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  1. Nanomaterial cytotoxicity is composition, size, and cell type dependent

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    Sohaebuddin Syed K

    2010-08-01

    Full Text Available Abstract Background Despite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs] with differing size (MWCNTs of different diameters 50 nm; but same length 0.5-2 μm to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT bronchiolar epithelial cells. Results Following characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination. Conclusions Nanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the

  2. Dependence of herpes simplex virus type 1-induced cell fusion on cell type

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    Bzik, D.J.; Person, S.

    1981-04-15

    Syncytial mutants of herpes simplex virus type 1 (HSV-1), such as syn20, cause extensive fusion of human embryonic lung (HEL) cells but only a small amount of fusion of human epidermoid carcinoma No. 2 (HEp-2) cells. In order to determine the cellular basis of this difference in fusion, sparse cultures of syn20-infected HEL or HEp-2 cells, previously labeled with (/sup 3/H)thymidine, were surrounded with uninfected, unlabeled HEL or HEp-2 cells. The fusion of radioactive with nonradioactive cells was determined at different times after infection using radioautography. The major difference in the fusion capacity of HEL and HEp-2 cells was not due to a difference in cell-surface receptors for a fusion factor in the two cell types. The process of infection of HEp-2 cells did not cause the plasma membranes of the cells to become refractory to fusion, because syn20-infected HEL cells fused equally well with either uninfected or infected HEp-2 cells. In a mixed infection with equal numbers of MP and its nonsyncytial parent, mP, extensive fusion was observed for infected HEL cells and significantly less fusion was observed for infected African green monkey (CV-1), baby hamster kidney (BHK-21), and HEp-2 cells.

  3. Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

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    Yu J. Shen

    2015-04-01

    Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  4. Dependence of Early and Late Chromosomal Aberrations on Radiation Quality and Cell Types

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    Lu, Tao; Zhang, Ye; Krieger, Stephanie; Yeshitla, Samrawit; Goss, Rosalin; Bowler, Deborah; Kadhim, Munira; Wilson, Bobby; Rohde, Larry; Wu, Honglu

    2017-01-01

    Exposure to radiation induces different types of DNA damage, increases mutation and chromosome aberration rates, and increases cellular transformation in vitro and in vivo. The susceptibility of cells to radiation depends on genetic background and growth condition of cells, as well as types of radiation. Mammalian cells of different tissue types and with different genetic background are known to have different survival rate and different mutation rate after cytogenetic insults. Genomic instability, induced by various genetic, metabolic, and environmental factors including radiation, is the driving force of tumorigenesis. Accurate measurements of the relative biological effectiveness (RBE) is important for estimating radiation-related risks. To further understand genomic instability induced by charged particles and their RBE, we exposed human lymphocytes ex vivo, human fibroblast AG1522, human mammary epithelial cells (CH184B5F5/M10), and bone marrow cells isolated from CBA/CaH(CBA) and C57BL/6 (C57) mice to high energy protons and Fe ions. Normal human fibroblasts AG1522 have apparently normal DNA damage response and repair mechanisms, while mammary epithelial cells (M10) are deficient in the repair of DNA DSBs. Mouse strain CBA is radio-sensitive while C57 is radio-resistant. Metaphase chromosomes at different cell divisions after radiation exposure were collected and chromosome aberrations were analyzed as RBE for different cell lines exposed to different radiations at various time points up to one month post irradiation.

  5. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

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    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  6. Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction.

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    Hong, Soyoung; Song, Seung-Joon; Lee, Jae Yeon; Jang, Hwanseok; Choi, Jaesoon; Sun, Kyung; Park, Yongdoo

    2013-08-01

    The fabrication of patterned microstructures within three-dimensional (3D) matrices is a challenging subject in tissue engineering and regenerative medicine. A 3D, free-moving bioprinting system was developed and hydrogels were patterned by varying the process parameters of z-axis moving velocity and ejection velocity. The patterning of hydrogel based microfibers in a 3D matrigel was achieved with dimensions of 4.5 mm length and widths from 79 to 200 μm. Hyaluronan-based hydrogels mixed with fibroblasts (L929), mouse endothelial cells (MS1), or human mesenchymal stem cells (hMSCs) were patterned using a 3D moving axis bioprinter and cell behavior was monitored in culture for up to 16 days. L929 and MS1 cells and hMSCs in patterned hydrogel revealed cell-cell interactions and a morphological dependency on cell types. HMSCs formed spheres through cell aggregation, while L929 cells increased in cellular mass without cell aggregation and MS1 dispersed into the matrix instead of aggregating. The aggregation of hMSCs was attenuated by treatment with Rho kinase (ROCK) inhibitor and cadherin antibody. This reflected the close relationship between cell aggregation and migration with RhoA and cell-cell adhesion molecules. Angiogenic-specific gene expression profiles showed that expression of CD105 decreased to 22% in the ROCK inhibitor group compared to control group. These results showed that cell-based patterns in a 3D matrix are highly dependent on both cell aggregation and migration over time.

  7. Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

    Science.gov (United States)

    Lachmann, Sylvie; Jevons, Amy; De Rycker, Manu; Casamassima, Adele; Radtke, Simone; Collazos, Alejandra; Parker, Peter J

    2011-01-01

    The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

  8. Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

    Directory of Open Access Journals (Sweden)

    Sylvie Lachmann

    Full Text Available The mammalian protein kinase N (PKN family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

  9. Recombinase-Dependent Mouse Lines for Chemogenetic Activation of Genetically Defined Cell Types

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    Natale R. Sciolino

    2016-06-01

    Full Text Available Chemogenetic technologies, including the mutated human Gq-coupled M3 muscarinic receptor (hM3Dq, have greatly facilitated our ability to directly link changes in cellular activity to altered physiology and behavior. Here, we extend the hM3Dq toolkit with recombinase-responsive mouse lines that permit hM3Dq expression in virtually any cell type. These alleles encode a fusion protein designed to increase effective expression levels by concentrating hM3Dq to the cell body and dendrites. To illustrate their broad utility, we targeted three different genetically defined cell populations: noradrenergic neurons of the compact, bilateral locus coeruleus and two dispersed populations, Camk2a+ neurons and GFAP+ glia. In all three populations, we observed reproducible expression and confirmed that activation of hM3Dq is sufficient to dose-dependently evoke phenotypic changes, without extreme phenotypes associated with hM3Dq overexpression. These alleles offer the ability to non-invasively control activity of diverse cell types to uncover their function and dysfunction at any developmental stage.

  10. Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

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    Yu, Yongbo; Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei

    2016-11-01

    The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

  11. Ozone-Induced Type 2 Immunity in Nasal Airways. Development and Lymphoid Cell Dependence in Mice.

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    Ong, Chee Bing; Kumagai, Kazuyoshi; Brooks, Phillip T; Brandenberger, Christina; Lewandowski, Ryan P; Jackson-Humbles, Daven N; Nault, Rance; Zacharewski, Timothy R; Wagner, James G; Harkema, Jack R

    2016-03-01

    Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.

  12. Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

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    Parr Rebecca D

    2011-06-01

    Full Text Available Abstract Background Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM, the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types. Methods Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls. Results Only full-length (FL, endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells. Conclusions The intracellular transport of NSP4 to

  13. Ozone-Induced Nasal Type 2 Immunity in Mice Is Dependent on Innate Lymphoid Cells.

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    Kumagai, Kazuyoshi; Lewandowski, Ryan; Jackson-Humbles, Daven N; Li, Ning; Van Dyken, Steven J; Wagner, James G; Harkema, Jack R

    2016-06-01

    Epidemiological studies suggest that elevated ambient concentrations of ozone are associated with activation of eosinophils in the nasal airways of atopic and nonatopic children. Mice repeatedly exposed to ozone develop eosinophilic rhinitis and type 2 immune responses. In this study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced eosinophilic rhinitis by using lymphoid-sufficient C57BL/6 mice, Rag2(-/-) mice that are devoid of T cells and B cells, and Rag2(-/-)Il2rg(-/-) mice that are depleted of all lymphoid cells including ILCs. The animals were exposed to 0 or 0.8 ppm ozone for 9 consecutive weekdays (4 h/d). Mice were killed 24 hours after exposure, and nasal tissues were selected for histopathology and gene expression analysis. ILC-sufficient C57BL/6 and Rag2(-/-) mice exposed to ozone developed marked eosinophilic rhinitis and epithelial remodeling (e.g., epithelial hyperplasia and mucous cell metaplasia). Chitinase-like proteins and alarmins (IL-33, IL-25, and thymic stromal lymphopoietin) were also increased morphometrically in the nasal epithelium of ozone-exposed C57BL/6 and Rag2(-/-) mice. Ozone exposure elicited increased expression of Il4, Il5, Il13, St2, eotaxin, MCP-2, Gob5, Arg1, Fizz1, and Ym2 mRNA in C57BL/6 and Rag2(-/-) mice. In contrast, ozone-exposed ILC-deficient Rag2(-/-)Il2rg(-/-) mice had no nasal lesions or overexpression of Th2- or ILC2-related transcripts. These results indicate that ozone-induced eosinophilic rhinitis, nasal epithelial remodeling, and type 2 immune activation are dependent on ILCs. To the best of our knowledge, this is the first study to demonstrate that ILCs play an important role in the nasal pathology induced by repeated ozone exposure.

  14. Cytotoxicity Induced by Engineered Silver Nanocrystallites is Dependent on Surface Coatings and Cell Types

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    Suresh, Anil K [ORNL; Pelletier, Dale A [ORNL; Wang, Wei [ORNL; Morrell-Falvey, Jennifer L [ORNL; Doktycz, Mitchel John [ORNL

    2012-01-01

    Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial recognition and are used extensively in biomedical applications such as wound dressing, surgical instruments and as bone substitute material. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Although numerous factors including the composition, size, shape, surface charge and capping molecule of nanoparticles are known to influence the cell cytotoxicity, our results demonstrate for the first time that surface coatings are a major determinant in eliciting the potential cytotoxicity and cell interactions of silver nanoparticles. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles were poly (diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated) and oleate-Ag with zeta potentials +45 5 mV, -12 2 mV, -42 5 mV and -45 5 mV respectively; the particles were thoroughly purified so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response and membrane damage caused by these four different silver nanoparticles were evaluated using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. From a toxicity perspective, our results clearly indicated that the cytotoxicity was depend on various factors such as synthesis procedure, surface coat or surface charge and the cell-type for the different silver nanoparticles that were investigated. Poly (diallyldimethylammonium) chloride -Ag was found to be the most toxic, followed by biogenic-Ag and oleate-Ag, whereas uncoated-Ag was found to be least toxic to both

  15. Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

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    Coulter JA

    2012-06-01

    Full Text Available Jonathan A Coulter,1 Suneil Jain,2 Karl T Butterworth,2 Laura Taggart,2 Glenn Dickson,2 Stephen J McMahon,3 Wendy Hyland,1 Mark F Muir,3 Coleman Trainor,2 Alan Hounsell,2,4 Joe M O'Sullivan,2,4 Giuseppe Schettino,2 Fred Currell,3 David G Hirst,1 Kevin M Prise21School of Pharmacy, McClay Research Centre, 2Centre for Cancer Research and Cell Biology, 3School of Mathematics and Physics, Queens University Belfast, 4Belfast Health and Social Care Trust, Belfast, IrelandBackground: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.Methods: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.Results: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.Conclusion: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic

  16. Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production.

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    Urata, Shuzo; Yasuda, Jiro

    2015-07-01

    Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that co-expression of glycoprotein (GPC) altered the requirements for cholesterol but not the L-domains and host factor, Tsg101, for Z-induced virus-like particle (VLP) production. In particular, the cholesterol requirement for VLP production was cell-type-dependent. In addition, GPC was found to be important for co-localization of Z with CD63, a late endosomal marker. We also found that the N-terminal region (aa 3-10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.

  17. Regulatory Domain Selectivity in the Cell-Type Specific PKN-Dependence of Cell Migration

    OpenAIRE

    Sylvie Lachmann; Amy Jevons; Manu De Rycker; Adele Casamassima; Simone Radtke; Alejandra Collazos; Peter J Parker

    2011-01-01

    The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relativel...

  18. Trophic significance of solitary cells of the prymnesiophyte Phaeocystis globosa depends on cell type

    DEFF Research Database (Denmark)

    Dutz, Jörg; Koski, Marja

    2006-01-01

    experiments, revealed that neither the production of transparent exopolymer particles and chitinous threads nor toxicity can explain the observed response. The cohesion of the threads into pentagonal stars was observed only in the avoided mesoflagellate and might cause a mechanical hindrance for the ingestion...... of mesoflagellates. Our results suggest that grazing loss and trophic transfer efficiency might be overestimated when solitary cells are treated as a single functional group with regard to their trophic position....

  19. Vestibular evoked myogenic potentials are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.

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    Lue, June-Horng; Day, An-Shiou; Cheng, Po-Wen; Young, Yi-Ho

    2009-01-01

    This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 +/- 1.2/4000 microm(2) in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 +/- 1.8/4000 microm(2)). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.

  20. GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

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    Lin, Tao; Meng, Lingjun [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States); Tsai, Robert Y.L., E-mail: rtsai@ibt.tamhsc.edu [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States)

    2011-10-22

    Highlights: {yields} Strong synergy between mycophenolic acid (MPA) and 5-FU in MDA-MB-231 cells. {yields} Cell type-dependent synergy between MPA and anti-proliferative agents. {yields} The synergy of MPA on 5-FU is recapitulated by RNA polymerase-I inhibition. {yields} The synergy of MPA on 5-FU requires the expression of nucleostemin. -- Abstract: Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

  1. Cell Type-Dependent RNA Recombination Frequency in the Japanese Encephalitis Virus

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    Wei-Wei Chiang

    2014-01-01

    Full Text Available Japanese encephalitis virus (JEV is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

  2. Cell type-dependent RNA recombination frequency in the Japanese encephalitis virus.

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    Chiang, Wei-Wei; Chuang, Ching-Kai; Chao, Mei; Chen, Wei-June

    2014-01-01

    Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

  3. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules.

    Science.gov (United States)

    Kim, Sun-Hee; Bang, So Hee; Kang, So Yeong; Park, Ki Dae; Eom, Jun Ho; Oh, Il Ung; Yoo, Si Hyung; Kim, Chan-Wha; Baek, Sun Young

    2015-02-01

    Human amniotic membrane-derived stromal cells (hAMSC) are candidates for cell-based therapies. We examined the characteristics of hAMSC including the interaction between hAMSC and breast cancer cells, MCF-7, and MDA-MB-231. Human amniotic membrane-derived stromal cells showed typical MSC properties, including fibroblast-like morphology, surface antigen expression, and mesodermal differentiation. To investigate cell-cell interaction via secreted molecules, we cultured breast cancer cells in hAMSC-conditioned medium (hAMSC-CM) and analyzed their proliferation, migration, and secretome profiles. MCF-7 and MDA-MB-231 cells exposed to hAMSC-CM showed increased proliferation and migration. However, in hAMSC-CM, MCF-7 cells proliferated significantly faster than MDA-MB-231 cells. When cultured in hAMSC-CM, MCF-7 cells migrated faster than MDA-MB-231 cells. Two cell types showed different profiles of secreted factors. MCF-7 cells expressed much amounts of IL-8, GRO, and MCP-1 in hAMSC-CM. Human amniotic membrane-derived stromal cells interact with breast cancer cells through secreted molecules. Factors secreted by hAMSCs promote the proliferation and migration of MCF-7 breast cancer cells. For much safe cell-based therapies using hAMSC, it is necessary to study carefully about interaction between hAMSC and cancer cells.

  4. Acute doxorubicin insult in the mouse ovary is cell- and follicle-type dependent.

    Directory of Open Access Journals (Sweden)

    Elon C Roti Roti

    Full Text Available Primary ovarian insufficiency (POI is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours than granulosa cells (measurable at 4 hours, as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10-12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult

  5. Guarded dependent type theory with coinductive types

    DEFF Research Database (Denmark)

    Bizjak, Aleš; Grathwohl, Hans Bugge; Clouston, Ranald;

    2015-01-01

    We present guarded dependent type theory, gDTT, an extensional dependent type theory with a later' modality and clock quantifiers for programming and proving with guarded recursive and coinductive types. The later modality is used to ensure the productivity of recursive definitions in a modular...

  6. Input- and Cell-Type-Specific Endocannabinoid-Dependent LTD in the Striatum

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    Yu-Wei Wu

    2015-01-01

    Full Text Available Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1 receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.

  7. Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions.

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    Matthew L Katz

    Full Text Available Sensory stimuli are encoded by diverse kinds of neurons but the identities of the recorded neurons that are studied are often unknown. We explored in detail the firing patterns of eight previously defined genetically-identified retinal ganglion cell (RGC types from a single transgenic mouse line. We first introduce a new technique of deriving receptive field vectors (RFVs which utilises a modified form of mutual information ("Quadratic Mutual Information". We analysed the firing patterns of RGCs during presentation of short duration (~10 second complex visual scenes (natural movies. We probed the high dimensional space formed by the visual input for a much smaller dimensional subspace of RFVs that give the most information about the response of each cell. The new technique is very efficient and fast and the derivation of novel types of RFVs formed by the natural scene visual input was possible even with limited numbers of spikes per cell. This approach enabled us to estimate the 'visual memory' of each cell type and the corresponding receptive field area by calculating Mutual Information as a function of the number of frames and radius. Finally, we made predictions of biologically relevant functions based on the RFVs of each cell type. RGC class analysis was complemented with results for the cells' response to simple visual input in the form of black and white spot stimulation, and their classification on several key physiological metrics. Thus RFVs lead to predictions of biological roles based on limited data and facilitate analysis of sensory-evoked spiking data from defined cell types.

  8. The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves.

    Science.gov (United States)

    Corpas, Francisco J; Fernández-Ocaña, Ana; Carreras, Alfonso; Valderrama, Raquel; Luque, Francisco; Esteban, Francisco J; Rodríguez-Serrano, María; Chaki, Mounira; Pedrajas, José R; Sandalio, Luisa M; del Río, Luis A; Barroso, Juan B

    2006-07-01

    Superoxide dismutase (SOD) is a key antioxidant enzyme present in prokaryotic and eukaryotic cells as a first line of defense against the accumulation of superoxide radicals. In olive leaves, the SOD enzymatic system was characterized and was found to be comprised of three isozymes, an Mn-SOD, an Fe-SOD and a CuZn-SOD. Transcript expression analysis of whole leaves showed that the three isozymes represented 82, 17 and 0.8% of the total SOD expressed, respectively. Using the combination of laser capture microdissection (LCM) and real-time quantitative reverse transcription-PCR (RT-PCR), the expression of these SOD isozymes was studied in different cell types of olive leaves, including spongy mesophyll, palisade mesophyll, xylem and phloem. In spongy mesophyll cells, the isozyme proportion was similar to that in whole leaves, but in the other cells the proportion of expressed SOD isozymes was different. In palisade mesophyll cells, Fe-SOD was the most abundant, followed by Mn-SOD and CuZn-SOD, but in phloem cells Mn-SOD was the most prominent isozyme, and Fe-SOD was present in trace amounts. In xylem cells, only the Mn-SOD was detected. On the other hand, the highest accumulation of superoxide radicals was localized in vascular tissue which was the tissue with the lowest level of SOD transcripts. These data show that in olive leaves, each SOD isozyme has a different gene expression depending on the cell type of the leaf.

  9. Cell-type-specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing.

    Science.gov (United States)

    Sun, Yanjun; Nguyen, Amanda Q; Nguyen, Joseph P; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M; Xu, Xiangmin

    2014-04-10

    We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC), the medial septum (MS), and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.

  10. Investigation of Neuronal Cell Type-Specific Gene Expression of Ca2+/Calmodulin-dependent Protein Kinase II.

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    Mima Kazuko

    2002-01-01

    Full Text Available The promoter activity of the rat Ca2+/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-flanking region of &agr; and &bgr; isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5'-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types.

  11. Guarded dependent type theory with coinductive types

    DEFF Research Database (Denmark)

    Bizjak, Aleš; Grathwohl, Hans Bugge; Clouston, Ranald

    2016-01-01

    , type based, way. Clock quantifiers are used for controlled elimination of the later modality and for encoding coinductive types using guarded recursive types. Key to the development of gDTT are novel type and term formers involving what we call delayed substitutions’. These generalise the applicative...... functor rules for the later modality considered in earlier work, and are crucial for programming and proving with dependent types. We show soundness of the type theory with respect to a denotational model....

  12. Manipulation of Intestinal Epithelial Cell Function by the Cell Contact-Dependent Type III Secretion Systems of Vibrio parahaemolyticus

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    Nicky eO'Boyle

    2014-01-01

    Full Text Available Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signalling proteins, thereby bringing about changes in the regulation of cellular behaviour. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favours bacterial colonization and persistence of V. parahaemolyticus in the host.

  13. Fibronectin upregulates cGMP-dependent protein kinase type Iβ through C/EBP transcription factor activation in contractile cells.

    Science.gov (United States)

    Chamorro-Jorganes, Aranzazu; Calleros, Laura; Griera, Mercedes; Saura, Marta; Luengo, Alicia; Rodriguez-Puyol, D; Rodriguez-Puyol, M

    2011-03-01

    The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

  14. Temporal properties of network-mediated responses to repetitive stimuli are dependent upon retinal ganglion cell type

    Science.gov (United States)

    Im, Maesoon; Fried, Shelley I.

    2016-04-01

    Objective. To provide artificially-elicited vision that is temporally dynamic, retinal prosthetic devices will need to repeatedly stimulate retinal neurons. However, given the diversity of physiological types of retinal ganglion cells (RGCs) as well as the heterogeneity of their responses to electric stimulation, temporal properties of RGC responses have not been adequately investigated. Here, we explored the cell type dependence of network-mediated RGC responses to repetitive electric stimulation at various stimulation rates. Approach. We examined responses of ON and OFF types of RGCs in the rabbit retinal explant to five consecutive stimuli with varying inter-stimulus intervals (10-1000 ms). Each stimulus was a 4 ms long monophasic sinusoidal cathodal current, which was applied epiretinally via a conical electrode. Spiking activity of targeted RGCs was recorded using a cell-attached patch electrode. Main results. ON and OFF cells had distinct responses to repetitive stimuli. Consistent with earlier studies, OFF cells always generated reduced responses to subsequent stimuli compared to responses to the first stimulus. In contrast, a new stimulus to ON cells suppressed all pending/ongoing responses from previous stimuli and initiated its own response that was remarkably similar to the response from a single stimulus in isolation. This previously unreported ‘reset’ behavior was observed exclusively and consistently in ON cells. These contrasts between ON and OFF cells created a range of stimulation rates (4-7 Hz) that maximized the ratio of the responses arising in ON versus OFF cells. Significance. Previous clinical testing reported that subjects perceive bright phosphenes (ON responses) and also prefer stimulation rates of 5-7 Hz. Our results suggest that responses of ON cells are weak at high rates of stimulation (> ˜7 Hz) due to the reset while responses of OFF cells are strong at low rates (cells more closely match physiological patterns (Im and Fried 2015

  15. Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

    DEFF Research Database (Denmark)

    Ohki, Makiko; Ohki, Yuichi; Ishihara, Makoto

    2010-01-01

    tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool...

