, PAK1, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAK1 in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), after more than 8 hours of detachment. Endogenous Akt, PAK1, and BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAK1 or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Co-overexpression of active PAK1 with dominant-negative Akt, or of active Akt with dominant-negative PAK1, still suppresses anoikis. Thus, Akt and PAK1 enhance survival through pathways that are at least partially independent. PAK1-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAK1 increased the cleavage of the upstream caspase 9, while constitutively active PAK1 inhibited caspase 9 activation. These results support a role for activated PAK1 in the suppression of anoikis in MCF10A epithelial cells.

  1. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

  2. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

  3. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  4. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma. PMID:27535933

  5. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

  6. Telomerase activity in non-small cell lung cancer

    PubMed Central

    Dobija-Kubica, Katarzyna; Bruliński, Krzysztof; Rogoziński, Paweł; Wiczkowski, Andrzej; Gawrychowska, Agata; Gawrychowski, Jacek

    2016-01-01

    Introduction High telomerase activity has been detected in the majority of malignant neoplasms including lung cancer. The purpose of the study was to attempt to use telomerase activity as a prognostic factor in patients with non-small cell lung cancer (NSCLC). Material and methods Telomerase activity was analyzed in 47 tissue specimens taken from patients with NSCLC. The control group consisted of 30 specimens of non-cancerous lung parenchyma. Telomerase activity was measured by means of the telomeric repeat amplification protocol (TRAP). Results Telomerase activity in the neoplastic tissue was significantly higher than in the lung parenchyma that was free from neoplastic infiltration. There was no significant association between telomerase activity and age, gender, tobacco smoking, histological type of the tumor, or staging (pTNM). No association was found between the level of telomerase activity in NSCLC specimens and the two-year survival rate of patients (p = 0.326). A higher level of telomerase activity in poorly differentiated tumors (G3) as compared to moderately differentiated tumors (G2) was detected (p = 0.008). A positive association was identified between telomerase activity in pulmonary parenchyma free from tumor infiltration and the presence of leukocyte infiltration (p = 0.0001). Conclusions No association was found between the level of telomerase activity in NSCLC specimens and the two-year survival rate of patients. The study has revealed a positive association between telomerase activity and the grade of differentiation (G) in NSCLC. PMID:27212973

  7. Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

    PubMed

    Puente, Lawrence G; Mireau, Laura R; Lysechko, Tara L; Ostergaard, Hanne L

    2005-06-01

    Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.

  8. FGF2 activates TRPC and Ca(2+) signaling leading to satellite cell activation.

    PubMed

    Liu, Yewei; Schneider, Martin F

    2014-01-01

    Satellite cells, as stem cells of adult skeletal muscle, are tightly associated with the differentiated muscle fibers and remain quiescent in the absence of muscle damage. In response to an injury, the quiescent satellite cell is activated by soluble factors, including FGFs released from injured myofibers. Using immunostaining, we here first show that TRPC1 channels are highly expressed in satellite cells attached to muscle fibers. Since CD34, a traditional stem cell marker, was recently found to be expressed in skeletal muscle satellite cells we labeled living satellite cells in their physiological niche associated with host FDB fibers using anti-CD34-FITC antibody. We then monitored intra-cellular calcium in anti-CD34-FITC labeled satellite cells attached to muscle fibers using the calcium sensitive dye X rhod-1 which has little fluorescence cross talk with FITC. FGF2 increased intracellular calcium in satellite cells, which was antagonized by the TRPC channel blocker SKF 96365. Immunostaining showed that NFATc3 is highly expressed in satellite cells, but not in host FDB fibers. Elevation of intracellular calcium by FGF2 is accompanied by nuclear translocation of NFATc3 and NFATc2 and by an increase in the number of MyoD positive cells per muscle fiber, both of which were attenuated by TRPC blocker SKF 96365. Our results suggest a novel pathway of satellite cell activation where FGF2 enhances calcium influx through a TRPC channel, and the increased cytosolic calcium leads to both NFATc3 and NFATc2 nuclear translocation and enhanced number of MyoD positive satellite cells per muscle fiber.