  16. Frequency-dependent, cell type-divergent signaling in the hippocamposeptal projection.

    Science.gov (United States)

    Mattis, Joanna; Brill, Julia; Evans, Suzanne; Lerner, Talia N; Davidson, Thomas J; Hyun, Minsuk; Ramakrishnan, Charu; Deisseroth, Karl; Huguenard, John R

    2014-08-27

    Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

  17. Cytotoxicity of β-D-glucose/sucrose-coated silver nanoparticles depends on cell type, nanoparticles concentration and time of incubation

    Science.gov (United States)

    Vergallo, Cristian; Panzarini, Elisa; Carata, Elisabetta; Ahmadi, Meysam; Mariano, Stefania; Tenuzzo, Bernardetta Anna; Dini, Luciana

    2016-06-01

    G2 cells exposed to AgNPs. The results indicate that HepG2 cells are more responsive to AgNPs treatment than PBLs. It is generally believed that cellular oxidative stress induces toxicity of NPs; however, in this study we did not detect any AgNPs-induced oxidative stress in HepG2, thus suggesting an alternative mechanism of toxicity in this cell line. In the whole, our findings suggest that AgNPs-GS-induced toxicity strictly depends on cell type, NPs amount and time of treatment.

  18. Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type

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    Dam Elisabeth

    2009-03-01

    Full Text Available Abstract Background HIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA and TRIM5α can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A – CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5α by the target cells. Results Viral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5α expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity. Conclusion Multiple

  19. NRF2 Oxidative Stress Induced by Heavy Metals is Cell Type Dependent

    Science.gov (United States)

    Exposure to metallic environmental toxicants has been demonstrated to induce a variety of oxidative stress responses in mammalian cells. The transcription factor Nrf2 is activated in response to oxidative stress and coordinates the expression of antioxidant gene products. In this...

  20. Drug-dependent functionalization of wild-type and mutant p53 in cisplatin-resistant human ovarian tumor cells.

    Science.gov (United States)

    Bhatt, Michelle; Ivan, Cristina; Xie, Xiaolei; Siddik, Zahid H

    2016-12-26

    Cisplatin (cis-Pt) resistance in tumor cells from p53 dysfunction is a significant clinical problem. Although mutation can inhibit p53 function, >60% of p53 mutants retain normal function according to literature reports. Therefore, we examined the status of p53 in cisplatin-resistant ovarian tumor models and its functional response to cis-Pt and the mechanistically-distinct non-cross-resistant oxaliplatin (oxali-Pt). Relative to sensitive A2780 cells harboring wild-type p53, the 2780CP/Cl-16, OVCAR-10, Hey and OVCA-433 cell lines were 10- to 30-fold resistant to cis-Pt, but was substantially circumvented by oxali-Pt. Mutant p53 in 2780CP/Cl-16 (p53V172F) and OVCAR-10 (p53V172F and p53G266R) cells, predicted as non-functional in p53 database, displayed attenuated response to cis-Pt, as did the polymorphic p53P72R (functionally equivalent to wild-type p53) in HEY and OVCA-433 cell lines. However, p53 was robustly activated by oxali-Pt in all cell lines, with resultant drug potency confirmed as p53-dependent by p53 knockout using CRISPR/Cas9 system. This p53 activation by oxali-Pt was associated with phosphorylation at Ser20 by MEK1/2 based on inhibitor and kinase studies. Cis-Pt, however, failed to phosphorylate Ser20 due to downregulated Chk2, and its clinical impact validated by reduced overall survival of ovarian cancer patients according to TCGA database. In conclusion, cis-Pt resistance occurs in both wild-type and mutant p53 ovarian cancer cells, but is associated with loss of Ser20 phosphorylation. However, these mutant p53, like polymorphic p53, are functional and activated by oxali-Pt-induced Ser20 phosphorylation. Thus, the potential exists for repurposing oxali-Pt or similar drugs against refractory cancers harboring wild-type or specific mutant p53.

  1. Cell-Type-Specific Circuit Connectivity of Hippocampal CA1 Revealed through Cre-Dependent Rabies Tracing

    Directory of Open Access Journals (Sweden)

    Yanjun Sun

    2014-04-01

    Full Text Available We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC, the medial septum (MS, and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.

  2. Hormonal, metabolic and cardiovascular responses to hypoglycaemia in Type 1 (insulin-dependent) diabetes with and without residual B cell function

    DEFF Research Database (Denmark)

    Madsbad, S; Hilsted, J; Krarup, T;

    1982-01-01

    Hormonal, metabolic and cardiovascular responses to insulin induced hypoglycaemia were investigated in seven Type 1 (insulin-dependent) diabetic patients with residual B cell function, eight Type 1 diabetic patients without B cell function and six healthy subjects. No differences were found betwe...

  3. Types of Stem Cells

    Science.gov (United States)

    ... Stem Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... Learn About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

  4. Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

    Science.gov (United States)

    Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

    2013-01-01

    The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

  5. CD4+ type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes

    DEFF Research Database (Denmark)

    Kadri, Nadir; Korpos, Eva; Gupta, Shashank

    2012-01-01

    Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry ...

  6. 25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner

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    Cantarutti Alyssa

    2010-11-01

    Full Text Available Abstract Background 25-hydroxycholesterol (25-OHC is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis. Methods Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC were treated with 25-OHC and tested for several different cellular parameters. Results We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2 expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex. Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex. These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity. Conclusions Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

  7. Topographically induced self-deformation of the nuclei of cells: dependence on cell type and proposed mechanisms.

    Science.gov (United States)

    Davidson, Patricia M; Fromigué, Olivia; Marie, Pierre J; Hasirci, Vasif; Reiter, Günter; Anselme, Karine

    2010-03-01

    Osteosarcoma-derived cell lines (SaOs-2, MG63) have recently been shown to deform their nucleus considerably in response to surface topography. Such a deformation had not been described previously. Here we present results on additional cell lines, including cancerous (OHS4, U2OS), immortalized (F/STRO-1(+)A and FHSO6) and healthy cells (HOP). The cancerous cells were found to deform extensively, the immortalized cells showed small deformations, whereas the healthy cells showed deformation only at short incubation times. These results suggest a strong link between the malignant transformation of cells and the state of the cytoskeletal network. We propose mechanisms to explain the deformation in which the cytoskeleton either pushes down on the nucleus during spreading or pulls it down upon adhesion to the pillars.

  8. Genetic fate mapping of type-1 stem cell-dependent increase in newborn hippocampal neurons after electroconvulsive seizures.

    Science.gov (United States)

    Weber, Tillmann; Baier, Vera; Lentz, Katharina; Herrmann, Elke; Krumm, Bertram; Sartorius, Alexander; Kronenberg, Golo; Bartsch, Dusan

    2013-12-01

    Electroconvulsive therapy (ECT) is a uniquely effective treatment for major depressive disorder. An increase in hippocampal neurogenesis is implicated in the recovery from depression. We used an inducible genetic mouse model in which only GFAP-expressing stem-like cells (type-1 cells) and their progeny are selectively labeled with the reporter protein β-galactosidase to track the process of neurogenesis in the dentate gyrus over 3 months following electroconvulsive seizures (ECS), the mouse equivalent of ECT. All ECS protocols tested induced a transient increase in type-1 cell divisions. While this led to an expansion of the type-1 cell pool after high-frequency ECS sessions for 5 consecutive days (5-ECS), asymmetric divisions drove neurogenesis by giving rise to Doublecortin (DCX)-expressing neuroblasts that matured into NeuN+ neurons. Significantly, the increase in newly generated DCX+ and NeuN+ cells after 5-ECS could be traced back to proliferating type-1 cells. Low-frequency continuation ECS (c-ECS) consisting of five single ECS sessions administered every 2 weeks resulted in a similar increase in newborn neurons as the high-frequency 5-ECS protocol. Moreover, the combination of 5-ECS and c-ECS led to a further significant increase in newborn neurons, suggesting a cellular mechanism responsible for the propitious effects of high-frequency ECT followed by continuation ECT in severely depressed patients. The ability of high- and low-frequency ECS to induce normally quiescent type-1 cells to proliferate and generate new neurons sets it apart from other antidepressant treatments and may underlie the superior clinical efficacy of ECT.

  9. Proliferative responses to altered 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 expression in human breast cancer cells are dependent on endogenous expression of 17HSD type 1 and the oestradiol receptors.

    Science.gov (United States)

    Jansson, A; Gunnarsson, C; Stål, O

    2006-09-01

    The primary source of oestrogen in premenopausal women is the ovary but, after menopause, oestrogen biosynthesis in peripheral tissue is the exclusive site of formation. An enzyme group that affects the availability of active oestrogens is the 17beta-hydroxysteroid dehydrogenase (17HSD) family. In breast cancer, 17HSD type 1 and type 2 have been mostly investigated and seem to be the principal 17HSD enzymes involved thus far. The question whether 17HSD type 1 or type 2 is of greatest importance in breast tumour development is still not clear. The aim of this study was to investigate how the loss of 17HSD type 2 expression, using siRNA in the non-tumour breast epithelial cells HMEC (human mammal epithelial cells) and MCF10A, and gain of 17HSD type 2 expression, using transient transfection in the breast cancer derived cell lines MCF7 and T47D, affect oestradiol conversion and proliferation rate measured as S-phase fraction. We further investigated how this was related to the endogenous expression of 17HSD type 1 and oestradiol receptors in the examined cell lines. The oestradiol level in the medium changed significantly in the MCF7 transfected cells and the siRNA-treated HMEC cells, but not in T47D or MCF10A. The S-phase fraction decreased in the 17HSD type 2-transfected MCF7 cells and the siRNA-treated HMEC cells. The results seemed to be dependent on the endogenous expression of 17HSD type 1 and the oestradiol receptors. In conclusion, we found that high or low levels of 17HSD type 2 affected the oestradiol concentration significantly. However, the response was dependent on the endogenous expression of 17HSD type 1. Expression of 17HSD type 1 seems to be dominant to 17HSD type 2. Therefore, it may be important to investigate a ratio between 17HSD type 1 and 17HSD type 2.

  10. CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response

    Directory of Open Access Journals (Sweden)

    Abdul S. Qadir

    2017-03-01

    Full Text Available Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs. We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1 and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95high-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95.

  11. A comparative study on collagen type I and hyaluronic acid dependent cell behavior for osteochondral tissue bioprinting.

    Science.gov (United States)

    Park, Ju Young; Choi, Jong-Cheol; Shim, Jin-Hyung; Lee, Jung-Seob; Park, Hyoungjun; Kim, Sung Won; Doh, Junsang; Cho, Dong-Woo

    2014-09-01

    Bioprinting is a promising technique for engineering composite tissues, such as osteochondral tissues. In this study, as a first step toward bioprinting-based osteochondral tissue regeneration, we systematically examined the behavior of chondrocytes and osteoblasts to hyaluronic acid (HA) and type I collagen (Col-1) hydrogels. First, we demonstrated that cells on hydrogels that were comprised of major native tissue extracellular matrix (ECM) components (i.e. chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels) exhibited better proliferation and cell function than cells on non-native ECM hydrogels (i.e., chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels). In addition, cells located near their native ECM hydrogels migrated towards them. Finally, we bioprinted three-dimensional (3D) osteochondral tissue-mimetic structures composed of two compartments, osteoblast-encapsulated Col-1 hydrogels and chondrocyte-encapsulated HA hydrogels, and found viability and functions of each cell type were well maintained within the 3D structures up to 14 days in vitro. These results suggest that with proper choice of hydrogel materials, bioprinting-based approaches can be successfully applied for osteochondral tissue regeneration.

  12. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    DEFF Research Database (Denmark)

    Yang, Hsiao Yin; Vonk, Lucienne A.; Licht, Ruud;

    2014-01-01

    application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20......% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day...

  13. Cell type-dependent induction of DNA damage by 1800 MHz radiofrequency electromagnetic fields does not result in significant cellular dysfunctions.

    Directory of Open Access Journals (Sweden)

    Shanshan Xu

    Full Text Available BACKGROUND: Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity. OBJECTIVES: To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation. METHODS: Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay. RESULTS: Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs, but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level. CONCLUSIONS: RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.

  14. Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues.

    Science.gov (United States)

    Fortier, Guillaume Marceau; Gauvin, Robert; Proulx, Maryse; Vallée, Maud; Fradette, Julie

    2013-04-01

    Mesenchymal cells are central to connective tissue homeostasis and are widely used for tissue-engineering applications. Dermal fibroblasts and adipose-derived stromal cells (ASCs) allow successful tissue reconstruction by the self-assembly approach of tissue engineering. This method leads to the production of multilayered tissues, devoid of exogenous biomaterials, that can be used as stromal compartments for skin or vesical reconstruction. These tissues are formed by combining cell sheets, generated through cell stimulation with ascorbic acid, which favours the cell-derived production/organization of matrix components. Since media motion can impact on cell behaviour, we investigated the effect of dynamic culture on mesenchymal cells during tissue reconstruction, using the self-assembly method. Tissues produced using ASCs in the presence of a wave-like movement were nearly twice thicker than under standard conditions, while no difference was observed for tissues produced from dermal fibroblasts. The increased matrix deposition was not correlated with an increased proliferation of ASCs, or by higher transcript levels of fibronectin or collagens I and III. A 30% increase of type V collagen mRNA was observed. Interestingly, tissues engineered from dermal fibroblasts featured a four-fold higher level of MMP-1 transcripts under dynamic conditions. Mechanical properties were similar for tissues reconstructed using dynamic or static conditions. Finally, cell sheets produced using ASCs under dynamic conditions could readily be manipulated, resulting in a 2 week reduction of the production time (from 5 to 3 weeks). Our results describe a distinctive property of ASCs' response to media motion, indicating that their culture under dynamic conditions leads to optimized tissue engineering.

  15. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    NARCIS (Netherlands)

    Yang, Hsiao-yin; Vonk, Lucienne A; Licht, Ruud; van Boxtel, Antonetta M G; Bekkers, Joris E J; Kragten, Angela H M; Hein, San; Varghese, Oommen P; Howard, Kenneth A; Öner, F Cumhur; Dhert, Wouter J A; Creemers, Laura B

    2014-01-01

    The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical appl

  16. Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

    Science.gov (United States)

    Vasilopoulou, E.; Loubière, L.S.; Lash, G.E.; Ohizua, O.; McCabe, C.J.; Franklyn, J.A.; Kilby, M.D.; Chan, S.Y.

    2014-01-01

    , TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote

  17. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

    Science.gov (United States)

    Huynh, Julie M; Dang, Hope; Munoz-Tucker, Isabel A; O'Ketch, Marvin; Liu, Ian T; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-02-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

  18. Influence of AQP1 on cell adhesion, migration, and tumor sphere formation in malignant pleural mesothelioma is substratum- and histological-type dependent.

    Science.gov (United States)

    Jagirdar, Rajesh M; Apostolidou, Eleni; Molyvdas, Paschalis Adam; Gourgoulianis, Konstantinos I; Hatzoglou, Chrissi; Zarogiannis, Sotirios G

    2016-03-15

    Malignant pleural mesothelioma (MPM) is an aggressive cancer. MPM cells express aquaporin-1 (AQP1) that in other cancers has been shown to participate in the tumor metastasis processes. However, in MPM patients AQP1 overexpression is an independent prognostic factor favoring survival. In this study we aimed at evaluating the role of AQP1 in cell adhesion, migration, and tumor sphere formation in nonmalignant mesothelial cells (MeT-5A) and in epithelioid (M14K) and sarcomatoid (ZL34) MPM cell lines. We used fibronectin (FN) or homologous cell-derived extracellular martrix (ECM) substratum to investigate the role of AQP1 in these experimental phenotypes, inhibiting AQP1 by 10(-5) M mercury chloride (MC). Deposited ECM during cell culture exhibited significant concentration differences among cell types. ZL34 cell adhesion was significantly higher than MeT-5A or M14K cells on FN and ECM. MeT-5A and M14K cell adhesion on FN was sensitive to AQP1 inhibition, whereas AQP1 inhibition on ECM was limited to M14K cells. Wound healing in ZL34 cells was significantly higher than MeT-5A and M14K cells on FN and ECM. AQP1 inhibition significantly lowered cell migration in ZL34 cells on FN and ECM. Sphere formation was not dependent on FN or ECM in the media. AQP1 inhibition in FN media reduced sphere formation in M14K cells, whereas, in ECM, all three cell types were sensitive to AQP1 inhibition.

  19. Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.

    Science.gov (United States)

    Strobel, M; Pförtner, H; Tuchscherr, L; Völker, U; Schmidt, F; Kramko, N; Schnittler, H-J; Fraunholz, M J; Löffler, B; Peters, G; Niemann, S

    2016-09-01

    Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction.

  20. Adolescent maturation of inhibitory inputs onto cingulate cortex neurons is cell-type specific and TrkB dependent

    Directory of Open Access Journals (Sweden)

    Angela eVandenberg

    2015-02-01

    Full Text Available The maturation of inhibitory circuits during adolescence may be tied to the onset of mental health disorders such as schizophrenia. Neurotrophin signaling likely plays a critical role in supporting inhibitory circuit development and is also implicated in psychiatric disease. Within the neocortex, subcircuits may mature at different times and show differential sensitivity to neurotrophin signaling. We measured miniature inhibitory and excitatory postsynaptic currents (mIPSC and mEPSCs in Layer 5 cell-types in the mouse anterior cingulate across the periadolescent period. We differentiated cell-types mainly by Thy1 YFP transgene expression and also retrobead injection labeling in the contralateral cingulate and ipsilateral pons. We found that YFP- neurons and commissural projecting neurons had lower frequency of mIPSCs than neighboring YFP+ neurons or pons projecting neurons in juvenile mice (P21-25. YFP- neurons and to a lesser extent commissural projecting neurons also showed a significant increase in mIPSC amplitude during the periadolescent period (P21-25 vs. P40-50, which was not seen in YFP+ neurons or pons projecting neurons. Systemic disruption of tyrosine kinase receptor B (TrkB signaling during P23-50 in TrkBF616A mice blocked developmental changes in mIPSC amplitude, without affecting miniature excitatory post synaptic currents (mEPSCs. Our data suggest that the maturation of inhibitory inputs onto layer 5 pyramidal neurons is cell-type specific. These data may inform our understanding of adolescent brain development across species and aid in identifying candidate subcircuits that may show greater vulnerability in mental illness.

  1. Types of voltage—dependent calcium channels involved in high potassium depolarization—induced amylase secretion in the exocrine pancreatic tumour cell line AR4—2J

    Institute of Scientific and Technical Information of China (English)

    CUIZONGJIE

    1998-01-01

    In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium,Attached cells when stimulated with high potassium secreted large amount of amylase.High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation.High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel anatagonists with an order of potency as follows:nifedipine>ω-agatoxin IVA>ω-conotoxin GVIA.In contrast,the L-type calcium channel anatagonist nifedipine almost completely inhibited potassium-induced amylase secretion,whereas the N-type channel antagonist ω-conotoxin GVIA was without effect.The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect,but this inhibition was not significant at the level of amylase secretion.In conclusion,the AR4-2J cell line posesses different voltage-dependent calcium channels(L,P,N)with the L-type predominantly involved in depolarization induced amylase secretion.

  2. Modeling the cell-type dependence of diffusion-limited intracellular ice nucleation and growth during both vitrification and slow freezing

    Science.gov (United States)

    Yang, Geer; Zhang, Aili; Xu, Lisa X.; He, Xiaoming

    2009-06-01

    In this study, a set of models for predicting the diffusion-limited ice nucleation and growth inside biological cells were established. Both the heterogeneous and homogeneous nucleation mechanisms were considered in the models. Molecular mobility including viscosity and mutual diffusion coefficient of aqueous cryoprotectant (i.e., glycerol here) solutions was estimated using models derived from the free volume theory for glass transition, which makes it possible to predict the two most important physical properties (i.e., viscosity and mutual diffusion coefficient) over wide ranges of temperature and concentration as encountered in cryopreservation. After being verified using experimental data, the models were used to predict the critical cooling rate (defined as the cooling rate required so that the crystallized volume is less than 0.1% of the cell volume) as a function of the initial glycerol concentration in a number of cell types with different sizes. For slowing freezing, it was found that the required critical cooling rate is cell-type dependent with influences from cell size and the ice nucleation and water transport parameters. In general, the critical cooling rate does not change significantly with the initial glycerol concentration used and tends to be higher for smaller cells. For vitrification, the required critical cooling rate does change significantly with the initial glycerol concentration used and tends to decrease with the decrease in cell size. However, the required critical cooling rate can be similar for cells with very different sizes. It was further found that the thermodynamic and kinetic parameters for intracellular ice formation associated with different cells rather than the cell size per se significantly affect the critical cooling rates required for vitrification. For all cell types, it was found that homogeneous nucleation dominates at ultrafast cooling rates and/or high glycerol concentrations, whereas heterogeneous nucleation becomes

  3. Structural Dependence of Electronic Properties in A-A-D-A-A-Type Organic Solar Cell Material

    Directory of Open Access Journals (Sweden)

    Ram S. Bhatta

    2015-01-01

    Full Text Available Small conjugated molecules (SCMs are promising candidates for organic photovoltaic (OPV devices because of their structural simplicity, well control over synthetic reproducibility, and low purification cost. However, industrial development of SCM-based OPV devices requires improving their performance, which in turn relies on the fundamental understanding of structural dependence of electronic properties of SCMs. Herein, we report the structural and electronic properties of the BCNDTS molecule as a model system for acceptor-acceptor-donor-acceptor-acceptor (A-A-D-A-A type SCMs, using density functional theory (DFT and time-dependent DFT methods. Systematic calculations of two-dimensional potential energy surfaces, molecular electrostatic potential surfaces, ground state frontier molecular orbital energies, and the vertical excitation energies are performed. We found that the lowest energy conformation of the BCNDTS molecule is planar. The planar conformation favors the lowest ground state and the excited state energies as well as the strongest oscillator strength. The present results suggest that SCMs containing central dithienosilole cores connected with 2,1,3-benzothiadiazole groups have potential to be an efficient electron donor for OPV devices.

  4. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line.

    Science.gov (United States)

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan

    2008-05-01

    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

  5. Herpes Simplex Virus Type-1 (HSV-1 Entry into Human Mesenchymal Stem Cells Is Heavily Dependent on Heparan Sulfate

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    Samiksha Choudhary

    2011-01-01

    Full Text Available Hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type-1 (HSV-1. The purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hMSCs to HSV-1 infection and identify the major entry receptor. Productive virus infection in hMSCs was confirmed by replication and plaque formation assays using a syncytial HSV-1 KOS (804 virus. To examine the significance of entry receptors, RT-PCR and antibody-blocking assays were performed. RT-PCR data showed the expression of gD receptors: nectin-1, 3-O sulfotransferase-3 (3-OST-3, and HVEM. Antibody-blocking assay together with heparinase treatment suggested an important role for HS and 3-O-sulfated heparan sulfate (3-OS HS, but not nectin-1 or HVEM, in mediating HSV-1 entry and spread in hMSCs. Taken together, our results provide strong evidence demonstrating that HSV-1 is capable of infecting hMSCs and HS and 3-OS HS serve as its entry receptors during this process.

  6. Cell density- and quorum sensing-dependent expression of type VI secretion system 2 in Vibrio parahaemolyticus.

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    Li Wang

    Full Text Available BACKGROUND: Vibrio parahaemolyticus AphA and OpaR are the two master quorum sensing (QS regulators that are abundantly expressed at low cell density (LCD and high cell density (HCD, respectively, with a feature of reciprocally gradient production of them with transition between LCD and HCD. The type VI secretion system 2 (T6SS2 gene cluster can be assigned into three putative operons, namely VPA1027-1024, VPA1043-1028, and VPA1044-1046. T6SS2 contributes to adhesion of V. parahaemolyticus to host cells. METHODOLOGY/PRINCIPAL FINDINGS: OpaR box-like sequences were found within the upstream promoter regions of all the above three operons, while none of AphA box-like elements could be identified for them. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that OpaR bound to the promoter regions of these three operons to stimulate their transcription, while AphA negatively regulated their transcription most likely through acting on OpaR. This regulation led to a gradient increase of T6SS2 transcription with transition from LCD to HCD. CONCLUSIONS/SIGNIFICANCE: V. parahaemolyticus OpaR and AphA positively and negatively regulate T6SS2 expression, respectively, leading to a gradient elevation of T6SS2 expression with transition from LCD to HCD. T6SS2 genes are thus assigned as the QS regulon members in V. parahaemolyticus.

  7. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

    Science.gov (United States)

    Matsumoto, Masaru; Nakajima, Wataru; Seike, Masahiro; Gemma, Akihiko; Tanaka, Nobuyuki

    2016-04-29

    Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

  8. Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner

    Science.gov (United States)

    Detje, Claudia N.; Riebesehl, Nina; Lienenklaus, Stefan; Steinmann, Eike; Kalinke, Ulrich; Pietschmann, Thomas

    2016-01-01

    Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN

  9. Poly(ethylene glycol-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells.

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    Rikuo Ochi

    Full Text Available Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol cholesteryl ether (PEG-cholesterol is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca(2+ channel currents (I(Ca,L recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of ICa,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-β-cyclodextrin (MβCD is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MβCD:cholesterol complex (cholesterol (MβCD caused inhibition of I(Ca,L but did not augment voltage-dependent inactivation. Incubation with MβCD increased I(Ca,L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MβCD of the cells initially pretreated with PEG-cholesterol, increased I(Ca,L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I(Ca,L more strongly as compared with cholesterol (MβCD. Poly(ethylene glycol conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I(Ca,L.

  10. Cell-type and state-dependent synchronization among rodent areas S1BF, V1, perirhinal cortex and hippocampus CA1

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    Martin eVinck

    2016-01-01

    Full Text Available Beta and gamma rhythms have been hypothesized to be involved in global and local coordination of neuronal activity, respectively. Here, we investigated how cells in rodent area S1BF are entrained by rhythmic fluctuations at various frequencies within the local area and in connected areas, and how this depends on behavioral state and cell type. We performed simultaneous extracellular field and unit recordings in four connected areas of the freely moving rat (S1BF, V1M, perirhinal cortex, CA1. S1BF spiking activity was strongly entrained by both beta and gamma S1BF oscillations, which were associated with deactivations and activations, respectively. We identified multiple classes of fast spiking and excitatory cells in S1BF, which showed prominent differences in rhythmic entrainment and in the extent to which phase locking was modulated by behavioral state. Using an additional dataset acquired by whole-cell recordings in head-fixed mice, these cell classes could be compared with identified phenotypes showing gamma rhythmicity in their membrane potential. We next examined how S1BF cells were entrained by rhythmic fluctuations in connected brain areas. Gamma-synchronization was detected in all four areas, however we did not detect significant gamma coherence among these areas. Instead, we only found long-range coherence in the theta-beta range among these areas. In contrast to local S1BF synchronization, we found long-range S1BF-spike to CA1-LFP synchronization to be homogeneous across inhibitory and excitatory cell types. These findings suggest distinct, cell-type contributions of low and high-frequency synchronization to intra- and inter-areal neuronal interactions.

  11. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

    Science.gov (United States)

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-07-05

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

  12. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

    Directory of Open Access Journals (Sweden)

    Cavazzoni Andrea

    2012-12-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  13. Increase in antioxidant activity by sheep/goat whey protein through nuclear factor-like 2 (Nrf2) is cell type dependent.

    Science.gov (United States)

    Kerasioti, Efthalia; Stagos, Dimitrios; Tzimi, Aggeliki; Kouretas, Dimitrios

    2016-11-01

    The aim of the present study was to investigate the molecular mechanisms through which sheep/goat whey protein exerts its antioxidant activity. Thus, it was examined whey protein's effects on the expression of transcription factor, nuclear factor-like 2 (Nrf2) and on the expression and activity of a number of antioxidant and phase II enzymes, superoxide dismutase (SOD), catalase (CAT), heme oxygenase 1 (HO-1), synthase glutamyl cysteine (GCS) and glutathione-s-transferase (GST), in muscle C2C12 and EA.hy926 endothelial cells. C2C12 and EA.hy926 cells were treated with sheep/goat whey protein (0.78 and 3.12 mg/ml) and incubated for 3, 6, 12, 18 and 24 h. Whey protein increased significantly the expression of Nrf2 only in EA.hy926 cells. Also, the expression of SOD, HO-1, CAT and the activity of SOD, CAT and GST were increased significantly in both cells types. The expression of GCS was increased significantly only in C2C12 cells. Sheep/goat whey protein was shown for the first time to exert its antioxidant activity through Nrf2-dependent mechanism in endothelial cells and Nrf2-independent mechanism in muscle cells. Thus, Nrf2 could be a target for food supplements containing whey protein in order to prevent oxidative stress damages and diseases related to endothelium.

  14. Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor.

    Science.gov (United States)

    Parkash, Jai

    2008-08-01

    The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues

  15. Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

    Science.gov (United States)

    Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

    2010-01-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

  16. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    Science.gov (United States)

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  17. An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses.

    Science.gov (United States)

    Pettersen, Frank O; Torheim, Eirik A; Dahm, Anders E A; Aaberge, Ingeborg S; Lind, Andreas; Holm, Malin; Aandahl, Einar M; Sandset, Per M; Taskén, Kjetil; Kvale, Dag

    2011-07-01

    Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E(2) following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8(+) T cells (-24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8(+) T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3(+) CD4(+) CD25(+) CD127(lo/-) Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.

  18. Differential resistance of human embryonic stem cells and somatic cell types to hydrogen peroxide-induced genotoxicity may be dependent on innate basal intracellular ROS levels.

    Science.gov (United States)

    Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Poonepalli, Anuradha; Hande, Manoor Prakash; Cao, Tong

    2015-01-01

    Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H₂O₂-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H₂O₂ for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H₂O₂.

  19. CELL TYPE-DEPENDENT AND DIFFERENTIATION STAGE-DEPENDENT EXPRESSION OF PML DOMAINS IN RAT, DETECTED BY MONOCLONAL-ANTIBODY HIS55

    NARCIS (Netherlands)

    LAM, YW; AMMERLAAN, W; O, WS; KROESE, F; OPSTELTEN, D

    1995-01-01

    Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In iso

  20. Reduction of conventional dendritic cells during Plasmodium infection is dependent on activation induced cell death by type I and II interferons.

    Science.gov (United States)

    Tamura, Takahiko; Kimura, Kazumi; Yui, Katsuyuki; Yoshida, Shigeto

    2015-12-01

    Dendritic cells (DCs) play critical roles in innate and adaptive immunity and in pathogenesis during the blood stage of malaria infection. The mechanisms underlying DC homeostasis during malaria infection are not well understood. In this study, the numbers of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the spleens after lethal rodent malaria infection were examined, and were found to be significantly reduced. Concomitant with up-regulation of maturation-associated molecules, activation of caspase-3 was significantly increased, suggesting induction of cell death. Studies using neutralizing antibody and gene-deficient mice showed that type I and II interferons were critically involved in activation induced cell death of cDCs during malaria infection. These results demonstrate that DCs rapidly disappeared following IFN-mediated DC activation, and that homeostasis of DCs was significantly impaired during malaria infection.

  1. Cell type and gender-dependent differential regulation of the p202 and Aim2 proteins: implications for the regulation of innate immune responses in SLE.

    Science.gov (United States)

    Panchanathan, Ravichandran; Duan, Xin; Arumugam, Muthuvel; Shen, Hui; Liu, Hongzhu; Choubey, Divaker

    2011-10-01

    Upon sensing cytosolic double-stranded DNA (dsDNA), the murine Aim2 (encoded by the Aim2 gene) protein forms an inflammasome and promotes the secretion of proinflammatory cytokines, such as IL-1β and IL-18. In contrast, the p202 protein (encoded by the Ifi202 gene) does not form an inflammasome. Previously, we have reported that the interferon (IFN) and female sex hormone-induced increased nuclear levels of p202 protein in immune cells are associated with increased susceptibility to develop a lupus-like disease. However, signaling pathways that regulate the expression of Aim2 protein remain unknown. Here we report that the expression of Aim2 gene is induced in bone marrow-derived macrophages (BMDMs) by IFN-α treatment and the expression is, in part, STAT1-dependent. However, treatment of splenic T or B cells with IFN-α or their stimulation, which induced the expression of Ifi202 gene, did not induce the expression of Aim2 gene. Furthermore, treatment of cells with the male hormone androgen increased levels of Aim2 mRNA and protein. Moreover, treatment of murine macrophage cell lines (RAW264.7 and J774A.1) with IFN-α differentially induced the expression of Aim2 and p202 proteins and regulated their sub-cellular localization. Additionally, activation of Toll-like receptors (TLR3, 4, and 9) in BMDMs and cell lines also differentially regulated the expression of Aim2 and Ifi202 genes. Our observations demonstrate that cell type and gender-dependent factors differentially regulate the expression of the Aim2 and p202 proteins, thus, suggesting opposing roles for these two proteins in innate immune responses in lupus disease.

  2. HIV Triggers a cGAS-Dependent, Vpu- and Vpr-Regulated Type I Interferon Response in CD4+ T Cells

    Directory of Open Access Journals (Sweden)

    Jolien Vermeire

    2016-10-01

    Full Text Available Several pattern-recognition receptors sense HIV-1 replication products and induce type I interferon (IFN-I production under specific experimental conditions. However, it is thought that viral sensing and IFN induction are virtually absent in the main target cells of HIV-1 in vivo. Here, we show that activated CD4+ T cells sense HIV-1 infection through the cytosolic DNA sensor cGAS and mount a bioactive IFN-I response. Efficient induction of IFN-I by HIV-1 infection requires proviral integration and is regulated by newly expressed viral accessory proteins: Vpr potentiates, while Vpu suppresses cGAS-dependent IFN-I induction. Furthermore, Vpr also amplifies innate sensing of HIV-1 infection in Vpx-treated dendritic cells. Our results identify cGAS as mediator of an IFN-I response to HIV-1 infection in CD4+ T cells and demonstrate that this response is modulated by the viral accessory proteins Vpr and Vpu. Thus, viral innate immune evasion is incomplete in the main target cells of HIV-1.

  3. The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death

    Science.gov (United States)

    Srisawat, Theera; Sukpondma, Yaowapa; Graidist, Potchanapond; Chimplee, Siriphon; Kanokwiroon, Kanyanatt

    2015-01-01

    Background: Vatica diospyroides type LS is a known source of valuable compounds for cancer treatment, however, in contrast little is known about therapeutic efficacy of type SS. Objective: This study focused on in vitro cytotoxicity of these fruit extracts, and the cell death mode they induce in breast cancer cells. Materials and Methods: Acetone extracts of fruit were tested for cytotoxicity against MCF-7 and MDA-MB-231 cell lines. The apoptosis and necrosis of these cells were quantified by fluorescence activated cell sorter (FACS) and western blot analyses. Results: After 72 h of treatment, the 50% growth inhibition concentrations (IC50) levels were 16.21 ± 0.13 µg/mL against MCF-7 and 30.0 ± 4.30 µg/mL against MDA-MB-231, indicating high and moderate cytotoxicity, respectively. From the FACS results, we estimate that the cotyledon extract at half IC50 produced 11.7% dead MCF-7 cells via apoptosis, whereas another concentrations both apoptosis and necrosis modes co-existed in a dose-dependent manner. In MDA-MB-231 cell line, only the apoptosis was induced by the pericarp extract in a dose-dependent manner. With the extracts at half IC50 concentration, in both cells, the expression of p21 decreased while that of Bax increased within 12–48 h of dosing, confirming apoptosis induced by time-dependent responses. Apoptosis dependent on p53 was found in MCF-7, whereas the mutant p53 of MDA-MB-231 cells was expressed. Conclusion: The results indicate that fruit extracts of V. diospyroides have cytotoxic effects against MCF-7 and MDA-MB-231 cells via apoptosis pathway in a dose-dependent manner. This suggests that the extracts could provide active ingredients for the development, targeting breast cancer therapy. PMID:26109760

  4. Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Fernando eBaisón-Olmo

    2015-04-01

    Full Text Available Salmonella enterica expresses two virulence-related type III secretion systems (T3SSs encoded in Salmonella pathogenicity island 1 (SPI1 and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, in spite of not having a role in invasion or intracellular proliferation in cultured mammalian cells. In vitro, expression of sseK1 was higher in media mimicking intracellular conditions, when SPI2 was induced, but it was also significant under SPI1 inducing conditions. A detailed analysis of translocation of SseK1 into host cells unveiled that it was a substrate of both, T3SS1 and T3SS2, although with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages or fibroblasts. The regulation of the expression of sseK1 was examined using lacZ and bioluminescent lux fusions. The two-component system PhoQ/PhoP is a positive regulator of this gene. A combination of sequence analysis, directed mutagenesis and electrophoretic mobility shift assays showed that phosphorylated PhoP binds directly to the promoter region of sseK1 and revealed a PhoP binding site located upstream of the predicted -35 hexamer of this promoter.

  5. Enteroendocrine cell types revisited

    DEFF Research Database (Denmark)

    Engelstoft, Maja S; Egerod, Kristoffer Lihme; Lund, Mari L

    2013-01-01

    The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell...... biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type...... and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa....

  6. Antiproliferative effect of Antrodia camphorata polysaccharides encapsulated in chitosan-silica nanoparticles strongly depends on the metabolic activity type of the cell line

    Science.gov (United States)

    Kong, Zwe-Ling; Chang, Jenq-Sheng; Chang, Ke Liang B.

    2013-09-01

    Chitosan molecules interact with silica and encapsulate the Antrodia camphorata extract (ACE) polysaccharides to form composite nanoparticles. The nanoparticle suspensions of ACE polysaccharides encapsulated in silica-chitosan and silica nanoparticles approach an average particle size of 210 and 294 nm in solution, respectively. The encapsulation efficiencies of ACE polysaccharides are 66 and 63.5 %, respectively. Scanning electron micrographs confirm the formation of near-spherical nanoparticles. ACE polysaccharides solution had better antioxidative capability than ACE polysaccharides encapsulated in silica or silica-chitosan nanoparticles suspensions. The antioxidant capacity of nanoparticles increases with increasing dissolution time. The antitumor effects of ACE polysaccharides, ACE polysaccharides encapsulated in silica, or silica-chitosan nanoparticles increased with increasing concentration of nanoparticles. This is the first report demonstrating the potential of ACE polysaccharides encapsulated in chitosan-silica nanoparticles for cancer chemoprevention. Furthermore, this study suggests that antiproliferative effect of nanoparticle-encapsulated bioactive could significantly depend on the metabolic activity type of the cell line.

  7. Antiproliferative effect of Antrodia camphorata polysaccharides encapsulated in chitosan-silica nanoparticles strongly depends on the metabolic activity type of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Zwe-Ling, E-mail: kongzl@mail.ntou.edu.tw; Chang, Jenq-Sheng; Chang, Ke Liang B. [National Taiwan Ocean University, Department of Food Science (China)

    2013-09-15

    Chitosan molecules interact with silica and encapsulate the Antrodia camphorata extract (ACE) polysaccharides to form composite nanoparticles. The nanoparticle suspensions of ACE polysaccharides encapsulated in silica-chitosan and silica nanoparticles approach an average particle size of 210 and 294 nm in solution, respectively. The encapsulation efficiencies of ACE polysaccharides are 66 and 63.5 %, respectively. Scanning electron micrographs confirm the formation of near-spherical nanoparticles. ACE polysaccharides solution had better antioxidative capability than ACE polysaccharides encapsulated in silica or silica-chitosan nanoparticles suspensions. The antioxidant capacity of nanoparticles increases with increasing dissolution time. The antitumor effects of ACE polysaccharides, ACE polysaccharides encapsulated in silica, or silica-chitosan nanoparticles increased with increasing concentration of nanoparticles. This is the first report demonstrating the potential of ACE polysaccharides encapsulated in chitosan-silica nanoparticles for cancer chemoprevention. Furthermore, this study suggests that antiproliferative effect of nanoparticle-encapsulated bioactive could significantly depend on the metabolic activity type of the cell line.

  8. Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKKε- and CYLD-dependent Bcl3 activation.

    Science.gov (United States)

    Gringhuis, Sonja I; Kaptein, Tanja M; Wevers, Brigitte A; Mesman, Annelies W; Geijtenbeek, Teunis B H

    2014-05-28

    Carbohydrate-specific signalling through DC-SIGN provides dendritic cells with plasticity to tailor immunity to the nature of invading microbes. Here we demonstrate that recognition of fucose-expressing extracellular pathogens like Schistosoma mansoni and Helicobacter pylori by DC-SIGN favors T helper cell type-2 (TH2) responses via activation of atypical NF-κB family member Bcl3. Crosstalk between TLR and DC-SIGN signalling results in TLR-induced MK2-mediated phosphorylation of LSP1, associated with DC-SIGN, upon fucose binding. Subsequently, IKKε and CYLD are recruited to phosphorylated LSP1. IKKε activation is pivotal for suppression of CYLD deubiquitinase activity and subsequent nuclear translocation of ubiquitinated Bcl3. Bcl3 activation represses TLR-induced proinflammatory cytokine expression, while enhancing interleukin-10 (IL-10) and TH2-attracting chemokine expression, shifting TH differentiation from TH1 to TH2 polarization. Thus, DC-SIGN directs adaptive TH2 immunity to fucose-expressing pathogens via an IKKε-CYLD-dependent signalling pathway leading to Bcl3 activation, which might be targeted in vaccination strategies or to prevent aberrant inflammation and allergy.

  9. Practical Reflection and Metaprogramming for Dependent Types

    DEFF Research Database (Denmark)

    Christiansen, David Raymond

    2016-01-01

    to program in these embedded languages. Interpreters and compilers must always take these invariants into account at each stage, and authors of embedded languages must work hard to relieve users of the burden of proving these properties. Idris is a dependently typed functional programming language whose...

  10. Targeted Intra-arterial Transplantation of Stem Cells to the Injured CNS is More Effective than Intravenous Administration - Engraftment is Dependent on Cell Type and Adhesion Molecule Expression

    DEFF Research Database (Denmark)

    Lundberg, Johan; Södersten, Erik; Sundström, Erik

    2011-01-01

    Stem cell transplantation procedures using intraparenchymal injections cause tissue injury in addition to associated surgical risks. Intra-venous cell administration give engraftment in parenchymal lesions although the method has low efficacy and specificity. In pathological conditions...... with inflammation, such as traumatic brain injury, there is a transient up-regulation of ICAM-1 and VCAM-1 which might provide enviromental cues for migration of stem cells from blood to parenchyma. The aim of this study was to i) analyze the effect of intra-arterial administration on cellular engraftment, ii......) compare engraftment and side effects between three different stem cell systems and iii) analyze gene expression in these three systems....

  11. A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells

    Directory of Open Access Journals (Sweden)

    Furr Samantha R

    2011-08-01

    Full Text Available Abstract Background The rapid onset of potentially lethal neuroinflammation is a defining feature of viral encephalitis. Microglia and astrocytes are likely to play a significant role in viral encephalitis pathophysiology as they are ideally positioned to respond to invading central nervous system (CNS pathogens by producing key inflammatory mediators. Recently, DNA-dependent activator of IFN regulatory factor (DAI has been reported to function as an intracellular sensor for DNA viruses. To date, the expression and functional role of DAI in the inflammatory responses of resident CNS cells to neurotropic DNA viruses has not been reported. Methods Expression of DAI and its downstream effector molecules was determined in C57BL/6-derived microglia and astrocytes, either at rest or following exposure to herpes simplex virus type 1 (HSV-1 and/or murine gammaherpesvirus-68 (MHV-68, by immunoblot analysis. In addition, such expression was studied in ex vivo microglia/macrophages and astrocytes from uninfected animals or mice infected with HSV-1. Inflammatory cytokine production by glial cultures following transfection with a DAI specific ligand (B-DNA, or following HSV-1 challenge in the absence or presence of siRNA directed against DAI, was assessed by specific capture ELISA. The production of soluble neurotoxic mediators by HSV-1 infected glia following DAI knockdown was assessed by analysis of the susceptibility of neuron-like cells to conditioned glial media. Results We show that isolated microglia and astrocytes constitutively express DAI and its effector molecules, and show that such expression is upregulated following DNA virus challenge. We demonstrate that these resident CNS cells express DAI in situ, and show that its expression is similarly elevated in a murine model of HSV-1 encephalitis. Importantly, we show B-DNA transfection can elicit inflammatory cytokine production by isolated glial cells and DAI knockdown can significantly reduce

  12. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Fujiwara, Yuki [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Yamaguchi, Hideki [Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-city, Saitama 332-0012 (Japan); Nakamura, Yoshikazu; Fukami, Kiyoko [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan)

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  13. The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, Adayapalam T.; Berni, Andrea; Marimuthu, Kodumudi M. [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy); Palitti, Fabrizio [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy)], E-mail: palitti@unitus.it

    2008-07-03

    In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G{sub 2}, S and G{sub 1} phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G{sub 1} irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G{sub 1} irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G{sub 1} in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.