  9. Hydrogenated Amorphous Silicon Germanium Active Layer for Top Cell of a Multi Junction Cell Structure.

    PubMed

    Cho, Jaehyun; Iftiquar, S M; Kim, Minbum; Park, Jinjoo; Jung, Junhee; Kim, Jiwoong; Yi, Junsin

    2016-05-01

    Intrinsic hydrogenated amorphous silicon-germanium (a-SiGe:H) alloy is generally used in the bottom cell because of its low band gap. The a-SiGe:H has a higher photo conductivity in comparison to the a-Si:H; thus, it is expected that the a-SiGe:H can show better short circuit current density than that of the a-Si:H based solar cell. Therefore, we optimized a-SiGe:H active layer that can be a suitable choice for the front cell of a multi junction.solar cell. Furthermore, we carried out a comparative study of the solar cells that have a-SiGe:H and a-Si:H as respective active layers. The a-SiGe:H based solar cells show higher short circuit current density, while the a-Si:H based cells show higheropen circuit voltage. The current-voltage characteristics of these cells are as follows: (a) V(oc) = 770 mV, J(sc) = 15.0 mA/cm2, FF = 64.5%, and η = 7.47% for a-SiGe:H based cell; and (b) V(oc) = 826 mV, J(sc) = 13.63 mA/cm2, FF = 72.0%, and η = 8.1% for a-Si:H based cell.

  10. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  11. Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

    PubMed

    Nagl, S; Haas, M; Lahmer, G; Büttner-Herold, M; Grabenbauer, G G; Fietkau, R; Distel, L V

    2016-05-01

    Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells. PMID:27467940

  12. ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED ARSENICALS

    EPA Science Inventory

    ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED TRIVALENT ARSENICALS. Z Drobna1, I Jaspers2, D J Thomas3 and M Styblo1. 1Department of Pediatrics; 2Center for Environmental Medicine and Lung Biology, University of North Carolina at Chapel Hill, NC, USA; 3US EPA, RTP, NC, USA.

  13. Decorin binds myostatin and modulates its activity to muscle cells

    SciTech Connect

    Miura, Takayuki; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito; Hennebry, Alex; Berry, Carole J.; Sharma, Mridula; Kambadur, Ravi; Nishimura, Takanori . E-mail: nishi@anim.agr.hokudai.ac.jp

    2006-02-10

    Myostatin, a member of TGF-{beta} superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-{beta} and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn{sup 2+} greater than 10 {mu}M, but not in the absence of Zn{sup 2+}. Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K {sub D}) of 2.02 x 10{sup -8} M and 9.36 x 10{sup -9} M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.

  14. Aurora A kinase activity influences calcium signaling in kidney cells.

    PubMed

    Plotnikova, Olga V; Pugacheva, Elena N; Golemis, Erica A

    2011-06-13

    Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD.

  15. Dextromethorphan Inhibits Activations and Functions in Dendritic Cells

    PubMed Central

    Chen, Der-Yuan; Song, Pei-Shan; Hong, Jau-Shyong; Chu, Ching-Liang; Pan, I-Horng; Chen, Yi-Ming; Lin, Ching-Hsiung; Lin, Sheng-Hao; Lin, Chi-Chen

    2013-01-01

    Dendritic cells (DCs) play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM), a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs) was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS), proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF-κB translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs). These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases. PMID:23781253

  16. Hox proteins: sculpting body parts by activating localized cell death.

    PubMed

    Alonso, Claudio R

    2002-11-19

    Hox proteins shape animal structures by eliciting different developmental programs along the anteroposterior body axis. A recent study reveals that the Drosophila Hox protein Deformed directly activates the cell-death-promoting gene reaper to maintain the boundaries between distinct head segments.

  17. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

    PubMed Central

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent. PMID:26858704

  18. Dextromethorphan inhibits activations and functions in dendritic cells.

    PubMed

    Chen, Der-Yuan; Song, Pei-Shan; Hong, Jau-Shyong; Chu, Ching-Liang; Pan, I-Horng; Chen, Yi-Ming; Lin, Ching-Hsiung; Lin, Sheng-Hao; Lin, Chi-Chen

    2013-01-01

    Dendritic cells (DCs) play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM), a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs) was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS), proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN- γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF- κ B translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs). These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases. PMID:23781253

  19. Blue light-activated hypocrellin B damages ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Jiang, Y.; Leung, A. W. N.; Xiang, J. Y.; Xu, C. S.