  14. Improved pancreatic beta-cell function in type 2 diabetic patients after lifestyle-induced weight loss is related to glucose-dependent insulinotropic polypeptide

    DEFF Research Database (Denmark)

    Solomon, Thomas; Haus, Jacob M; Kelly, Karen R;

    2010-01-01

    Restoration of insulin secretion is critical for the treatment of type 2 diabetes. Exercise and diet can alter glucose-induced insulin responses, but whether this is due to changes in beta-cell function per se is not clear. The mechanisms by which lifestyle intervention may modify insulin secreti...

  15. Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

    Science.gov (United States)

    Faul, Elizabeth J; Wanjalla, Celestine N; Suthar, Mehul S; Gale, Michael; Wirblich, Christoph; Schnell, Matthias J

    2010-07-22

    As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

  16. L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells.

    Science.gov (United States)

    Toselli, Mauro; Taglietti, Vanni

    2005-05-01

    Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

  17. Cosmic Web Type Dependence of Halo Clustering

    CERN Document Server

    Fisher, J D

    2016-01-01

    We use the Millennium simulation to show that halo clustering varies significantly with cosmic web type. Halos are classified as node, filament, sheet and void halos based on the eigenvalue decomposition of the velocity shear tensor. This classification allows us to examine the clustering of halos as a function of web type in different mass ranges. We find that node halos show positive bias for all mass ranges probed, even for 10^11 and 10^12 Msun/h mass bins where the clustering of the entire halo sample is anti-biased. In all mass bins filament halos show negligible bias, whereas void and sheet halos are anti-biased. The zero-crossing of the void and sheet correlation functions occur at much smaller scales Mpc/h when compared to 5the same correlation functions for the entire halo sample. Our results suggest that the mass dependence of halo clustering is rooted in the composition of web types in the mass bin. The substantial fraction of node type halos for halo masses 2 x 10^13 Msun/h leads to positive bias....

  18. Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

    Directory of Open Access Journals (Sweden)

    Elizabeth J Faul

    Full Text Available As with many viruses, rabies virus (RABV infection induces type I interferon (IFN production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC in order to differentiate which pattern recognition receptor(s (PRR is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

  19. Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells.

    Science.gov (United States)

    Pratt, Evan P S; Salyer, Amy E; Guerra, Marcy L; Hockerman, Gregory H

    2016-01-05

    We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

  20. Cell Type-dependent Gene Transcription Profile in Three Dimensional Human Skin Tissue Model Exposed to Low Doses of Ionizing Radiation: Implications for Medical Exposures

    Energy Technology Data Exchange (ETDEWEB)

    Freiin von Neubeck, Claere H.; Shankaran, Harish; Karin, Norman J.; Kauer, Paula M.; Chrisler, William B.; Wang, Xihai; Robinson, Robert J.; Waters, Katrina M.; Tilton, Susan C.; Sowa, Marianne B.

    2012-04-17

    The concern over possible health risks from exposures to low doses of ionizing radiation has been driven largely by the increase in medical exposures, the routine implementation of X-ray backscatter devices for airport security screening, and, most recently, the nuclear incident in Japan. Due to a paucity of direct epidemiological data at very low doses, cancer risk must be estimated from high dose exposure scenarios. However, there is increasing evidence that low and high dose exposures result in different signaling events and may have different mechanisms of cancer induction. We have examined the radiation induced temporal response of an in vitro three dimensional (3D) human skin tissue model using microarray-based transcriptional profiling. Our data shows that exposure to 100 mGy of X-rays is sufficient to affect gene transcription. Cell type specific analysis showed significant changes in gene expression with the levels of > 1400 genes altered in the dermis and > 400 genes regulated in the epidermis. The two cell types rarely exhibited overlapping responses at the mRNA level. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements validated the microarray data in both regulation direction and value. Key pathways identified relate to cell cycle regulation, immune responses, hypoxia, reactive oxygen signaling, and DNA damage repair. We discuss in particular the role of proliferation and emphasizing how the disregulation of cellular signaling in normal tissue may impact progression towards radiation induced secondary diseases.

  1. Size-dependent rheology of type-I collagen networks.

    Science.gov (United States)

    Arevalo, Richard C; Urbach, Jeffrey S; Blair, Daniel L

    2010-10-20

    We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.

  2. Evidence that Vpu modulates HIV-1 Gag-envelope interaction towards envelope incorporation and infectivity in a cell type dependent manner.

    Directory of Open Access Journals (Sweden)

    Archana Gautam

    Full Text Available The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env incorporation defect caused by a Gag matrix (MA mutation (L30E was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC and monocyte-derived macrophages (MDM. However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.

  3. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    Science.gov (United States)

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  4. The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner

    Directory of Open Access Journals (Sweden)

    Bontems Sébastien

    2007-10-01

    Full Text Available Abstract Background Varicella Zoster Virus Immediate Early 63 protein (IE63 has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. Results In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-κB dependent genes such as IL-8, ICAM-1, and IκBα, it modulates transcription of these genes upon TNFα induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. Conclusion While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-κB dependent genes by the accelerated resynthesis of the inhibitor IκBα.

  5. Vitamin D dependent rickets type I

    Directory of Open Access Journals (Sweden)

    Chan Jong Kim

    2011-02-01

    Full Text Available Vitamin D is present in two forms, ergocalciferol (vitamin D2 produced by plants and cholecalciferol (vitamin D3 produced by animal tissues or by the action of ultraviolet light on 7-dehydrocholesterol in human skin. Both forms of vitamin D are biologically inactive pro-hormones that must undergo sequential hydroxylations in the liver and the kidney before they can bind to and activate the vitamin D receptor. The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH2D], plays an essential role in calcium and phosphate metabolism, bone growth, and cellular differentiation. Renal synthesis of 1,25(OH2D from its endogenous precursor, 25-hydroxyvitamin D (25OHD, is the rate-limiting and is catalyzed by the 1?#7016;ydroxylase. Vitamin D dependent rickets type I (VDDR-I, also referred to as vitamin D 1?#7016;ydroxylase deficiency or pseudovitamin D deficiency rickets, is an autosomal recessive disorder characterized clinically by hypotonia, muscle weakness, growth failure, hypocalcemic seizures in early infancy, and radiographic findings of rickets. Characteristic laboratory features are hypocalcemia, increased serum concentrations of parathyroid hormone (PTH, and low or undetectable serum concentrations of 1,25(OH2D despite normal or increased concentrations of 25OHD. Recent advances have showed in the cloning of the human 1?#7016;ydroxylase and revealed mutations in its gene that cause VDDR-I. This review presents the biology of vitamin D, and 1?#7016;ydroxylase mutations with clinical findings.

  6. In vitro RBE-LET dependence for multiple particle types

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Overgaard, Jens; Bassler, Niels

    2011-01-01

    Background. In vitro RBE values for various high LET radiation types have been determined for many different cell types. Occasionally it is criticized that RBE for a given endpoint cannot be single-value dependent on LET alone, but also on particle species, due to the different dose deposition...... profiles on microscopic scale. Hence LET is not sufficient as a predictor of RBE, and this is one of the motivations for development of radiobiological models which explicitly depend on the detailed particle energy spectrum of the applied radiation field. The aim of the present study is to summarize...... points were collected from 20, four and four publications for V79, CHO and T1, respectively, in total covering 228 RBE values from a broad range of particle species. Results and discussion. All RBE-LET data points demonstrate surprising agreement within the general error band formed by the numerous data...

  7. Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

    Science.gov (United States)

    Lindert, Susanne; Wickert, Lucia; Sawitza, Iris; Wiercinska, Eliza; Gressner, Axel M; Dooley, Steven; Breitkopf, Katja

    2005-05-01

    Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.

  8. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    Science.gov (United States)

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

  9. MEH-PPV and PCBM Solution Concentration Dependence of Inverted-Type Organic Solar Cells Based on Eosin-Y-Coated ZnO Nanorod Arrays

    Directory of Open Access Journals (Sweden)

    Riski Titian Ginting

    2013-01-01

    Full Text Available The influence of polymer solution concentration on the performance of chlorobenzene- (CB- and chloroform- (CF- based inverted-type organic solar cells has been investigated. The organic photoactive layers consisted of poly(2-methoxy-5-(2-ethyl hexyloxy-1,4-phenylenevinylene (MEH-PPV and (6,6-phenyl C61 butyric acid methyl ester (PCBM were spin coated from CF with concentrations of 4, 6, and 8 mg/mL and from CB with concentrations of 6, 8, and 10 mg/mL onto Eosin-Y-coated ZnO nanorod arrays (NRAs. Fluorine doped tin oxide (FTO and silver (Ag were used as electron collecting electrode and hole collecting electrode, respectively. Experimental results showed that the short circuit current density and power conversion efficiency increased with decrease of solution concentration for both CB and CF devices, which could be attributed to reducing charge recombination in thinner photoactive layer and larger contact area between the rougher photoactive layer and Ag contact. However, the open circuit voltage decreased with decreasing solution concentration due to increase of leakage current from ZnO NRAs to Ag as the ZnO NRAs were not fully covered by the polymer blend. The highest power conversion efficiencies of 0.54 ± 0.10% and 0.87 ± 0.15% were achieved at the respective lowest solution concentrations of CB and CF.

  10. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    Science.gov (United States)

    Márkus, Nóra M; Hasel, Philip; Qiu, Jing; Bell, Karen F S; Heron, Samuel; Kind, Peter C; Dando, Owen; Simpson, T Ian; Hardingham, Giles E

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

  11. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    Directory of Open Access Journals (Sweden)

    Nóra M Márkus

    Full Text Available Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs, however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes, differing neuronal subtype (CA3 vs. CA1 hippocampus and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

  12. The Early Activation of CD8+ T Cells Is Dependent on Type I IFN Signaling following Intramuscular Vaccination of Adenovirus Vector

    Directory of Open Access Journals (Sweden)

    Masahisa Hemmi

    2014-01-01

    Full Text Available Few of the vaccines in current use can induce antigen- (Ag- specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m. vaccination of an adenovirus vector (Adv can induce Ag-specific cytotoxic T lymphocytes (CTLs in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs, which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.

  13. RNA cell typing and DNA profiling of mixed samples: can cell types and donors be associated?

    Science.gov (United States)

    Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia

    2013-09-01

    Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling.

  14. Regulation of the tumor marker Fascin by the viral oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) depends on promoter activation and on a promoter-independent mechanism.

    Science.gov (United States)

    Mohr, Caroline F; Gross, Christine; Bros, Matthias; Reske-Kunz, Angelika B; Biesinger, Brigitte; Thoma-Kress, Andrea K

    2015-11-01

    Adult T-cell leukemia/lymphoma is a highly infiltrative neoplasia of CD4(+) T-lymphocytes that occurs in about 5% of carriers infected with the deltaretrovirus human T-cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax perturbs cellular signaling pathways leading to upregulation of host cell factors, amongst them the actin-bundling protein Fascin, an invasion marker of several types of cancer. However, transcriptional regulation of Fascin by Tax is poorly understood. In this study, we identified a triple mode of transcriptional induction of Fascin by Tax, which requires (1) NF-κB-dependent promoter activation, (2) a Tax-responsive region in the Fascin promoter, and (3) a promoter-independent mechanism sensitive to the Src family kinase inhibitor PP2. Thus, Tax regulates Fascin by a multitude of signals. Beyond, using Tax-expressing and virus-transformed lymphocytes as a model system, our study is the first to identify the invasion marker Fascin as a novel target of PP2, an inhibitor of metastasis.

  15. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Directory of Open Access Journals (Sweden)

    Yu-Yo Sun

    Full Text Available The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α through inhibition of prolyl hydrolase 2 (PHD2 and activation of the phosphatidylinositide-3 kinase (PI3K/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo and vascular endothelial growth factor (VEGF, two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  16. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Science.gov (United States)

    Sun, Yu-Yo; Lin, Shang-Hsuan; Lin, Hung-Cheng; Hung, Chia-Chi; Wang, Chen-Yu; Lin, Yen-Chu; Hung, Kuo-Sheng; Lien, Cheng-Chang; Kuan, Chia-Yi; Lee, Yi-Hsuan

    2013-01-01

    The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α) through inhibition of prolyl hydrolase 2 (PHD2) and activation of the phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo) and vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  17. Cholesterol-Dependent Energy Transfer between Fluorescent Proteins—Insights into Protein Proximity of APP and BACE1 in Different Membranes in Niemann-Pick Type C Disease Cells

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    2012-11-01

    Full Text Available Förster resonance energy transfer (FRET -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP and amyloid precursor protein-mRFP (APP-mRFP in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer’s disease (AD pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1−/−, exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC, were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT and CHO-NPC1−/− cells (EPI-illumination microscopy, as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM, were performed. Additionally, generalized polarization (GP measurements of CHO-WT and CHO-NPC1−/− cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1−/− cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1−/−. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

  18. Variant brain-derived neurotrophic factor (BDNF) (Met66) alters the intracellular trafficking and activity-dependent secretion of wild-type BDNF in neurosecretory cells and cortical neurons.

    Science.gov (United States)

    Chen, Zhe-Yu; Patel, Paresh D; Sant, Gayatree; Meng, Chui-Xiang; Teng, Kenneth K; Hempstead, Barbara L; Lee, Francis S

    2004-05-05

    Brain-derived neurotrophic factor (BDNF) plays a critical role in nervous system and cardiovascular development and function. Recently, a common single nucleotide polymorphism in the bdnf gene, resulting in a valine to methionine substitution in the prodomain (BDNF(Met)), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders in humans heterozygous for the variant BDNF. When expressed by itself in hippocampal neurons, less BDNF(Met) is secreted in an activity-dependent manner. The nature of the cellular defect when both BDNF(Met) and wild-type BDNF (BDNF(Val)) are present in the same cell is not known. Given that this is the predominant expression profile in humans, we examined the effect of coexpressed BDNF(Met) on BDNF(Val) intracellular trafficking and processing. Our data indicate that abnormal trafficking of BDNF(Met) occurred only in neuronal and neurosecretory cells and that BDNF(Met) could alter the intracellular distribution and activity-dependent secretion of BDNF(Val). We determined that, when coexpressed in the same cell, approximately 70% of the variant BDNF forms BDNF(Val).BDNF(Met) heterodimers, which are inefficiently sorted into secretory granules resulting in a quantitative decreased secretion. Finally, we determined the form of BDNF secreted in an activity-dependent manner and observed no differences in the forms of BDNF(Met) or the BDNF(Val).BDNF(Met) heterodimer compared with BDNF(Val). Together, these findings indicate that components of the regulated secretory machinery interacts specifically with a signal in the BDNF prodomain and that perturbations in BDNF trafficking may lead to selective impairment in CNS function.

  19. [Cell therapy for type I diabete].

    Science.gov (United States)

    Sokolova, I B

    2009-01-01

    Cell therapy is a modern and promising approach to type I diabetes mellitus treatment. Nowadays a wide range of cells is used in laboratory experiments and clinical studies, including allogeneic and xenogeneic cells of Langergance islets, bone marrow cells, haematopoietic stem cells, mesenchymal stem cells, and cord blood stem cells. Any type of the cells named could correct the status of the patients to a certain extent. However, full recovery after cell therapy has not been achieved yet.

  20. Personality types and nicotine Dependency among medical sciences students

    Directory of Open Access Journals (Sweden)

    H. Bakshi

    2014-01-01

    Full Text Available Smoking has recently become a major public health threat among the youth of today in Iran. Many clinicians and researchers hypothesized that tobacco-related disorders are maintained by the ability of nicotine to regulate positive and negative mood states. Moreover, some research indicates that there is no correlation between personality type, cigarette smoking, and heart disease, while some others mention that people with personality type A are more inclined towards smoking and related diseases. Thus, to test this hypothesis, we have studied possible correlations between psychological personality and tobacco-dependency among university students in the central part of Iran. In the current study, the most prevalent personality type was B (56.8%, with A (43.2%. Regarding smoking status, 17.5% (70 of the students were smokers and 82.5% (330 non-smokers; moreover, our results showed 66.7% (47 of smokers had low dependency and 33.3% (23 were physically dependent on nicotine. Concerning the difference between smokers and non-smokers based on their personality type, the results showed that 51.4% smokers had type A personality and 59.9% non-smokers were type B. There were also statistical differences between personality type and tobacco usage in students (p<0.05. We also found statistical differences between physical dependency and personality type; that is, 67.3% of smoking students who were physically dependent on nicotine had A type personality (p<0.05. The results suggest that there are several psychological types having higher association with tobacco use than other types. It poses some additional challenges for students’ support services to address mental health problems. The personality type in our study turned out to be an important factor influencing the nicotine dependency of the students.

  1. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  2. The selection and function of cell type-specific enhancers.

    Science.gov (United States)

    Heinz, Sven; Romanoski, Casey E; Benner, Christopher; Glass, Christopher K

    2015-03-01

    The human body contains several hundred cell types, all of which share the same genome. In metazoans, much of the regulatory code that drives cell type-specific gene expression is located in distal elements called enhancers. Although mammalian genomes contain millions of potential enhancers, only a small subset of them is active in a given cell type. Cell type-specific enhancer selection involves the binding of lineage-determining transcription factors that prime enhancers. Signal-dependent transcription factors bind to primed enhancers, which enables these broadly expressed factors to regulate gene expression in a cell type-specific manner. The expression of genes that specify cell type identity and function is associated with densely spaced clusters of active enhancers known as super-enhancers. The functions of enhancers and super-enhancers are influenced by, and affect, higher-order genomic organization.

  3. Size-Dependent Rheology of Type-I Collagen Networks

    OpenAIRE

    Arevalo, Richard C.; Urbach, Jeffrey S.; Blair, Daniel L.

    2010-01-01

    We investigate the system size dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by t...

  4. Involvement of IRF4 dependent dendritic cells in T cell dependent colitis

    DEFF Research Database (Denmark)

    Pool, Lieneke; Rivollier, Aymeric Marie Christian; Agace, William Winston

    of chronic intestinal inflammation remains unclear. In the current study we used the CD45RBhi T cell transfer model of colitis to determine the role of IRF4 dependent DCs in intestinal inflammation. In this model naïve CD4+ T cells when transferred into RAG-/- mice, proliferate and expand in response...... these results suggest an important role for Irf4 dependent DCs in T cell driven colitis....

  5. Type II (noninsulin-dependent) diabetes: new treatment options.

    Science.gov (United States)

    Bodzin, B J

    1997-01-01

    Type II diabetes (noninsulin-dependent diabetes mellitus [NIDDM]) is a common primary and secondary diagnosis in home care patients. This article describes the pathophysiology of NIDDM, the new drugs that have been released for treatment, and the nursing implications inherent in using these new medications.

  6. A CAHN-HILLIARD TYPE EQUATION WITH GRADIENT DEPENDENT POTENTIAL

    Institute of Scientific and Technical Information of China (English)

    Yin Jingxue; Huang Rui

    2008-01-01

    We investigate a Cahn-Hilliard type equation with gradient dependent potential. After establishing the existence and uniqueness, we pay our attention mainly to the regularity of weak solutions by means of the energy estimates and the theory of Campanato Spaces.

  7. Age-dependent arginine phosphokinase activity changes in male vestigial and wild-type Drosophila melanogaster.

    Science.gov (United States)

    Baker, G T

    1975-01-01

    The activity of arginine phosphokinase, an important muscle enzyme in insects, was investigated with age in vestigial-winged and wild-type Drosophila melanogaster. Identical patterns of age-dependent activity changes were observed in the vestigial-winged flies as in the wild-type, even though vestigial-winged flies exhibit a 50% mortality approximately two thirds that of the wild-type as well as being incapable of flight. Results indicate that the age-dependent changes in arginine phosphokinase activity are intrinsically regulated within the cells of the flight muscle.

  8. Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+)Channel, and It is functionally important in renal afferent arterioles

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2000-01-01

    in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-alpha(1A) antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22+/-4% (n=6) inhibition of inward...... Ca(2+) current by omega-Agatoxin IVA (10(-8) mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca(2+) in afferent arterioles with fluorescence-imaging microscopy showed 32+/-9% (n=10) inhibition of the K(+)-induced rise in Ca(2...... preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca(2+) influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles....

  9. ATP-dependent potassium channels and type 2 diabetes mellitus.

    Science.gov (United States)

    Bonfanti, Dianne Heloisa; Alcazar, Larissa Pontes; Arakaki, Priscila Akemi; Martins, Laysa Toschi; Agustini, Bruna Carla; de Moraes Rego, Fabiane Gomes; Frigeri, Henrique Ravanhol

    2015-05-01

    Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

  10. Studies on TCM Syndrome Types of Heroin-Dependence

    Institute of Scientific and Technical Information of China (English)

    杜万君; 郭崧

    2004-01-01

    @@ Great advances have been achieved in abstinence treatment on heroin-dependence and prevention of drug-reusing, and a variety of traditional Chinese medicine (TCM) drugs or preparations have been developed and applied in clinic in recent years. The Zheng Tong Ning Granules (正通宁颗粒冲剂), for example, has been tested on healthy volunteers and proved to be more tolerable than clonidine1. However, they are designed only for certain symptoms appeared in a certain stage of the abstinence treatment. A comprehensive study on TCM syndrome types of heroin-dependence and the relevant treatment are still lacking. Following is our pioneer attempt made in this field.

  11. Dependent Types for Multi-Rate Flows in Synchronous Programming

    Directory of Open Access Journals (Sweden)

    William Blair

    2017-02-01

    Full Text Available Synchronous programming languages emerged in the 1980s as tools for implementing reactive systems, which interact with events from physical environments and often must do so under strict timing constraints. In this report, we encode inside ATS various real-time primitives in an experimental synchronous language called Prelude, where ATS is a statically typed language with an ML-like functional core that supports both dependent types (of DML-style and linear types. We show that the verification requirements imposed on these primitives can be formally expressed in terms of dependent types in ATS. Moreover, we modify the Prelude compiler to automatically generate ATS code from Prelude source. This modified compiler allows us to solely rely on typechecking in ATS to discharge proof obligations originating from the need to typecheck Prelude code. Whereas ATS is typically used as a general purpose programming language, we hereby demonstrate that it can also be conveniently used to support some forms of advanced static checking in languages equipped with less expressive types.

  12. Crizotinib induces PUMA-dependent apoptosis in colon cancer cells.

    Science.gov (United States)

    Zheng, Xingnan; He, Kan; Zhang, Lin; Yu, Jian

    2013-05-01

    Oncogenic alterations in MET or anaplastic lymphoma kinase (ALK) have been identified in a variety of human cancers. Crizotinib (PF02341066) is a dual MET and ALK inhibitor and approved for the treatment of a subset of non-small cell lung carcinoma and in clinical development for other malignancies. Crizotinib can induce apoptosis in cancer cells, whereas the underlying mechanisms are not well understood. In this study, we found that crizotinib induces apoptosis in colon cancer cells through the BH3-only protein PUMA. In cells with wild-type p53, crizotinib induces rapid induction of PUMA and Bim accompanied by p53 stabilization and DNA damage response. The induction of PUMA and Bim is mediated largely by p53, and deficiency in PUMA or p53, but not Bim, blocks crizotinib-induced apoptosis. Interestingly, MET knockdown led to selective induction of PUMA, but not Bim or p53. Crizotinib also induced PUMA-dependent apoptosis in p53-deficient colon cancer cells and synergized with gefitinib or sorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and therapeutic responses to crizotinib in xenograft models. These results establish a critical role of PUMA in mediating apoptotic responses of colon cancer cells to crizotinib and suggest that mechanisms of oncogenic addiction to MET/ALK-mediated survival may be cell type-specific. These findings have important implications for future clinical development of crizotinib.

  13. Cell-Type-Specific Optogenetics in Monkeys.

    Science.gov (United States)

    Namboodiri, Vijay Mohan K; Stuber, Garret D

    2016-09-08

    The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys.

  14. Contact-dependent Stimulation and Inhibition of Dendritic Cells by Natural Killer Cells

    OpenAIRE

    Piccioli, Diego; Sbrana, Silverio; Melandri, Emiliano; Valiante, Nicholas M.

    2002-01-01

    Natural killer (NK) cells and dendritic cells (DCs) are two distinct cell types of innate immunity. It is known that the in vitro interaction of human NK cells with autologous DCs results in DC lysis. Here we show that contact-dependent interactions between activated human NK cells and immature DCs (iDCs) provides a “control switch” for the immune system. At low NK/DC ratios, this interaction dramatically amplifies DC responses, whereas at high ratios it completely turns off their responses. ...

  15. Localized Calcineurin Confers Ca2+-Dependent Inactivation Upon Neuronal L-Type Ca2+ Channels

    OpenAIRE

    2012-01-01

    Excitation-driven entry of Ca2+ through L-type voltage-gated Ca2+ channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of CaV1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI), which stro...

  16. Does the stellar disc flattening depend on the galaxy type?

    CERN Document Server

    Mosenkov, A V; Reshetnikov, V P; Bizyaev, D V; Kautsch, S J

    2015-01-01

    We analyze the dependence of the stellar disc flatness on the galaxy morphological type using 2D decomposition of galaxies from the reliable subsample of the Edge-on Galaxies in SDSS (EGIS) catalogue. Combining these data with the retrieved models of the edge-on galaxies from the Two Micron All Sky Survey (2MASS) and the Spitzer Survey of Stellar Structure in Galaxies (S$^4$G) catalogue, we make the following conclusions: (1) The disc relative thickness $z_0/h$ in the near- and mid-infrared passbands correlates weakly with morphological type and does not correlate with the bulge-to-total luminosity ratio $B/T$ in all studied bands. (2) Applying an 1D photometric profile analysis overestimates the disc thickness in galaxies with large bulges making an illusion of the relationship between the disc flattening and the ratio $B/T$. (3) In our sample the early-type disc galaxies (S0/a) have both flat and "puffed" discs. The early spirals and intermediate-type galaxies have a large scatter of the disc flatness, whic...

  17. A multiwell platform for studying stiffness-dependent cell biology.

    Directory of Open Access Journals (Sweden)

    Justin D Mih

    Full Text Available Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

  18. Involvement of IRF4 dependent dendritic cells in T cell dependent colitis

    DEFF Research Database (Denmark)

    Pool, Lieneke; Rivollier, Aymeric Marie Christian; Agace, William Winston

    in genetically susceptible individuals and pathogenic CD4+ T cells, which accumulate in the inflamed mucosa, are believed to be key drivers of the disease. While dendritic cells (DCs) are important in the priming of intestinal adaptive immunity and tolerance their role in the initiation and perpetuation...... of chronic intestinal inflammation remains unclear. In the current study we used the CD45RBhi T cell transfer model of colitis to determine the role of IRF4 dependent DCs in intestinal inflammation. In this model naïve CD4+ T cells when transferred into RAG-/- mice, proliferate and expand in response...... to bacterial derived luminal antigen, localize to the intestinal mucosa and induce colitis. Adoptive transfer of naïve T cells into CD11cCre.IRF4fl/fl.RAG-1-/- mice resulted in reduced monocyte recruitment to the intestine and mesenteric lymph nodes (MLN) compared to Cre- controls. Inflammatory cytokines...

  19. Practical Programming with Higher-Order Encodings and Dependent Types

    DEFF Research Database (Denmark)

    Poswolsky, Adam; Schürmann, Carsten

    2008-01-01

    Higher-order abstract syntax (HOAS) refers to the technique of representing variables of an object-language using variables of a meta-language. The standard first-order alternatives force the programmer to deal with superficial concerns such as substitutions, whose implementation is often routine......, tedious, and error-prone. In this paper, we describe the underlying calculus of Delphin. Delphin is a fully implemented functional-programming language supporting reasoning over higher-order encodings and dependent types, while maintaining the benefits of HOAS. More specifically, just as representations...... utilizing HOAS free the programmer from concerns of handling explicit contexts and substitutions, our system permits programming over such encodings without making these constructs explicit, leading to concise and elegant programs. To this end our system distinguishes bindings of variables intended...

  20. Gap Dependent Rheology in Type I Collagen Gels

    Science.gov (United States)

    Arevalo, Richard; Urbach, Jeffrey; Blair, Daniel

    2010-03-01

    Branched type I collagen fiber networks provide extracellular support in mammalian tissues. The intricate network structure can succumb to partial or complete tearing under sufficient applied strain. Under small shear strains, in vitro collagen gels exhibit strain-stiffening while maintaining overall network integrity. Higher shear strains lead to network failure through discrete yielding events. We perform rheology and confocal-rheology experiments to fully elucidate the strain-stiffening and yielding behavior in these highly nonlinear materials. We apply continuous shear strains to collagen gels confined within the rheometer at fixed gaps. We observe that sheared collagen in the strain-stiffening and yielding regime has an apparent modulus that is strongly dependent on the collagen thickness. Moreover, we demonstrate that network yielding is universally controlled by the ratio of the collagen thickness to the mesh size. These results have broad implications for the interpretation of rheological data of extracellular matrix proteins and for the design of biomimetic scaffolds.

  1. Industrial n-type solar cells with >20% cell efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Romijn, I.G.; Anker, J.; Burgers, A.R.; Gutjahr, A.; Koppes, M.; Kossen, E.J.; Lamers, M.W.P.E.; Heurtault, Benoit; Saynova-Oosterling, D.S.; Tool, C.J.J. [ECN Solar Energy, Petten (Netherlands)

    2013-03-15

    To realize high efficiencies at low costs, ECN has developed the n-Pasha solar cell concept. The n-Pasha cell concept is a bifacial solar cell concept on n-Cz base material, with which average efficiencies of above 20% have been demonstrated. In this paper recent developments at ECN to improve the cost of ownership (lower Euro/Wp) of the n-Pasha cell concept are discussed. Two main drivers for the manufacturing costs of n-type solar cells are addressed: the n-type Cz silicon material and the silver consumption. We show that a large resistivity range between 2 and 8 cm can be tolerated for high cell efficiency, and that the costs due to the silver metallization can be significantly reduced while increasing the solar cell efficiency. Combining the improved efficiency and cost reduction makes the n-Pasha cell concept a very cost effective solution to manufacture high efficient solar cells and modules.

  2. Temperature Dependences on Various Types of Photovoltaic (PV) Panel

    Science.gov (United States)

    Audwinto, I. A.; Leong, C. S.; Sopian, K.; Zaidi, S. H.

    2015-09-01

    Temperature is one of the key roles in PV technology performance, since with the increases of temperature the open-circuit voltage will drop accordingly so do the electrical efficiency and power output generation. Different types of Photovoltaic (PV) panels- silicon solar panels and thin film solar panels; mono-crystalline, poly-crystalline, CIS, CIGS, CdTe, back-contact, and bi-facial solar panel under 40°C to 70°C approximately with 5°C interval have been comparatively analyzed their actual performances with uniformly distribution of light illumination from tungsten halogen light source, ±500W/m2. DC-Electronic Load and Data Logger devices with “Lab View” data program interface were used to collect all the necessary parameters in this study. Time needed to achieve a certain degree of temperature was recorded. Generally, each of the panels needed 15 minutes to 20 minutes to reach 70°C. Halogen based light source is not compatible in short wave-length in response to thin-film solar cell. Within this period of times, all the panels are facing a performance loss up to 15%. Other parameters; Pmax, Vmax, Imax, Voc, Isc, Rserries, Rshunt, Fillfactor were collected as study cases. Our study is important in determining Photovoltaic type selection and system design as for study or industrial needed under different temperature condition.

  3. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    Science.gov (United States)

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  4. B cell autophagy mediates TLR7-dependent autoimmunity and inflammation.

    Science.gov (United States)

    Weindel, Chi G; Richey, Lauren J; Bolland, Silvia; Mehta, Abhiruchi J; Kearney, John F; Huber, Brigitte T

    2015-01-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5(f/f)). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.

  5. The expression of hyperpolarization activated cyclic nucleotide gated (HCN channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation

    Directory of Open Access Journals (Sweden)

    Page Carly

    2008-08-01

    Full Text Available Abstract Background Aim of this study was to test the hypothesis that levels of hyperpolarization activated cyclic nucleotide gated channels 1 to 4 (HCN1-4 are linked to the reproductive age of the ovary. Methods Young, adult, and reproductively aged ovaries were collected from Sprague-Dawley rats. RT-PCR and western blot analysis of ovaries was performed to investigate the presence of mRNA and total protein for HCN1-4. Immunohistochemistry with semiquantitative H score analysis was performed using whole ovarian histologic sections. Results RT-PCR analysis showed the presence of mRNA for HCN1-4. Western blot analysis revealed HCN1-3 proteins in all ages of ovarian tissues. Immunohistochemistry with H score analysis demonstrated distinct age-related changes in patterns of HCN1-3 in the oocytes, granulosa cells, theca cells, and corpora lutea. HCN4 was present only in the oocytes, with declining levels during the reproduction lifespan. Conclusion The evidence presented here demonstrates cell-type and developmental age patterns of HCN1-4 channel expression in rat ovaries. Based on this, we hypothesize that HCN channels have functional significance in rat ovaries and may have changing roles in reproductive aging.

  6. Fuel cells - Fundamentals and types: Unique features

    Science.gov (United States)

    Selman, J. R.

    An overview of the working principles, thermodynamic efficiencies, types, and engineering aspects of fuel cells is presented. It is noted that fuel cells are distinguished from other direct energy conversion devices by the existence of charge separation at the electrodes involving ions in an electrolyte. The electrical energy produced by a fuel cell is shown to be equal to the change in the free energy of the reactants, and thermodynamic balances of reactions in different fuel cells are provided. The production of electricity in the discharge mode involves a spontaneous reaction of overproduction of electrons at the anode and consumption of the electrons at the cathode, with the total ionic current being equal to the electronic current in the external circuit. Attention is given to the operations and problems of acid, alkaline, molten carbonate, and solid oxide fuel cells, in addition to applications of electro-organic fuel cells.

  7. Stress factor – dependent differences in molecular mechanisms of premature cell senescence

    Directory of Open Access Journals (Sweden)

    Petrova N. V.

    2015-10-01

    Full Text Available Cell senescence is an established cell stress response in the form of a permanent proliferation arrest accompanied by a complex phenotype. Senescent cells share several crucial features, such as lack of DNA synthesis, increased senescence-associated β-galactosidase activity and upregulation of cyclin-dependent kinase inhibitors. Most of these universal senescence markers are indicative not only for cell senescence but for other types of growth arrest as well. Along with ubiquitous markers, cell senescence has accessory characteristics, which mostly depend on senescence-inducing stimulus and/or cell type. Here, we review main markers and mechanisms involved in the induction of cell senescence with a focus on stress factor-dependent differences in signaling pathways activated in senescence.

  8. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

    Science.gov (United States)

    Hoorweg, Kerim; Narang, Priyanka; Li, Zhi; Thuery, Anne; Papazian, Natalie; Withers, David R; Coles, Mark C; Cupedo, Tom

    2015-11-01

    Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs.

  9. Glycosynapses: microdomains controlling carbohydrate-dependent cell adhesion and signaling

    Directory of Open Access Journals (Sweden)

    Senitiroh Hakomori

    2004-09-01

    Full Text Available The concept of microdomains in plasma membranes was developed over two decades, following observation of polarity of membrane based on clustering of specific membrane components. Microdomains involved in carbohydrate-dependent cell adhesion with concurrent signal transduction that affect cellular phenotype are termed "glycosynapse". Three types of glycosynapse have been distinguished: "type 1" having glycosphingolipid associated with signal transducers (small G-proteins, cSrc, Src family kinases and proteolipids; "type 2" having O-linked mucin-type glycoprotein associated with Src family kinases; and "type 3" having N-linked integrin receptor complexed with tetraspanin and ganglioside. Different cell types are characterized by presence of specific types of glycosynapse or their combinations, whose adhesion induces signal transduction to either facilitate or inhibit signaling. E.g., signaling through type 3 glycosynapse inhibits cell motility and differentiation. Glycosynapses are distinct from classically-known microdomains termed "caveolae", "caveolar membrane", or more recently "lipid raft", which are not involved in carbohydrate-dependent cell adhesion. Type 1 and type 3 glycosynapses are resistant to cholesterol-binding reagents, whereas structure and function of "caveolar membrane" or "lipid raft" are disrupted by these reagents. Various data indicate a functional role of glycosynapses during differentiation, development, and oncogenic transformation.O conceito de microdomínios em membrana plasmática foi desenvolvido há mais de duas décadas, após a observação da polaridade da membrana baseada no agrupamento de componentes específicos da membrana. Microdomínios envolvidos na adesão celular dependente de carboidrato, com transdução de sinal que afeta o fenótipo celular são denominados ''glicosinapses''. Três tipos de glicosinapse foram observados: ''tipo 1'' que possue glicoesfingolipídio associado com transdutores de sinal

  10. The quest for targets executing MYC-dependent cell transformation

    Directory of Open Access Journals (Sweden)

    Markus eHartl

    2016-06-01

    Full Text Available MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than forty upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, for determination which of the known, or yet unidentified targets are responsible for processing the oncogenic MYC program, further systematic and selective approaches are required. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets.Knowledge about essential MYC regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated

  11. Concentration dependent effects of tobacco particulates from different types of cigarettes on expression of drug metabolizing proteins, and benzo(a)pyrene metabolism in primary normal human oral epithelial cells.

    Science.gov (United States)

    Sacks, Peter G; Zhao, Zhong-Lin; Kosinska, Wieslawa; Fleisher, Kenneth E; Gordon, Terry; Guttenplan, Joseph B

    2011-09-01

    The ability of tobacco smoke (TS) to modulate phase I and II enzymes and affect metabolism of tobacco carcinogens is likely an important factor in its carcinogenicity. For the first time several types of TS particulates (TSP) were compared in different primary cultured human oral epithelial cells (NOE) for their abilities to affect metabolism of the tobacco carcinogen, (BaP) to genotoxic products, and expression of drug metabolizing enzymes. TSP from, reference filtered (2RF4), mentholated (MS), reference unfiltered, (IR3), ultra low tar (UL), and cigarettes that primarily heat tobacco (ECL) were tested. Cells pretreated with TSP concentrations of 0.2-10 μg/ml generally showed increased rates of BaP metabolism; those treated with TSP concentrations above 10 μg/ml showed decreased rates. Effects of TSPs were similar when expressed on a weight basis. Weights of TSP/cigarette varied in the order: MS≈IR3>2RF4>ECL>UL. All TSPs induced the phase I proteins, cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), phase II proteins, NAD(P)H dehydrogenase quinone 1 (NQO1), and microsomal glutathione S-transferase 1 (MGST1), and additionally, hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2), as assessed by qRT-PCR. The pattern of gene induction at probable physiological levels favored activation over detoxification.

  12. Infection of A549 human type II epithelial cells with Mycobacterium tuberculosis induces changes in mitochondrial morphology, distribution and mass that are dependent on the early secreted antigen, ESAT-6.

    Science.gov (United States)

    Fine-Coulson, Kari; Giguère, Steeve; Quinn, Frederick D; Reaves, Barbara J

    2015-10-01

    Pulmonary infection by Mycobacterium tuberculosis (Mtb) involves the invasion of alveolar epithelial cells (AECs). We used Mitotracker Red(®) to assess changes in mitochondrial morphology/distribution and mass from 6 to 48 h post infection (hpi) by confocal microscopy and flow cytometry in Mtb-infected A549 type II AECs. During early infection there was no effect on mitochondrial morphology, however, by 48 hpi mitochondria appeared fragmented and concentrated around the nucleus. In flow cytometry experiments, the median fluorescence intensity (MFI) decreased by 44% at 48 hpi; double-labelling using antibodies to the integral membrane protein COXIV revealed that these changes were due to a decrease in mitochondrial mass. These changes did not occur with the apathogenic strain, Mycobacterium bovis BCG. ESAT-6 is a virulence factor present in Mtb Erdman but lacking in M. bovis BCG. We performed similar experiments using Mtb Erdman, an ESAT-6 deletion mutant and its complement. MFI decreased at 48 hpi in the parent and complemented strains versus uninfected controls by 52% and 36% respectively; no decrease was detected in the deletion mutant. These results indicate an involvement of ESAT-6 in the perturbation of mitochondria induced by virulent Mtb in AECs and suggest mitophagy may play a role in the infection process.

  13. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways.

  14. Regulation of the type Mb sodium-dependent phosphate cotransporter expression in the intestine

    Institute of Scientific and Technical Information of China (English)

    Bin WANG; Yulong YIN

    2009-01-01

    Phosphate (Pi) plays important roles in growth, development, bone mineralization, energy metabolism, nucleic acid synthesis, cell signaling, and acid-base regulation. The rate of intestinal absorption of Pi is a major determinant of Pi homeostasis. The type lib sodium- dependent Pi cotransporter (NaPi-Iib) is responsible for intestinal Pi absorption. Many physiological factors regulate the rate of Pi absorption via modulating the expression of NaPi-Iib in the intestine. In this review, we summarize the role of these factors in the regulation of NaPi-Iib expression in the intestine.

  15. Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Hirai

    Full Text Available Human induced pluripotent stem cells (iPSCs can be divided into a leukemia inhibitory factor (LIF-dependent naïve type and a basic fibroblast growth factor (bFGF-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

  16. Cell type-specific bipolar cell input to ganglion cells in the mouse retina.

    Science.gov (United States)

    Neumann, S; Hüser, L; Ondreka, K; Auler, N; Haverkamp, S

    2016-03-01

    Many distinct ganglion cell types, which are the output elements of the retina, were found to encode for specific features of a visual scene such as contrast, color information or movement. The detailed composition of retinal circuits leading to this tuning of retinal ganglion cells, however, is apart from some prominent examples, largely unknown. Here we aimed to investigate if ganglion cell types in the mouse retina receive selective input from specific bipolar cell types or if they sample their synaptic input non-selectively from all bipolar cell types stratifying within their dendritic tree. To address this question we took an anatomical approach and immunolabeled retinae of two transgenic mouse lines (GFP-O and JAM-B) with markers for ribbon synapses and type 2 bipolar cells. We morphologically identified all green fluorescent protein (GFP)-expressing ganglion cell types, which co-stratified with type 2 bipolar cells and assessed the total number of bipolar input synapses and the proportion of synapses deriving from type 2 bipolar cells. Only JAM-B ganglion cells received synaptic input preferentially from bipolar cell types other than type 2 bipolar cells whereas the other analyzed ganglion cell types sampled their bipolar input most likely from all bipolar cell terminals within their dendritic arbor.

  17. Epidemiology of type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Green, Anders

    1999-01-01

    Recent estimates suggest that more than 100,000 inhabitants in the Middle East suffer from type 1 diabetes and that about 6000 subjects in the region develop the disease each year. This paper illustrates how epidemiological principles and methods may assist in a rational assessment of the public...... health impact of type 1 diabetes in the Middle East. Making a series of assumptions, it is estimated that the future prevalence of type 1 diabetes in the region will increase slightly, but that the increase may be more pronounced if the disease incidence is increasing and the prognosis improved....... It is recommended that more valid information is established on the basic epidemiological features of type 1 diabetes in the Middle East, as this will provide the basis of more rational planning of the current and future diabetes healthcare in the region....

  18. Cell cycle-dependent gene networks relevant to cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The analysis of sophisticated interplays between cell cycle-dependent genes in a disease condition is one of the largely unexplored areas in modern tumor biology research. Many cell cycle-dependent genes are either oncogenes or suppressor genes, or are closely asso- ciated with the transition of a cell cycle. However, it is unclear how the complicated relationships between these cell cycle-dependent genes are, especially in cancers. Here, we sought to identify significant expression relationships between cell cycle-dependent genes by analyzing a HeLa microarray dataset using a local alignment algorithm and constructed a gene transcriptional network specific to the cancer by assembling these newly identified gene-gene relationships. We further characterized this global network by partitioning the whole network into several cell cycle phase-specific sub-networks. All generated networks exhibited the power-law node-degree dis- tribution, and the average clustering coefficients of these networks were remarkably higher than those of pure scale-free networks, indi- cating a property of hierarchical modularity. Based on the known protein-protein interactions and Gene Ontology annotation data, the proteins encoded by cell cycle-dependent interacting genes tended to share the same biological functions or to be involved in the same biological processes, rather than interacting by physical means. Finally, we identified the hub genes related to cancer based on the topo- logical importance that maintain the basic structure of cell cycle-dependent gene networks.

  19. Illusory Licensing Effects across Dependency Types: ERP Evidence

    Science.gov (United States)

    Xiang, Ming; Dillon, Brian; Phillips, Colin

    2009-01-01

    A number of recent studies have argued that grammatical illusions can arise in the process of completing linguistic dependencies, such that unlicensed material is temporarily treated as licensed due to the presence of a potential licensor that is semantically appropriate but in a syntactically inappropriate position. A frequently studied case…

  20. Rate dependence of cell-to-cell variations of lithium-ion cells

    Science.gov (United States)

    An, Fuqiang; Chen, Lufan; Huang, Jun; Zhang, Jianbo; Li, Ping

    2016-10-01

    Lithium-ion cells are commonly used in a multicell configuration in power devices and electric vehicles, making the cell-to-cell variation (CtCV) a key factor to consider in system design and management. Previous studies on CtCV have two major limitations: the number of cells is usually less than one hundred, and the cells are usually commercial cells already subjected to cell-screenings. In this article, we first make a statistical analysis on the CtCV of 5473 fresh cells from an automotive battery manufacturer before the cell-screening process. Secondly, 198 cells are randomly selected from these 5473 cells and the rate dependence of the CtCV is examined, focusing on the correlations of capacity versus weight and capacity versus resistance, corresponding to thermodynamic and kinetic factors, respectively. The rate dependence of these two correlations is explained from a phenomenological model. Finally, eight cells from the 198 cells are further characterized with electrochemical impedance spectroscopy method to elucidate the kinetic origins of the CtCV.