    2011-10-01

    In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hyppcrellin B concentration was kept at 2.5 μM and light doses from 0.5-4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

  20. Clonal mast cell activation syndrome with anaphylaxis to sulfites.

    PubMed

    Cifuentes, Liliana; Ring, Johannes; Brockow, Knut

    2013-01-01

    Sulfites are rarely suspected as causative agents of immediate-type hypersensitivity. We report on a 49-year-old male patient who developed recurrent severe hypotension after food ingestion. A diagnosis of monoclonal mast cell activation syndrome was established. In the double-blind, placebo-controlled food challenge, the patient reacted to potassium metabisulfite with anaphylaxis.

  1. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

    PubMed

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

  2. Effects of that ATRA inhibits Nrf2-ARE pathway on glial cells activation after intracerebral hemorrhage

    PubMed Central

    Yin, Xiao-Ping; Zhou, Jun; Wu, Dan; Chen, Zhi-Ying; Bao, Bing

    2015-01-01

    Previous studies indicate that the Nrf2-ARE signaling pathway plays a neruo-protective role in glia cell, however, the mechanism was also elusive. This study aims to explore the inhibitive function of all-trans-retinoic (ATRA) on Nrf2-ARE pathway in intracerebral hemorrhage (ICH), and investigate the mechanism. In this study, the femoral artery injection method was employed to establish ICH model. The model rats were randomly divided into four groups, including Sham group, ICH group, ATRA group and DMSO group. The neurological scores were evaluated for the four groups at different time points. Hematoxylin-Eosin staining was used to stain the CD11b positive glia cells. Double immunofluorescence staining method was utilized to observe the co-expression of HO-1, NF-κB, Nrf2 and TNF-α and CD11b marker in glia cells. Western blot assay was used to detect the Nrf2 protein (total and binding Nrf2), HO-1, NF-κB and TNF-α proteins in every group. The results indicated that neurologiclal scores were significantly decreased in ATRA group compared to ICH gorup (P < 0.05). The glia cells were significantly activated and accumulated in ICH rats. ATRA significantly decreased co-expression of Nrf2, HO-1 and CD11b, and increased co-expression of NF-κB, TNF-α and CD11b of glia cells. ATRA significantly decreased total Nrf2 expression and increased binding Nrf2 expression in ATRA group compared to ICH group (P < 0.05). ATRA decreased anti-oxygen protein Nrf2 and HO-1, and increases inflammatory factors NF-κB and TNF-α. In conclusion, the application of ATRA could inhibit the neuro-protective function effectively by blocking the Nrf2-ARE pathway in glia cells. PMID:26617752

  3. Enrichment of epidermal stem cells of rats by Vario magnetic activated cell sorting system.

    PubMed

    Chen, Wei; Zhang, Wei-wei; Shi, Chunying; Lian, Xiaohua; Yi, Shanghong; Yang, Tian

    2013-09-01

    Epidermal stem cells (ESCs) play an important role in skin homeostasis, wound repair, and tumorigensis which have great potential in scientific research and clinical application. So, the efficient isolation of these infrequent stem cells is very important for researchers to solve the problem of low purity and insufficient quantity of stem cells in vitro. The aim of this study was to investigate a method for the enrichment of ESCs by magnetic activated cell sorting system. The isolation strategy was CD71 depletion followed by α6-integrin positive selection. The percentage of α6(bri)CD71(dim) cells in isolated cells was 94.59%. Transmission electron microscopy results revealed that α6(bri) CD71(dim) cells exhibited some typical characteristics like progenitor cells, such as big nucleus, obvious nucleolus, large nuclear-cytoplasm ratio, and few organelles in cytoplasm. When cultured in vitro, the α6(bri)CD71(dim) cells had greater proliferating potential and higher colony-forming ability, and high levels of epidermal stem cell markers were expressed in our positive cells. ESCs have been successfully isolated from neonatal epidermis using Vario MACS and cultured in vitro. This isolation method is simple, fast, and inexpensive, providing an important tool for tissue engineering and cell transplantation studies.