  1. The Cloninger Type I/Type II Typology: Configurations and Personality Profiles in Socially Stable Alcohol Dependent Patients

    Directory of Open Access Journals (Sweden)

    Peter Wennberg

    2014-01-01

    Full Text Available Many attempts have been made to derive alcohol use typologies or subtypes of alcohol dependence and this study aimed at validating the type I/type II typology in a treatment sample of socially stable alcohol dependent males and females. A second aim was to compare the two types with respect to their temperament profiles. Data was part of a larger ongoing longitudinal study, the Gothenburg Alcohol Research Project, and included 269 alcohol dependent males and females recruited from three treatment centers. The results showed that type II alcoholism occurred as a more homogenous type than type I alcoholism, and type I alcoholism seemed too heterogeneous to be summarized into one single type. When adapting a strict classification, less than a third of the study population could be classified in accordance with the typology, suggesting that the typology is not applicable, at least in socially stable individuals with alcohol dependence. The results also showed that type II alcoholics showed higher levels of novelty seeking than did the individuals that were classified as type I alcoholics. Quite surprisingly, the individuals classified as type II alcoholics also showed higher levels of harm avoidance than did the individuals that were classified as type I alcoholics.

  2. Exercise-Dependent Regulation of NK Cells in Cancer Protection

    DEFF Research Database (Denmark)

    Idorn, Manja; Hojman, Pernille

    2016-01-01

    Natural killer (NK) cells are the most responsive immune cells to exercise, displaying an acute mobilization to the circulation during physical exertion. Recently, exercise-dependent mobilization of NK cells was found to play a central role in exercise-mediated protection against cancer. Here, we...... a mechanistic explanation for the protective effect of exercise on cancer, and we propose that exercise represents a potential strategy as adjuvant therapy in cancer, by improving NK cell recruitment and infiltration in solid tumors....

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

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

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

  4. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration

    OpenAIRE

    Howe, Alan K.; Baldor, Linda C.; Hogan, Brian P.

    2005-01-01

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures ...

  5. The Morphological Type Dependence of K-band Luminosity Functions

    CERN Document Server

    Devereux, Nick; Willner, S P; Ashby, M L N; Willmer, C N A

    2009-01-01

    Differential 2.2um (K-band) luminosity functions are presented for a complete sample of 1570 nearby Vgsr < 3000 km/s, where Vgsr is the velocity measured with respect to the Galactic standard of rest), bright (K < 10 mag), galaxies segregated by visible morphology. The K-band luminosity function for late-type spirals follows a power law that rises towards low luminosities whereas the K-band luminosity functions for ellipticals, lenticulars and bulge-dominated spirals are peaked with a fall off at both high and low luminosities. However, each morphological type (E, S0, S0/a-Sab, Sb-Sbc, Sc-Scd) contributes approximately equally to the overall K-band luminosity density in the local universe, and by inference, the stellar mass density as well.

  6. Identity information content depends on the type of facial movement

    Science.gov (United States)

    Dobs, Katharina; Bülthoff, Isabelle; Schultz, Johannes

    2016-09-01

    Facial movements convey information about many social cues, including identity. However, how much information about a person’s identity is conveyed by different kinds of facial movements is unknown. We addressed this question using a recent motion capture and animation system, with which we animated one avatar head with facial movements of three types: (1) emotional, (2) emotional in social interaction and (3) conversational, all recorded from several actors. In a delayed match-to-sample task, observers were best at matching actor identity across conversational movements, worse with emotional movements in social interactions, and at chance level with emotional facial expressions. Model observers performing this task showed similar performance profiles, indicating that performance variation was due to differences in information content, rather than processing. Our results suggest that conversational facial movements transmit more dynamic identity information than emotional facial expressions, thus suggesting different functional roles and processing mechanisms for different types of facial motion.

  7. The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

    Science.gov (United States)

    Vogelweith, Fanny; Moret, Yannick; Monceau, Karine; Thiéry, Denis; Moreau, Jérôme

    2016-05-01

    Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes.

  8. Genomic Typing of Red Cell Antigens

    Science.gov (United States)

    2011-09-01

    Antigen‐Matched  Red  Cells  for  Sickle  Cell  Anemia   Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...Storry JR &  Delaney, M. CASE  REPORT OF TESTING AND MANAGEMENT OF BOMBAY  (OH)  PREGNANCY . XXXIst  International  Congress of the ISBT, Berlin, Germany...JK null Phenotype (Manuscript in preparation).  5. Delaney M et al: A Genetic Impossibility? Case report of Bombay (Oh)  pregnancy  with Group AB

  9. Moving hot cell for LMFBR type reactor

    Energy Technology Data Exchange (ETDEWEB)

    Kanbe, Mitsuru

    1994-09-16

    A moving hot cell for an LMFBR type reactor is made movable on a reactor operation floor between a position just above the reactor container and a position retreated therefrom. Further, it comprises an overhung portion which can incorporate a spent fuel just thereunder, and a crane for moving a fuel assembly between a spent fuel cask and a reactor container. Further, an opening/closing means having a shielding structure is disposed to the bottom portion and the overhung portion thereof, to provide a sealing structure, in which only the receiving port for the spent fuel cask faces to the inner side, and the cask itself is disposed at the outside. Upon exchange of fuels, the movable hot cell is placed just above the reactor to take out the spent fuels, so that a region contaminated with primary sodium is limited within the hot cell. On the other hand, upon maintenance and repair for equipments, the hot cell is moved, thereby enabling to provide a not contaminated reactor operation floor. (N.H.).

  10. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism

    Directory of Open Access Journals (Sweden)

    J Kang

    2014-11-01

    Full Text Available Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP-linked basic fibroblast growth factor (FGF2-immobilised polystyrene surface (PS-MBP-FGF2 was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC behaviour in response to two different mechanisms of cell adhesion; (i FGF2-heparan sulphate proteoglycan (HSPG-mediated adhesion vs. (ii fibronectin (FN-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  11. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism.

    Science.gov (United States)

    Kang, J; Park, H M; Kim, Y W; Kim, Y H; Varghese, S; Seok, H K; Kim, Y G; Kim, S H

    2014-11-25

    Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP)-linked basic fibroblast growth factor (FGF2)-immobilised polystyrene surface (PS-MBP-FGF2) was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC) behaviour in response to two different mechanisms of cell adhesion; (i) FGF2-heparan sulphate proteoglycan (HSPG)-mediated adhesion vs. (ii) fibronectin (FN)-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS)-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  12. A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

    Directory of Open Access Journals (Sweden)

    Amy B. Howell

    2011-03-01

    Full Text Available This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP staining, while the secretion of interleukin-8 (IL-8 and matrix metalloproteinases (MMPs was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

  13. Endonuclease specificity and sequence dependence of type IIS restriction enzymes.

    Science.gov (United States)

    Lundin, Sverker; Jemt, Anders; Terje-Hegge, Finn; Foam, Napoleon; Pettersson, Erik; Käller, Max; Wirta, Valtteri; Lexow, Preben; Lundeberg, Joakim

    2015-01-01

    Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  14. Endonuclease specificity and sequence dependence of type IIS restriction enzymes.

    Directory of Open Access Journals (Sweden)

    Sverker Lundin

    Full Text Available Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI. We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  15. The Dependence of Galaxy Type on Host Halo Mass

    CERN Document Server

    Weinmann, S M; Yang, X; Mo, H J; Weinmann, Simone M.; Bosch, Frank C. van den; Yang, Xiaohu

    2006-01-01

    We examine the relation between galaxy properties and environment in the SDSS DR2, quantifying environment in terms of the mass of the host halo, which is obtained with a new iterative group finder. We find that galaxy type fractions scale strongly and smoothly with halo mass, but, at fixed mass, not with luminosity. We compare these findings with the semi-analytical galaxy formation model of Croton et al. (2006). The discrepancies we find can be explained with an oversimplified implementation of strangulation, the neglect of tidal stripping, and shortcomings in the treatments of dust extinction and/or AGN feedback.

  16. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

    Science.gov (United States)

    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  17. Frequency- dependent cell responses to an electromagnetic stimulus

    Science.gov (United States)

    Taghian, Toloo; Sheikh, Abdul; Narmoneva, Daria; Kogan, Andrei

    2013-03-01

    External electric field (EF) acting on cells in the ionic environment can trigger a variety of mechanical and chemical cell responses that regulate cell functions, such as adhesion, migration and cell signaling; thus manipulation of EF can be used in therapeutic applications. To optimize this process, realistic studies of EF interaction with cells are essential. We have developed a combined theoretical-experimental approach to study cell response to the external EF in the native configuration. The cell is modeled as a membrane-enclosed hemisphere which is cultured on a substrate and is surrounded by electrolyte. Maxwell's equations are solved numerically (ANSYS-HFSS) to obtain 3D EF distribution inside and near the cell subjected to an external EF. Theoretical results indicate that the cell response is frequency dependent, where at low frequency EF is excluded from the cell interior while EF penetration into the cell increases for higher frequencies. In both regimes the spatial distribution and strength of induced EF in membrane varies with frequency. Experimental results are consistent with theoretical predictions and show frequency-dependent cell response, including both membrane-initiated and intracellular pathway activation and growth factor release. The authors acknowledge the financial support from the NSF (DMR-1206784 & DMR-0804199 to AK); the NIH (1R21 DK078814-01A1 to DN) and the University of Cincinnati (Interdisciplinary Faculty Research Support Grant to DN and AK).

  18. Pressure Dependent Wall Relaxation in Polarized $^3$He Gaseous Cells

    CERN Document Server

    Peng, C; Chu, P -H; Gao, H; Zhang, Y

    2013-01-01

    Pressure dependence of longitudinal relaxation time (T$_1$) due to the cell wall was observed previously at both room temperature and low temperature in valved Rb-coated refillable $^3$He gaseous cells in \\cite{Zheng2}. The diffusion of $^3$He from measurement cell through a capillary tube to the valve and the subsequent depolarization on the surface of the valve was proposed to possibly explain such a pressure dependence at room temperature \\cite{Saam}. In this paper, we investigate this diffusion effect through measurements of T$_1$ with newly designed Rb-coated Pyrex glass cells at 295 K as well as finite element analysis (FEA) studies. Both the experimental results and FEA studies show that the diffusion effect is insufficient to explain the observed linear pressure-dependent behavior of T$_1$.

  19. Auxin regulates SNARE-dependent vacuolar morphology restricting cell size.

    Science.gov (United States)

    Löfke, Christian; Dünser, Kai; Scheuring, David; Kleine-Vehn, Jürgen

    2015-03-05

    The control of cellular growth is central to multicellular patterning. In plants, the encapsulating cell wall literally binds neighbouring cells to each other and limits cellular sliding/migration. In contrast to its developmental importance, growth regulation is poorly understood in plants. Here, we reveal that the phytohormone auxin impacts on the shape of the biggest plant organelle, the vacuole. TIR1/AFBs-dependent auxin signalling posttranslationally controls the protein abundance of vacuolar SNARE components. Genetic and pharmacological interference with the auxin effect on vacuolar SNAREs interrelates with auxin-resistant vacuolar morphogenesis and cell size regulation. Vacuolar SNARE VTI11 is strictly required for auxin-reliant vacuolar morphogenesis and loss of function renders cells largely insensitive to auxin-dependent growth inhibition. Our data suggests that the adaptation of SNARE-dependent vacuolar morphogenesis allows auxin to limit cellular expansion, contributing to root organ growth rates.

  20. Effects of arsenic trioxide on voltage-dependent potassium channels and on cell proliferation of human multiple myeloma cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jin; WANG Wei; WEI Qing-fang; FENG Tie-ming; TAN Li-jun; YANG Bao-feng

    2007-01-01

    @@ Arsenic trioxide (ATO) can induce cellular apoptosis and inhibit the activities of multiple myeloma (MM)cells in vitro,1 but how it works is not very clear. Recent studies showed that ATO worked on the voltagedependent potassium channel and L-type calcium channel in myocardial cells,2-5 but the effect of ATO on ion channels of tumor cells was rarely reported. As the potassium channel plays an important role in controlling cell proliferation,6 we studied the effects of ATO on the voltage-dependent potassium current (Ikv) of the voltage-dependent potassium channel in an MM cell line,and probed into the relationship between changes of the Ikv caused by ATO and cell proliferation.

  1. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    Science.gov (United States)

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  2. Rapid changes in plasma androgens during insulin withdrawal in male type 1 (insulin-dependent) diabetics

    DEFF Research Database (Denmark)

    Madsbad, S; Gluud, C; Bennett, Patrick

    1986-01-01

    Plasma concentrations of testosterone, androstenedione and dihydrotestosterone were measured in 15 Type 1 (insulin-dependent) diabetics with (n = 8) and without (n = 7) B-cell function during 12 h of insulin withdrawal and compared with those of 8 normal subjects. Before insulin withdrawal...... no significant difference was found in androgen concentrations between the diabetic and the normal subjects. The normal diurnal profiles, with highest androgen concentrations in the morning before insulin withdrawal (08:00) and lowest concentrations at 20:00 h were maintained in the diabetics. However......, testosterone and dihydrotestosterone concentrations were lower in the diabetics after 4 h of insulin withdrawal and remained so throughout the study. The concentrations of androstenedione were not significantly different between diabetics and normal subjects except after 4 h of insulin withdrawal. Despite...

  3. Calcium dependent CAMTA1 in adult stem cell commitment to a myocardial lineage.

    Directory of Open Access Journals (Sweden)

    Barbara Muller-Borer

    Full Text Available The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+ oscillations in the stem cells that are synchronous with the Ca(2+ transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+ signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+ signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+-dependent intermediate, up-regulation of CAMTA1.

  4. T lymphocyte subsets in patients with newly diagnosed type 1 (insulin-dependent) diabetes

    DEFF Research Database (Denmark)

    Buschard, K; Röpke, C; Madsbad, S

    1983-01-01

    T lymphocyte subsets in peripheral blood from 11 newly diagnosed Type 1 (insulin-dependent) diabetic patients were studied prospectively at three time intervals: as soon as possible after diagnosis, 3 weeks and 5 months later. Lymphocytes were marked with monoclonal OKT antibodies and examined...... in a fluorescence-activated cell sorter. The percentage of T lymphocytes (OKT3) did not change significantly at the three study times. The percentage of helper/inducer T cells (OKT4) was high the first week after diagnosis, but decreased at the 5-month examination (p less than 0.05). The percentage of suppressor....../cytotoxic T cells (OKT8) was low at diagnosis but increased at 3 weeks (p less than 0.02) and 5 months (p less than 0.01). The ratio OKT4/OKT8 lymphocytes was 2.28 at diagnosis, decreasing to 1.77 at 3 weeks and 1.87 at 5 months, compared with 1.46 for 16 age-matched control subjects. There was no significant...

  5. Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

    Science.gov (United States)

    Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Sinha, Sunilima; Alexenko, Andrei P; Spate, Lee; Prather, Randall S; Roberts, R Michael

    2011-08-19

    The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

  6. A Higher—Order Unification Algorithm for Inductive Types and Dependent Types

    Institute of Scientific and Technical Information of China (English)

    谭庆平

    1997-01-01

    is paper presents a method to define a set of mutually recursive inductive types,and develops a higher-order unification algorithm for λП∑ extended with inductive types.The algorithm is an extension of Elliott's algorithm for λП∑.The notaiton of normal forms plays a vital role in higher-order unification.The weak head normal forms in the extended type theory is defined to reveal the ultimate “top level structures”of the fully normalized terms and types.Unification transformation rules are designed to deal with inductive types,a recursive operator and its reduction rule.The algorithm can construct recursive functions automatically.

  7. Cell fate determination by ubiquitin-dependent regulation of translation

    Science.gov (United States)

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  8. Distance-dependent homeostatic synaptic scaling mediated by A-type potassium channels

    Directory of Open Access Journals (Sweden)

    Hiroshi T Ito

    2009-11-01

    Full Text Available Many lines of evidence suggest that the efficacy of synapses on CA1 pyramidal neuron dendrites increases as a function of distance from the cell body. The strength of an individual synapse is also dynamically modulated by activity-dependent synaptic plasticity, which raises the question as to how a neuron can reconcile individual synaptic changes with the maintenance of the proximal-to-distal gradient of synaptic strength along the dendrites. As the density of A-type potassium channels exhibits a similar gradient from proximal (low-to-distal (high dendrites, the A-current may play a role in coordinating local synaptic changes with the global synaptic strength gradient. Here we describe a form of homeostatic plasticity elicited by conventional activity blockade (with TTX coupled with a block of the A-type potassium channel. Following A-type potassium channel inhibition for 12 hrs, recordings from CA1 somata revealed a significantly higher miniature excitatory postsynaptic current (mEPSC frequency, whereas in dendritic recordings, there was no change in mEPSC frequency. Consistent with mEPSC recordings, we observed a significant increase in AMPA receptor density in stratum pyramidale but not stratum radiatum. Based on these data, we propose that the differential distribution of A-type potassium channels along the apical dendrites may create a proximal-to-distal membrane potential gradient. This gradient may regulate AMPA receptor distribution along the same axis. Taken together, our results indicate that A-type potassium channels play an important role in controlling synaptic strength along the dendrites, which may help to maintain the computational capacity of the neuron.

  9. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Science.gov (United States)

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  10. Cell type-specific neuroprotective activity of untranslocated prion protein.

    Directory of Open Access Journals (Sweden)

    Elena Restelli

    Full Text Available BACKGROUND: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. PRINCIPAL FINDINGS: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. SIGNIFICANCE: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

  11. T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner.

    Science.gov (United States)

    Boes, Marianne; Bertho, Nicolas; Cerny, Jan; Op den Brouw, Marjolein; Kirchhausen, Tomas; Ploegh, Hidde

    2003-10-15

    Interaction of Ag-loaded dendritic cells with Ag-specific CD4 T cells induces the formation of long tubular class II MHC-positive compartments that polarize toward the T cell. We show involvement of a Toll-like receptor-mediated signal in this unusual form of intracellular class II MHC trafficking. First, wild-type dendritic cells loaded with LPS-free Ag failed to show formation of class II-positive tubules upon Ag-specific T cell engagement, but did so upon supplementation of the Ag with low concentrations of LPS. Second, Ag-loaded myeloid differentiation factor 88 -deficient dendritic cells failed to form these tubules upon interaction with T cells, regardless of the presence of LPS. Finally, inclusion of a cell-permeable peptide that blocks TNFR-associated factor 6 function, downstream of myeloid differentiation factor 88, blocked T cell-dependent tubulation. A Toll-like receptor-dependent signal is thus required to allow Ag-loaded dendritic cells to respond to T cell contact by formation of extended endosomal compartments. This activation does not result in massive translocation of class II MHC molecules to the cell surface.

  12. Dependent Neyman type A processes based on common shock Poisson approach

    Science.gov (United States)

    Kadilar, Gamze Özel; Kadilar, Cem

    2016-04-01

    The Neyman type A process is used for describing clustered data since the Poisson process is insufficient for clustering of events. In a multivariate setting, there may be dependencies between multivarite Neyman type A processes. In this study, dependent form of the Neyman type A process is considered under common shock approach. Then, the joint probability function are derived for the dependent Neyman type A Poisson processes. Then, an application based on forest fires in Turkey are given. The results show that the joint probability function of the dependent Neyman type A processes, which is obtained in this study, can be a good tool for the probabilistic fitness for the total number of burned trees in Turkey.

  13. Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

    Science.gov (United States)

    Phiboonchaiyanan, Preeyaporn Plaimee; Kiratipaiboon, Chayanin; Chanvorachote, Pithi

    2016-04-25

    Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients.

  14. Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks

    Science.gov (United States)

    Huang, S.; Ingber, D. E.

    2000-01-01

    Development of characteristic tissue patterns requires that individual cells be switched locally between different phenotypes or "fates;" while one cell may proliferate, its neighbors may differentiate or die. Recent studies have revealed that local switching between these different gene programs is controlled through interplay between soluble growth factors, insoluble extracellular matrix molecules, and mechanical forces which produce cell shape distortion. Although the precise molecular basis remains unknown, shape-dependent control of cell growth and function appears to be mediated by tension-dependent changes in the actin cytoskeleton. However, the question remains: how can a generalized physical stimulus, such as cell distortion, activate the same set of genes and signaling proteins that are triggered by molecules which bind to specific cell surface receptors. In this article, we use computer simulations based on dynamic Boolean networks to show that the different cell fates that a particular cell can exhibit may represent a preprogrammed set of common end programs or "attractors" which self-organize within the cell's regulatory networks. In this type of dynamic network model of information processing, generalized stimuli (e.g., mechanical forces) and specific molecular cues elicit signals which follow different trajectories, but eventually converge onto one of a small set of common end programs (growth, quiescence, differentiation, apoptosis, etc.). In other words, if cells use this type of information processing system, then control of cell function would involve selection of preexisting (latent) behavioral modes of the cell, rather than instruction by specific binding molecules. Importantly, the results of the computer simulation closely mimic experimental data obtained with living endothelial cells. The major implication of this finding is that current methods used for analysis of cell function that rely on characterization of linear signaling pathways or

  15. β-cell replacement sources for type 1 diabetes: a focus on pancreatic ductal cells.

    Science.gov (United States)

    Corritore, Elisa; Lee, Yong-Syu; Sokal, Etienne M; Lysy, Philippe A

    2016-08-01

    Thorough research on the capacity of human islet transplantation to cure type 1 diabetes led to the achievement of 3- to 5-year-long insulin independence in nearly half of transplanted patients. Yet, translation of this technique to clinical routine is limited by organ shortage and the need for long-term immunosuppression, restricting its use to adults with unstable disease. The production of new bona fide β cells in vitro was thus investigated and finally achieved with human pluripotent stem cells (PSCs). Besides ethical concerns about the use of human embryos, studies are now evaluating the possibility of circumventing the spontaneous tumor formation associated with transplantation of PSCs. These issues fueled the search for cell candidates for β-cell engineering with safe profiles for clinical translation. In vivo studies revealed the regeneration capacity of the exocrine pancreas after injury that depends at least partially on facultative progenitors in the ductal compartment. These stimulated subpopulations of pancreatic ductal cells (PDCs) underwent β-cell transdifferentiation through reactivation of embryonic signaling pathways. In vitro models for expansion and differentiation of purified PDCs toward insulin-producing cells were described using cocktails of growth factors, extracellular-matrix proteins and transcription factor overexpression. In this review, we will describe the latest findings in pancreatic β-cell mass regeneration due to adult ductal progenitor cells. We will further describe recent advances in human PDC transdifferentiation to insulin-producing cells with potential for clinical translational studies.