  4. M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells.

    PubMed

    Ferretti, Michela; Fabbiano, Cinzia; Di Bari, Maria; Conte, Claudia; Castigli, Emilia; Sciaccaluga, Miriam; Ponti, Donatella; Ruggieri, Paola; Raco, Antonino; Ricordy, Ruggero; Calogero, Antonella; Tata, Ada Maria

    2013-04-01

    Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

  5. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  6. Activity of quinone alkylating agents in quinone-resistant cells.

    PubMed

    Begleiter, A; Leith, M K

    1990-05-15

    The role of the quinone group in the antitumor activity of quinone alkylating agents, such as mitomycin C and 2,5-diaziridinyl-3,5-bis(carboethoxyamino)-1,4-benzoquinone, is still uncertain. The quinone group may contribute to antitumor activity by inducing DNA strand breaks through the formation of free radicals and/or by influencing the alkylating activity of the quinone alkylators. The cytotoxic activity and DNA damage produced by the model quinone alkylating agents, benzoquinone mustard and benzoquinone dimustard, were compared in L5178Y murine lymphoblasts sensitive and resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard. The resistant cell lines, L5178Y/HBM2 and L5178Y/HBM10, have increased concentrations of glutathione and elevated catalase, superoxide dismutase, glutathione S-transferase, and DT-diaphorase activity. L5178Y/HBM2 and L5178Y/HBM10 cells were 7.4- and 8.5-fold less sensitive to benzoquinone mustard and 1.7- and 4.3-fold less sensitive to benzoquinone dimustard, respectively, compared with sensitive cells, but showed no resistance to the non-quinone alkylating agent, aniline mustard. The formation of DNA double strand breaks by benzoquinone mustard was reduced by 2- and 8-fold in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, while double strand break formation by benzoquinone dimustard was reduced only in the L5178Y/HBM10 cells. The number of DNA-DNA cross-links produced by benzoquinone mustard was 3- and 6-fold lower, and the number produced by benzoquinone dimustard was 35% and 2-fold lower in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, compared with L5178Y parental cells. In contrast, cross-linking by aniline mustard was unchanged in sensitive and resistant cells. Dicoumarol, an inhibitor of DT-diaphorase, increased the cytotoxic activity of both benzoquinone mustard and benzoquinone dimustard in L5178Y/HBM10 cells. This study provides evidence that elevated DT-diaphorase activity in the resistant cells

  7. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

  8. Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

    PubMed Central

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms. PMID:24992192

  9. T-Cell Tumor Elimination as a Result of T-Cell Receptor-Mediated Activation

    NASA Astrophysics Data System (ADS)

    Ashwell, Jonathan D.; Longo, Dan L.; Bridges, Sandra H.

    1987-07-01

    It has recently been shown that activation of murine T-cell hybridomas with antigen inhibits their growth in vitro. The ``suicide'' of these neoplastic T cells upon stimulation with antigen suggested the possibility that activation via the antigen-specific receptor could also inhibit the growth of neoplastic T cells in vivo. To test this, mice were subcutaneously inoculated with antigen-specific T-cell hybridomas and then treated intraperitoneally with antigen. Administration of the appropriate antigen immediately after inoculation with the T-cell hybridoma abrogated tumor formation; antigen administered after tumors had become established decreased the tumor burden and, in a substantial fraction of animals, led to long-term survival. The efficacy of antigen therapy was due to both a direct inhibitory effect on tumor growth and the induction of host immunity. These studies demonstrate the utility of cellular activation as a means of inhibiting neoplastic T-cell growth in vivo and provide a rationale for studying the use of less selective reagents that can mimic the activating properties of antigen, such as monoclonal antibodies, in the treatment of T-cell neoplasms of unknown antigen specificity.

  10. Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis.

    PubMed

    Uehara, Tsuyoshi; Parzych, Katherine R; Dinh, Thuy; Bernhardt, Thomas G

    2010-04-21

    During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC(-) defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.