  16. A Web-Server of Cell Type Discrimination System

    Directory of Open Access Journals (Sweden)

    Anyou Wang

    2014-01-01

    Full Text Available Discriminating cell types is a daily request for stem cell biologists. However, there is not a user-friendly system available to date for public users to discriminate the common cell types, embryonic stem cells (ESCs, induced pluripotent stem cells (iPSCs, and somatic cells (SCs. Here, we develop WCTDS, a web-server of cell type discrimination system, to discriminate the three cell types and their subtypes like fetal versus adult SCs. WCTDS is developed as a top layer application of our recent publication regarding cell type discriminations, which employs DNA-methylation as biomarkers and machine learning models to discriminate cell types. Implemented by Django, Python, R, and Linux shell programming, run under Linux-Apache web server, and communicated through MySQL, WCTDS provides a friendly framework to efficiently receive the user input and to run mathematical models for analyzing data and then to present results to users. This framework is flexible and easy to be expended for other applications. Therefore, WCTDS works as a user-friendly framework to discriminate cell types and subtypes and it can also be expended to detect other cell types like cancer cells.

  17. Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

    Science.gov (United States)

    Chung, Shan; Lin, Yuwen L; Reed, Chae; Ng, Carl; Cheng, Zhijie Jey; Malavasi, Fabio; Yang, Jihong; Quarmby, Valerie; Song, An

    2014-05-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

  18. Temperature dependence of photovoltaic cells, modules, and systems

    Energy Technology Data Exchange (ETDEWEB)

    Emery, K.; Burdick, J.; Caiyem, Y. [National Renewable Energy Lab., Golden, CO (United States)] [and others

    1996-05-01

    Photovoltaic (PV) cells and modules are often rated in terms of a set of standard reporting conditions defined by a temperature, spectral irradiance, and total irradiance. Because PV devices operates over a wide range of temperatures and irradiances, the temperature and irradiance related behavior must be known. This paper surveys the temperature dependence of crystalline and thin-film, state-of-the-art, research-size cells, modules, and systems measured by a variety of methods. The various error sources and measurement methods that contribute to cause differences in the temperature coefficient for a given cell or module measured with various methods are discussed.

  19. Semitransparent organic solar cells with organic wavelength dependent reflectors

    NARCIS (Netherlands)

    Galagan, Y.O.; Debije, M.G.; Blom, P.W.M.

    2011-01-01

    Semitransparent organic solar cells employing solution-processable organic wavelength dependent reflectors of chiral nematic (cholesteric) liquid crystals are demonstrated. The cholesteric liquid crystal (CLC) reflects only in a narrow band of the solar spectrum and remains transparent for the remai

  20. Two Types of Pressure Dependence of Residual Resistivity in Doped Kondo Insulators

    Institute of Scientific and Technical Information of China (English)

    YUAN Yi-Zhe; LI Zheng-Zhong; XIAO Ming-Wen; XU Wang; XU Xiao-Hua

    2004-01-01

    The pressure dependence of the residual resistivity of the doped electron-type and hole-type Kondo insulators (KIs) are calculated within the framework of the slave-boson mean-field theory and the coherent potential approximation. It is shown that as the pressure increases, the resistivity increases and decreases for the dilute doping electron-type and hole-type KIs, respectively. These results are qualitatively in agreement with the experiments.

  1. Localized Castleman disease of plasma cell type in the abdomen

    Institute of Scientific and Technical Information of China (English)

    LU Zhi-hua; WU Mei

    2011-01-01

    Castleman disease is a relatively rare entity,with the hyaline-vascular type the predominant form.Although the plasma cell type is uncommon,it still comprises approximately 10% of cases of localized diseases.In addition,the abdomen is a rare site for involvement and localized Castleman disease of the plasma cell type in the abdomen is rare.The radiologic features of localized plasma cell type in the abdomen are mostly limited to case reports.In addition to the conventional imaging findings,we present some new imaging findings of localized plasma cell type in the abdomen.

  2. Path dependence of lithium ion cells aging under storage conditions

    Science.gov (United States)

    Su, Laisuo; Zhang, Jianbo; Huang, Jun; Ge, Hao; Li, Zhe; Xie, Fengchao; Liaw, Bor Yann

    2016-05-01

    This work investigates path dependence of lithium ion cells that are stored under static and non-static conditions. In the static storage tests, the levels of temperature and state of charge (SOC) are kept constant. The results of 12 tests from a combination of three temperatures and four SOCs show that, as expected, the cell ages faster at higher temperature and higher SOC. However, the cell aging mode, while consistent for all the evaluated temperatures, is different at 95% SOC from that at lower SOCs. In the non-static storage tests, the levels of temperature and SOC vary with time during the test process. The effect of the sequence of stress levels on cell aging is studied statistically using the statistical method of analysis of variation (ANOVA). It is found that cell capacity fade is path independent of both SOC and temperature, while cell resistance increase is path dependent on SOC and path independent of temperature. Finally, rate-based empirical aging models are adopted to fit the cell aging in the static storage tests. The aging model for capacity fade is demonstrated to be applicable to the non-static tests with errors between -3% and +3% for all the tested conditions over 180 days.

  3. Ryanodine Receptors Selectively Interact with L Type Calcium Channels in Mouse Taste Cells.

    Directory of Open Access Journals (Sweden)

    Michelle R Rebello

    Full Text Available WE REPORTED THAT RYANODINE RECEPTORS ARE EXPRESSED IN TWO DIFFERENT TYPES OF MAMMALIAN PERIPHERAL TASTE RECEPTOR CELLS: Type II and Type III cells. Type II cells lack voltage-gated calcium channels (VGCCs and chemical synapses. In these cells, ryanodine receptors contribute to the taste-evoked calcium signals that are initiated by opening inositol trisphosphate receptors located on internal calcium stores. In Type III cells that do have VGCCs and chemical synapses, ryanodine receptors contribute to the depolarization-dependent calcium influx.The goal of this study was to establish if there was selectivity in the type of VGCC that is associated with the ryanodine receptor in the Type III taste cells or if the ryanodine receptor opens irrespective of the calcium channels involved. We also wished to determine if the ryanodine receptors and VGCCs require a physical linkage to interact or are simply functionally associated with each other. Using calcium imaging and pharmacological inhibitors, we found that ryanodine receptors are selectively associated with L type VGCCs but likely not through a physical linkage.Taste cells are able to undergo calcium induced calcium release through ryanodine receptors to increase the initial calcium influx signal and provide a larger calcium response than would otherwise occur when L type channels are activated in Type III taste cells.

  4. Artesunate induces AIF-dependent apoptosis in A549 cells

    Science.gov (United States)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  5. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    Energy Technology Data Exchange (ETDEWEB)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  6. Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

    Science.gov (United States)

    Kobayashi, Junya; Saito, Yuichiro; Okui, Michiyo; Miwa, Noriko; Komatsu, Kenshi

    2015-04-01

    Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

  7. An Introduction to Programming and Proving with Dependent Types in Coq

    Directory of Open Access Journals (Sweden)

    Adam Chlipala

    2010-01-01

    Full Text Available Computer proof assistants vary along many dimensions. Among the mature implementations, the Coq system is distinguished by two key features. First, we have support for programming with dependent types in the tradition of type theory, based on dependent function types and inductive type families. Second, we have a domain-specific language for coding correct-by-construction proof automation. Though the Coq user community has grown quite large, neither of the aspects I highlight is widely used. In this tutorial, I aim to provide a pragmatic introduction to both, showing how they can bring significant improvements in productivity.

  8. Calcium signaling and T-type calcium channels in cancer cell cycling

    Institute of Scientific and Technical Information of China (English)

    James T Taylor; Xiang-Bin Zeng; Jonathan E Pottle; Kevin Lee; Alun R Wang; Stephenie G Yi; Jennifer A S Scruggs; Suresh S Sikka; Ming Li

    2008-01-01

    Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells,free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear;however, there is a growing body of evidence that suggests the T-type Ca2+ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca2+ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel isminimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca2+ channels are not expressed in epithelial cells, selective T-type Ca2+ channel blockers may be useful in the treatment of certain types of cancers.

  9. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

    Directory of Open Access Journals (Sweden)

    Larimore Kevin

    2012-06-01

    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  10. Temperature dependent electroreflectance study of CdTe solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Raadik, T., E-mail: taavi.raadik@ttu.ee [Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn (Estonia); Krustok, J.; Josepson, R.; Hiie, J. [Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn (Estonia); Potlog, T.; Spalatu, N. [Moldova State University, A. Mateevici str. 60, MD-2009 Chisinau (Moldova, Republic of)

    2013-05-01

    Cadmium telluride is a promising material for large scale photovoltaic applications. In this paper we study CdS/CdTe heterojunction solar cells with electroreflectance spectroscopy. Both CdS and CdTe layers in solar cells were grown sequentially without intermediate processing by the close-space sublimation method. Electroreflectance measurements were performed in the temperature range of T = 100–300 K. Two solar cells were investigated with conversion efficiencies of 4.1% and 9.6%. The main focus in this work was to study the temperature dependent behavior of the broadening parameter and the bandgap energy of CdTe thin film in solar cells. Room temperature bandgap values of CdTe were E{sub g} = 1.499 eV and E{sub g} = 1.481 eV for higher and lower efficiency solar cells, respectively. Measured bandgap energies are lower than for single crystal CdTe. The formation of CdTe{sub 1−x}S{sub x} solid solution layer on the surface of CdTe is proposed as a possible cause of lower bandgap energies. - Highlights: ► Temperature dependent electroreflectance measurements of CdS/CdTe solar cells ► Investigation of junction properties between CdS and CdTe ► Formation of CdTe{sub 1−} {sub x}S{sub x} solid solution layer in the junction area.

  11. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

    Science.gov (United States)

    Yagoda, Nicholas; von Rechenberg, Moritz; Zaganjor, Elma; Bauer, Andras J; Yang, Wan Seok; Fridman, Daniel J; Wolpaw, Adam J; Smukste, Inese; Peltier, John M; Boniface, J Jay; Smith, Richard; Lessnick, Stephen L; Sahasrabudhe, Sudhir; Stockwell, Brent R

    2007-06-14

    Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

  12. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V;

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  13. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    Science.gov (United States)

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  14. Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes

    DEFF Research Database (Denmark)

    Nielsen, Lotte B; Ploug, Kenneth B; Swift, Peter;

    2007-01-01

    OBJECTIVE: The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucos...

  15. Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells.

    Science.gov (United States)

    Kofler, David M; Marson, Alexander; Dominguez-Villar, Margarita; Xiao, Sheng; Kuchroo, Vijay K; Hafler, David A

    2014-06-01

    Prostaglandin E2 (PGE2) promotes Th17 expansion while otherwise inhibiting other CD4+ T cell subsets. Here, we identified a PGE2-dependent pathway that induces pathogenic Th17 cells in autoimmune disease and is regulated by the transcription factor RORC. Compared with other CD4+ cell types from healthy subjects, there is a surprising lack of the prostaglandin receptor EP2 on Th17 cells; therefore, we examined the hypothesis that RORγt, which is highly expressed in Th17 cells, mediates EP2 downregulation. Chromatin immunoprecipitation followed by DNA sequencing revealed that RORγt binds directly to Ptger2 (the gene encoding EP2 receptor) in Th17 cells isolated from WT mice. In Th17 cells isolated from humans, RORC repressed EP2 by directly silencing PTGER2 transcription, and knock down of RORC restored EP2 expression in Th17 cells. Compared with Th17 cells from healthy individuals, Th17 cells from patients with MS exhibited reduced RORC binding to the PTGER2 promoter region, resulting in higher EP2 levels and increased expression of IFN-γ and GM-CSF. Finally, overexpression of EP2 in Th17 cells from healthy individuals induced a specific program of inflammatory gene transcription that produced a pathogenic Th17 cell phenotype. These findings reveal that RORC directly regulates the effects of PGE2 on Th17 cells, and dysfunction of this pathway induces a pathogenic Th17 cell phenotype.

  16. Functional identification of islet cell types by electrophysiological fingerprinting

    Science.gov (United States)

    Zhang, Quan; Vergari, Elisa; Kellard, Joely A.; Rodriguez, Blanca; Ashcroft, Frances M.; Rorsman, Patrik

    2017-01-01

    The α-, β- and δ-cells of the pancreatic islet exhibit different electrophysiological features. We used a large dataset of whole-cell patch-clamp recordings from cells in intact mouse islets (N = 288 recordings) to investigate whether it is possible to reliably identify cell type (α, β or δ) based on their electrophysiological characteristics. We quantified 15 electrophysiological variables in each recorded cell. Individually, none of the variables could reliably distinguish the cell types. We therefore constructed a logistic regression model that included all quantified variables, to determine whether they could together identify cell type. The model identified cell type with 94% accuracy. This model was applied to a dataset of cells recorded from hyperglycaemic βV59M mice; it correctly identified cell type in all cells and was able to distinguish cells that co-expressed insulin and glucagon. Based on this revised functional identification, we were able to improve conductance-based models of the electrical activity in α-cells and generate a model of δ-cell electrical activity. These new models could faithfully emulate α- and δ-cell electrical activity recorded experimentally. PMID:28275121

  17. Proteolysis of synaptobrevin, syntaxin, and SNAP-25 in alveolar epithelial type II cells.

    Science.gov (United States)

    Zimmerman, U J; Malek, S K; Liu, L; Li, H L

    1999-10-01

    Synaptobrevin-2, syntaxin-1, and SNAP-25 were identified in rat alveolar epithelial type II cells by Western blot analysis. Synaptobrevin-2 was localized in the lamellar bodies, and syntaxin-1 and SNAP-25 were found in 0.4% Nonidet P40-soluble and -insoluble fractions, respectively, of the type II cells. When the isolated type II cells were stimulated for secretion with calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, these proteins were found to have been proteolyzed. Preincubation of cells with calpain inhibitor II (N-acetylleucylleucylmethionine), however, prevented the proteolysis. Treatment of the cell lysate with exogenous calpain resulted in a time-dependent decrease of these proteins. The data suggest that synaptobrevin, syntaxin, and SNAP-25 are subject to proteolytic modification by activated calpain in intact type II cells stimulated for secretion.

  18. Endothelial cell tumor growth is Ape/ref-1 dependent.

    Science.gov (United States)

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K; Gordillo, Gayle M

    2015-09-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

  19. GABAergic cell types in the lizard hippocampus.

    Science.gov (United States)

    Guirado, S; Dávila, J C

    1999-04-01

    The neurochemical classification of GABAergic cells in the lizard hippocampus resulted in a further division into four major, non-overlapping subtypes. Each GABAergic cell subtype displays specific targets on the principal hippocampal neurons. The synaptic targets of the GABA/neuropeptide subtype are the distal apical dendrites of principal neurons. Calretinin- and parvalbumin-containing GABAergic cells synapse on the cell body and proximal dendrites of principal cells. Calbindin is expressed in a distinct group of interneurons, the synapses of which are directed to the dendrites of principal neurons. Finally, another subtype displays NADPH-diaphorase activity, but its synaptic target has not been established.

  20. Double-stranded RNA induces biphasic STAT1 phosphorylation by both type I interferon (IFN)-dependent and type I IFN-independent pathways.

    Science.gov (United States)

    Dempoya, Junichi; Matsumiya, Tomoh; Imaizumi, Tadaatsu; Hayakari, Ryo; Xing, Fei; Yoshida, Hidemi; Okumura, Ken; Satoh, Kei

    2012-12-01

    Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system.

  1. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  2. Tungsten effects on phosphate-dependent biochemical pathways are species and liver cell line dependent.

    Science.gov (United States)

    Johnson, David R; Ang, Chooyaw; Bednar, Anthony J; Inouye, Laura S

    2010-08-01

    Tungsten, in the form of tungstate, polymerizes with phosphate, and as extensive polymerization occurs, cellular phosphorylation and dephosphorylation reactions may be disrupted, resulting in negative effects on cellular functions. A series of studies were conducted to evaluate the effect of tungsten on several phosphate-dependent intracellular functions, including energy cycling (ATP), regulation of enzyme activity (cytosolic protein tyrosine kinase [cytPTK] and tyrosine phosphatase), and intracellular secondary messengers (cyclic adenosine monophosphate [cAMP]). Rat noncancerous hepatocyte (Clone-9), rat cancerous hepatocyte (H4IIE), and human cancerous hepatocyte (HepG2) cells were exposed to 1-1000 mg/l tungsten (in the form of sodium tungstate) for 24 h, lysed, and analyzed for the above biochemical parameters. Cellular ATP levels were not significantly affected in any cell line. After 4 h, tungsten significantly decreased cytPTK activity in Clone-9 cells at >or= 18 mg/l, had no effect in H4IIE cells, and significantly increased cytPTK activity by 70% in HepG2 cells at >or= 2 mg/l. CytPTK displayed a slight hormetic response to tungsten after 24-h exposure yet returned to normal after 48-h exposure. Tungsten significantly increased cAMP by over 60% in Clone-9 cells at >or= 100 mg/l, significantly increased cAMP in H4IIE cells at only 100 mg/l, and significantly increased cAMP in HepG2 cells between 1-100 mg/l but at much more modest levels (8-20%). In conclusion, these data indicate that tungsten produces complex results that must be carefully interpreted in the context of their respective animal models, as well as the phenotype of the cell lines (i.e., normal vs. cancerous).

  3. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  4. Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

    Science.gov (United States)

    Mertsch, Sonja; Thanos, Solon

    2017-01-01

    For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

  5. Methacrylation induces rapid, temperature-dependent, reversible self-assembly of type-I collagen.

    Science.gov (United States)

    Drzewiecki, Kathryn E; Parmar, Avanish S; Gaudet, Ian D; Branch, Jonathan R; Pike, Douglas H; Nanda, Vikas; Shreiber, David I

    2014-09-23

    Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

  6. Hydrogen sulfide inhibits L-type calcium currents depending upon the protein sulfhydryl state in rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Rongyuan Zhang

    Full Text Available Hydrogen sulfide (H(2S is a novel gasotransmitter that inhibits L-type calcium currents (I (Ca, L. However, the underlying molecular mechanisms are unclear. In particular, the targeting site in the L-type calcium channel where H(2S functions remains unknown. The study was designed to investigate if the sulfhydryl group could be the possible targeting site in the L-type calcium channel in rat cardiomyocytes. Cardiac function was measured in isolated perfused rat hearts. The L-type calcium currents were recorded by using a whole cell voltage clamp technique on the isolated cardiomyocytes. The L-type calcium channel containing free sulfhydryl groups in H9C2 cells were measured by using Western blot. The results showed that sodium hydrosulfide (NaHS, an H(2S donor produced a negative inotropic effect on cardiac function, which could be partly inhibited by the oxidant sulfhydryl modifier diamide (DM. H(2S donor inhibited the peak amplitude of I( Ca, L in a concentration-dependent manner. However, dithiothreitol (DTT, a reducing sulfhydryl modifier markedly reversed the H(2S donor-induced inhibition of I (Ca, L in cardiomyocytes. In contrast, in the presence of DM, H(2S donor could not alter cardiac function and L type calcium currents. After the isolated rat heart or the cardiomyocytes were treated with DTT, NaHS could markedly alter cardiac function and L-type calcium currents in cardiomyocytes. Furthermore, NaHS could decrease the functional free sulfhydryl group in the L-type Ca(2+ channel, which could be reversed by thiol reductant, either DTT or reduced glutathione. Therefore, our results suggest that H(2S might inhibit L-type calcium currents depending on the sulfhydryl group in rat cardiomyocytes.

  7. In vivo Cytotoxicity of Type I CD20 Antibodies Critically Depends on Fc Receptor ITAM Signaling

    NARCIS (Netherlands)

    de Haij, Simone; Jansen, J. H. Marco; Boross, Peter; Beurskens, Frank J.; Bakema, Jantine E.; Bos, Desiree L.; Martens, Anton; Verbeek, J. Sjef; Parren, Paul W. H. I.; van de Winkel, Jan G. J.; Leusen, Jeanette H. W.

    2010-01-01

    Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking

  8. Reexamination of relations between the Myers-Briggs type indicator and field dependence-independence.

    Science.gov (United States)

    Schmidt, C P; McCutcheon, J W

    1988-12-01

    The Group Embedded Figures Test and the Myers-Briggs Type Indicator were administered to 210 undergraduate and graduate students. Bivariate relations between the embedded figures test and the Indicator scales of Extraversion-Introversion (EI), Thinking-Feeling (TF), and Judgment-Perception (JP) were nonsignificant while the relation between scores on embedded figures and Sensing-Intuition (SN) was statistically significant. ESFP, ISFJ, and ESFJ types were significantly more field-dependent than the INFP and ENTP types.

  9. POSH misexpression induces caspase-dependent cell death in Drosophila.

    Science.gov (United States)

    Lennox, Ashley L; Stronach, Beth

    2010-02-01

    POSH (Plenty of SH3 domains) is a scaffold for signaling proteins regulating cell survival. Specifically, POSH promotes assembly of a complex including Rac GTPase, mixed lineage kinase (MLK), MKK7, and Jun kinase (JNK). In Drosophila, genetic analysis implicated POSH in Tak1-dependent innate immune response, in part through regulation of JNK signaling. Homologs of the POSH signaling complex components, MLK and MKK7, are essential in Drosophila embryonic dorsal closure. Using a gain-of-function approach, we tested whether POSH plays a role in this process. Ectopic expression of POSH in the embryo causes dorsal closure defects due to apoptosis of the amnioserosa, but ectodermal JNK signaling is normal. Phenotypic consequences of POSH expression were found to be dependent on Drosophila Nc, the caspase-9 homolog, but only partially on Tak1 and not at all on Slpr and Hep. These results suggest that POSH may use different signaling complexes to promote cell death in distinct contexts.

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

    CERN Document Server

    Alsmeyer, Gerold

    2012-01-01

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

  11. Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner.

    Science.gov (United States)

    Kruglova, Anna A; Matveeva, Natalia M; Gridina, Maria M; Battulin, Nariman R; Karpov, Anton; Kiseleva, Elena V; Morozova, Ksenia N; Serov, Oleg L

    2010-06-01

    Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

  12. Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles.

    Science.gov (United States)

    Greulich, C; Diendorf, J; Gessmann, J; Simon, T; Habijan, T; Eggeler, G; Schildhauer, T A; Epple, M; Köller, M

    2011-09-01

    Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.

  13. Receptor-Dependent Coronavirus Infection of Dendritic Cells

    Science.gov (United States)

    Turner, Brian C.; Hemmila, Erin M.; Beauchemin, Nicole; Holmes, Kathryn V.

    2004-01-01

    In several mammalian species, including humans, coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in murine coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the murine coronavirus receptor, murine CEACAM1a, are susceptible to murine coronavirus infection by a receptor-dependent pathway. PMID:15113927

  14. Target cell-dependent normalization of transmitter release at neocortical synapses.

    Science.gov (United States)

    Koester, Helmut J; Johnston, Daniel

    2005-05-01

    The efficacy and short-term modification of neocortical synaptic connections vary with the type of target neuron. We investigated presynaptic Ca2+ and release probability at single synaptic contacts between pairs of neurons in layer 2/3 of the rat neocortex. The amplitude of Ca2+ signals in boutons of pyramids contacting bitufted or multipolar interneurons or other pyramids was dependent on the target cell type. Optical quantal analysis at single synaptic contacts suggested that release probabilities are also target cell-specific. Both the Ca2+ signal and the release probability of different boutons of a pyramid contacting the same target cell varied little. We propose that the mechanisms that regulate the functional properties of boutons of a pyramid normalize the presynaptic Ca2+ influx and release probability for all those boutons that innervate the same target cell.

  15. Behind the lines–actions of bacterial type III effector proteins in plant cells

    OpenAIRE

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Ty...

  16. Pathogenic memory type Th2 cells in allergic inflammation.

    Science.gov (United States)

    Endo, Yusuke; Hirahara, Kiyoshi; Yagi, Ryoji; Tumes, Damon J; Nakayama, Toshinori

    2014-02-01

    Immunological memory is a hallmark of adaptive immunity. Memory CD4 T helper (Th) cells are central to acquired immunity, and vaccines for infectious diseases are developed based on this concept. However, memory Th cells also play a critical role in the pathogenesis of various chronic inflammatory diseases, including asthma. We refer to these populations as 'pathogenic memory Th cells.' Here, we review recent developments highlighting the functions and characteristics of several pathogenic memory type Th2 cell subsets in allergic inflammation. Also discussed are the similarities and differences between pathogenic memory Th2 cells and recently identified type 2 innate lymphoid cells (ILC2), focusing on cytokine production and phenotypic profiles.

  17. Context-dependent role for chromatin remodeling component PBRM1/BAF180 in clear cell renal cell carcinoma

    Science.gov (United States)

    Murakami, A; Wang, L; Kalhorn, S; Schraml, P; Rathmell, W K; Tan, A C; Nemenoff, R; Stenmark, K; Jiang, B-H; Reyland, M E; Heasley, L; Hu, C-J

    2017-01-01

    A subset of clear cell renal cell carcinoma (ccRCC) tumors exhibit a HIF1A gene mutation, yielding two ccRCC tumor types, H1H2 type expressing both HIF1α and HIF2α, and H2 type expressing HIF2α, but not functional HIF1α protein. However, it is unclear how the H1H2 type ccRCC tumors escape HIF1's tumor-suppressive activity. The polybromo-1 (PBRM1) gene coding for the BAF180 protein, a component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, is inactivated in 40% ccRCCs, the function and mechanism of BAF180 mutation is unknown. Our previous study indicates that BAF180-containing SWI/SNF chromatin remodeling complex is a co-activator for transcription factor HIF to induce HIF target genes. Thus, our questions are if BAF180 is involved in HIF-mediated hypoxia response and if PBRM1/BAF180 mutation has any association with the HIF1A retention in H1H2 type ccRCC. We report here that BAF180 is mutated in H1H2 ccRCC cell lines and tumors, and BAF180 re-expression in H1H2 ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-suppressive role in these cells. However, BAF180 is expressed in HIF1-deficient H2 ccRCC cell lines and tumors, and BAF180 knockdown in H2 type ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-promoting activity in these cells. In addition, our data show that BAF180 functions as co-activator for HIF1- and HIF2-mediated transcriptional response, and BAF180's tumor-suppressive and -promoting activity in ccRCC cell lines depends on co-expression of HIF1 and HIF2, respectively. Thus, our studies reveal that BAF180 function in ccRCC is context dependent, and that mutation of PBRM1/BAF180 serves as an alternative strategy for ccRCC tumors to reduce HIF1 tumor-suppressive activity in H1H2 ccRCC tumors. Our studies define distinct functional subgroups of ccRCCs based on expression of BAF180, and suggest that BAF180 inhibition may be a novel therapeutic

  18. Localized calcineurin confers Ca2+-dependent inactivation on neuronal L-type Ca2+ channels.

    Science.gov (United States)

    Oliveria, Seth F; Dittmer, Philip J; Youn, Dong-ho; Dell'Acqua, Mark L; Sather, William A

    2012-10-31

    Excitation-driven entry of Ca(2+) through L-type voltage-gated Ca(2+) channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of Ca(V)1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to Ca(V)1.2 channels by A-kinase anchoring proteins (AKAPs). Ca(V)1.2 channels also undergo negative autoregulation via Ca(2+)-dependent inactivation (CDI), which strongly limits Ca(2+) entry. An abundance of evidence indicates that CDI relies upon binding of Ca(2+)/calmodulin (CaM) to an isoleucine-glutamine motif in the carboxy tail of Ca(V)1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca(2+)/CaM-activated phosphatase calcineurin (CaN) is scaffolded to Ca(V)1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity-mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase's autoinhibitory domain-interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca(2+)/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling.

  19. Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

    Science.gov (United States)

    Gerbe, François; Sidot, Emmanuelle; Smyth, Danielle J; Ohmoto, Makoto; Matsumoto, Ichiro; Dardalhon, Valérie; Cesses, Pierre; Garnier, Laure; Pouzolles, Marie; Brulin, Bénédicte; Bruschi, Marco; Harcus, Yvonne; Zimmermann, Valérie S; Taylor, Naomi; Maizels, Rick M; Jay, Philippe

    2016-01-14

    Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

  20. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

    Directory of Open Access Journals (Sweden)

    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  1. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  2. Dependence of nitrogen concentration in type Ib diamonds on synthesis temperature

    Institute of Scientific and Technical Information of China (English)

    TIAN Yu; JIA XiaoPeng; ZANG ChuanYi; LI ShangSheng; XIAO HongYu; ZHANG YaFei; HUANG GuoFeng; LI Rui; HAN QiGang; MA LiQiu; LI Yong; CHEN XiaoZhou; ZHANG Chong; MA HongAn

    2009-01-01

    Type Ib diamonds were grown by the temperature gradient method (TGM) at 5.5 GPa and 1500-1560 K in a china-type cubic anvil high pressure apparatus using Ni70Mn25Co5 alloy as solvent/catalyst. The concentration of nitrogen (CN) in type Ib diamonds synthesized at different synthesis temperatures was measured by a Fourier transform infrared (FTIR) spectrometer. The dependence of CN in diamond on synthesis temperature was studied. For the type Ib diamonds synthesized using Ni70Mn25Co5 as catalyst, its CN decreases along with the increase of synthesis temperature.

  3. Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    James Campbell

    2016-03-01

    Full Text Available One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.

  4. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

    Directory of Open Access Journals (Sweden)

    Cremer Thomas

    2005-12-01

    Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

  5. Local Time-Dependent Charging in a Perovskite Solar Cell.

    Science.gov (United States)

    Bergmann, Victor W; Guo, Yunlong; Tanaka, Hideyuki; Hermes, Ilka M; Li, Dan; Klasen, Alexander; Bretschneider, Simon A; Nakamura, Eiichi; Berger, Rüdiger; Weber, Stefan A L

    2016-08-03

    Efficient charge extraction within solar cells explicitly depends on the optimization of the internal interfaces. Potential barriers, unbalanced charge extraction, and interfacial trap states can prevent cells from reaching high power conversion efficiencies. In the case of perovskite solar cells, slow processes happening on time scales of seconds cause hysteresis in the current-voltage characteristics. In this work, we localized and investigated these slow processes using frequency-modulation Kelvin probe force microscopy (FM-KPFM) on cross sections of planar methylammonium lead iodide (MAPI) perovskite solar cells. FM-KPFM can map the charge density distribution and its dynamics at internal interfaces. Upon illumination, space charge layers formed at the interfaces of the selective contacts with the MAPI layer within several seconds. We observed distinct differences in the charging dynamics at the interfaces of MAPI with adjacent layers. Our results indicate that more than one process is involved in hysteresis. This finding is in agreement with recent simulation studies claiming that a combination of ion migration and interfacial trap states causes the hysteresis in perovskite solar cells. Such differences in the charging rates at different interfaces cannot be separated by conventional device measurements.

  6. MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells.

    Science.gov (United States)

    Kim, Sangmin; Lee, Jeongmin; Jeon, Myeongjin; Lee, Jeong Eon; Nam, Seok Jin

    2016-04-01

    Interleukin-8 (IL-8) serves as a prognostic marker for breast cancer, and its expression level correlates with metastatic breast cancer and poor prognosis. Here, we investigated the levels of IL-8 expression in a variety of breast cancer cells and the regulatory mechanism of IL-8 in triple-negative breast cancer (TNBC) cells. Our results showed that IL-8 expression correlated positively with overall survival in basal-type breast cancer patients. The levels of IL-8 mRNA expression and protein secretion were significantly increased in TNBC cells compared with non-TNBC cells. In addition, the invasiveness of the TNBC cells was dramatically increased by IL-8 treatment and then augmented invasion-related proteins such as matrix metalloproteinase (MMP)-2 or MMP-9. We observed that elevated IL-8 mRNA expression and protein secretion were suppressed by a specific MEK1/2 inhibitor, UO126. In contrast, the overexpression of constitutively active MEK significantly increased the level of IL-8 mRNA expression in BT474 non-TNBC cells. Finally, we investigated the effect of UO126 on the tumorigenecity of TNBC cells. Our results showed that anchorage-independent growth, cell invasion, and cell migration were also decreased by UO126 in TNBC cells. As such, we demonstrated that IL-8 expression is regulated through MEK/ERK-dependent pathways in TNBC cells. A diversity of MEK blockers, including UO126, may be promising for treating TNBC patients.

  7. CNK1 promotes invasion of cancer cells through NF-kappaB-dependent signaling.

    Science.gov (United States)

    Fritz, Rafael D; Radziwill, Gerald

    2010-03-01

    Hallmarks of cancer cells are uncontrolled proliferation, evasion of apoptosis, angiogenesis, cell invasion, and metastasis, which are driven by oncogenic activation of signaling pathways. Herein, we identify the scaffold protein CNK1 as a mediator of oncogenic signaling that promotes invasion in human breast cancer and cervical cancer cells. Downregulation of CNK1 diminishes the invasiveness of cancer cells and correlates with reduced expression of matrix metalloproteinase 9 (MMP-9) and membrane-type 1 MMP (MT1-MMP). Ectopic expression of CNK1 elevates MT1-MMP promoter activity in a NF-kappaB-dependent manner. Moreover, CNK1 cooperates with the NF-kappaB pathway, but not with the extracellular signal-regulated protein kinase pathway, to promote cell invasion. Mechanistically, CNK1 regulates the alternative branch of the NF-kappaB pathway because knockdown of CNK1 interferes with processing of NF-kappaB2 p100 to p52 and its localization to the nucleus. In agreement with this, the invasion of CNK1-depleted cells is less sensitive to RelB downregulation compared with the invasion of control cells. Moreover, CNK1-dependent MT1-MMP promoter activation is blocked by RelB siRNA. Thus, CNK1 is an essential mediator of an oncogenic pathway involved in invasion of breast and cervical cancer cells and is therefore a putative target for cancer therapy.

  8. Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

    Science.gov (United States)

    Holm, Christian K; Jensen, Søren B; Jakobsen, Martin R; Cheshenko, Natalia; Horan, Kristy A; Moeller, Hanne B; Gonzalez-Dosal, Regina; Rasmussen, Simon B; Christensen, Maria H.; Yarovinsky, Timur O; Rixon, Frazer J; Herold, Betsy C; Fitzgerald, Katherine A; Paludan, Søren R

    2012-01-01

    The innate immune system senses infection by detecting evolutionarily conserved molecules essential for microbial survival or abnormal location of molecules. Here we demonstrate the existence of a novel innate detection mechanism, which is induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon (IFN) response with expression of IFN-stimulated genes (ISGs), in vivo recruitment of leukocytes, and potentiation of Toll-like receptor 7 and 9 signaling. The fusion dependent response was dependent on stimulator of interferon genes (STING) but independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant cell formation. PMID:22706339

  9. The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

    Science.gov (United States)

    Cha, Min-Ji; Choi, Eunhyun; Lee, Seahyoung; Song, Byeong-Wook; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-02-15

    In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3β (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

  10. Type I IFN signaling in CD8– DCs impairs Th1-dependent malaria immunity

    Science.gov (United States)

    Haque, Ashraful; Best, Shannon E.; Montes de Oca, Marcela; James, Kylie R.; Ammerdorffer, Anne; Edwards, Chelsea L.; de Labastida Rivera, Fabian; Amante, Fiona H.; Bunn, Patrick T.; Sheel, Meru; Sebina, Ismail; Koyama, Motoko; Varelias, Antiopi; Hertzog, Paul J.; Kalinke, Ulrich; Gun, Sin Yee; Rénia, Laurent; Ruedl, Christiane; MacDonald, Kelli P.A.; Hill, Geoffrey R.; Engwerda, Christian R.

    2014-01-01

    Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cellular immune responses through activation of type I IFN signaling. Recent evidence suggests that immune suppression and susceptibility to the malaria parasite, Plasmodium, is mediated by type I IFN; however, it is unclear how type I IFN suppresses immunity to blood-stage Plasmodium parasites. During experimental severe malaria, CD4+ Th cell responses are suppressed, and conventional DC (cDC) function is curtailed through unknown mechanisms. Here, we tested the hypothesis that type I IFN signaling directly impairs cDC function during Plasmodium infection in mice. Using cDC-specific IFNAR1-deficient mice, and mixed BM chimeras, we found that type I IFN signaling directly affects cDC function, limiting the ability of cDCs to prime IFN-γ–producing Th1 cells. Although type I IFN signaling modulated all subsets of splenic cDCs, CD8– cDCs were especially susceptible, exhibiting reduced phagocytic and Th1-promoting properties in response to type I IFNs. Additionally, rapid and systemic IFN-α production in response to Plasmodium infection required type I IFN signaling in cDCs themselves, revealing their contribution to a feed-forward cytokine-signaling loop. Together, these data suggest abrogation of type I IFN signaling in CD8– splenic cDCs as an approach for enhancing Th1 responses against Plasmodium and other type I IFN–inducing pathogens. PMID:24789914

  11. A rationalization of the Type IV loading dependence in the Kärger-Pfeifer classification of self-diffusivities

    NARCIS (Netherlands)

    Krishna, R.; van Baten, J.M.

    2011-01-01

    Kärger and Pfeifer (1987) [1] have listed five different types of dependencies of the self-diffusivities, Di,self, on the loading, Θi, of guest molecules in zeolites. Of these five types, the Type IV dependence is particularly intriguing because it displays a maximum in the Di,self − Θi dependence f

  12. An EOQ model with time dependent Weibull deterioration and ramp type demand ,

    Directory of Open Access Journals (Sweden)

    Chaitanya Kumar Tripathy

    2011-04-01

    Full Text Available This paper presents an order level inventory system with time dependent Weibull deterioration and ramp type demand rate where production and demand are time dependent. The proposed model of this paper considers economic order quantity under two different cases. The implementation of the proposed model is illustrated using some numerical examples. Sensitivity analysis is performed to show the effect of changes in the parameters on the optimum solution.

  13. Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease

    Science.gov (United States)

    Maricic, Igor; Girardi, Enrico; Zajonc, Dirk M.; Kumar, Vipin

    2014-01-01

    Summary Lipids presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d, are recognized by natural killer T (NKT) cells, which can be broadly categorized into two subsets. The well-characterized type I NKT cells, express a semi-invariant T cell receptor (TCR) and can recognize both α- and β-linked glycolipids, whereas type II NKT cells are less well studied, express a relatively diverse TCR repertoire, and recognize β-linked lipids. Recent structural studies have shown a distinct mode of recognition of a self-glycolipid sulfatide bound to CD1d by a type II NKT TCR. To further characterize antigen recognition by these cells we have used the structural data and screened other small molecules able to bind to CD1d and activate type II NKT cells. Using plate-bound CD1d and APC-based antigen presentation assay we found that phospholipids such as lysophosphatidylcholine (LPC) can stimulate the sulfatide-reactive type II NKT hybridoma Hy19.3 in a CD1d-dependent manner. Using plasmon resonance studies we found that this type II NKT TCR binds with CD1d-bound LPC with micromolar affinities similar to that for sulfatide. Furthermore LPC-mediated activation of type II NKT cells leads to anergy induction in type I NKT cells and affords protection from ConA-induced hepatitis. These data indicate that, in addition to self-glycolipids, self-lysophospholipids are also recognized by type II NKT cells. Since lysophospholipids are involved during inflammation our findings have implications for not only understanding activation of type II NKT cells in physiological settings but also for the development of immune intervention in inflammatory diseases. PMID:25261475

  14. Functional cell types in taste buds have distinct longevities.

    Directory of Open Access Journals (Sweden)

    Isabel Perea-Martinez

    Full Text Available Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

  15. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

    Science.gov (United States)

    Lee, M M; Schiefelbein, J

    1999-11-24

    The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

  16. Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms.

    Science.gov (United States)

    Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang; Li, Rongshan

    2015-07-01

    IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN.

  17. LRS Bianchi Type-V Viscous Fluid Universe With a Time Dependent Cosmological Term $\\Lambda$

    CERN Document Server

    Pradhan, Anirudh; Singh, C V

    2007-01-01

    An LRS Bianchi type-V cosmological models representing a viscous fluid distribution with a time dependent cosmological term $\\Lambda$ is investigated. To get a determinate solution, the viscosity coefficient of bulk viscous fluid is assumed to be a power function of mass density. It turns out that the cosmological term $\\Lambda(t)$ is a decreasing function of time, which is consistent with recent observations of type Ia supernovae. Various physical and kinematic features of these models have also been explored.

  18. Irrelevance, Heterogeneous Equality, and Call-by-value Dependent Type Systems

    Directory of Open Access Journals (Sweden)

    Vilhelm Sjöberg

    2012-02-01

    Full Text Available We present a full-spectrum dependently typed core language which includes both nontermination and computational irrelevance (a.k.a. erasure, a combination which has not been studied before. The two features interact: to protect type safety we must be careful to only erase terminating expressions. Our language design is strongly influenced by the choice of CBV evaluation, and by our novel treatment of propositional equality which has a heterogeneous, completely erased elimination form.

  19. Migratory CD103+ dendritic cells suppress Helminth-driven Type 2 immunity through constitutive expression of IL-12

    Science.gov (United States)

    Batf3-dependent CD103+ and CD8alpha+ dendritic cells (DCs) play a central role in the development of type 1 immune responses. However, their role in type 2 immunity remains unclear. We found that Th2 cell responses were enhanced in Batf3-/- mice responding to helminth parasite antigens (Ag). As a r...

  20. Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion.

    Science.gov (United States)

    Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M; Froelich, Christopher J; Pardo, Julián

    2015-03-13

    Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

  1. Crystallinity dependent thermal degradation in organic solar cell

    Science.gov (United States)

    Lee, Hyunho; Sohn, Jiho; Tyagi, Priyanka; Lee, Changhee

    2017-01-01

    An operating solar cell undergoes solar heating; thus, the degradation study of organic photo-voltaic (OPV) with a thermal stress is required for their practical applications. We present a thermal degradation study on OPVs fabricated with photo-active polymers having different crystalline phase. Light intensity dependent analysis for different thermal stress duration is performed. In crystalline, the degradation majorly occurs due to drop in open-circuit voltage while in amorphous one it is due to drop in short-circuit current. Physical mechanism in both systems is explained and supported by the X-ray diffraction, morphological and optical characterization.

  2. TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression.

    Science.gov (United States)

    Simon, Melissa J; Gao, Shan; Kang, Woo Hyeun; Banta, Scott; Morrison, Barclay

    2009-09-01

    Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.

  3. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric

    2010-01-01

    . From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas...... fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly...

  4. TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth.

    Science.gov (United States)

    Haricharan, Svasti; Brown, Powel

    2015-06-23

    Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30-50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types.

  5. Type I Interferons and Natural Killer Cell Regulation in Cancer

    Science.gov (United States)

    Müller, Lena; Aigner, Petra; Stoiber, Dagmar

    2017-01-01

    Type I interferons (IFNs) are known to mediate antitumor effects against several tumor types and have therefore been commonly used in clinical anticancer treatment. However, how IFN signaling exerts its beneficial effects is only partially understood. The clinically relevant activity of type I IFNs has been mainly attributed to their role in tumor immune surveillance. Different mechanisms have been postulated to explain how type I IFNs stimulate the immune system. On the one hand, they modulate innate immune cell subsets such as natural killer (NK) cells. On the other hand, type I IFNs also influence adaptive immune responses. Here, we review evidence for the impact of type I IFNs on immune surveillance against cancer and highlight the role of NK cells therein.

  6. Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

    Science.gov (United States)

    Amin, Shayista; Thywissen, Andreas; Heinekamp, Thorsten; Saluz, Hans Peter; Brakhage, Axel A

    2014-07-01

    Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells.

  7. Sporadic Creutzfeldt-Jakob disease: the extent of microglia activation is dependent on the biochemical type of PrPSc.

    Science.gov (United States)

    Puoti, Gianfranco; Giaccone, Giorgio; Mangieri, Michela; Limido, Lucia; Fociani, Paolo; Zerbi, Pietro; Suardi, Silvia; Rossi, Giacomina; Iussich, Selina; Capobianco, Raffaella; Di Fede, Giuseppe; Marcon, Gabriella; Cotrufo, Roberto; Filippini, Graziella; Bugiani, Orso; Tagliavini, Fabrizio

    2005-10-01

    In prion-related encephalopathies, microglial activation occurs early and is dependent on accumulation of disease-specific forms of the prion protein (PrPSc) and may play a role in nerve cell death. Previously, we found that different types of PrPSc (i.e. type 1 and type 2) coexisted in approximately 25% of patients with sporadic Creutzfeldt-Jakob disease (CJD); and a close relationship was detected between PrPSc type, the pattern of PrP immunoreactivity, and extent of spongiform degeneration. To investigate whether microglial reaction is related to the biochemical type and deposition pattern of PrPSc, we carried out a neuropathologic and biochemical study on 26 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. By quantitative analysis, we demonstrated that strong microglial activation was associated with type 1 PrPSc and diffuse PrP immunoreactivity, whereas type 2 PrPSc and focal PrP deposits were accompanied by mild microglia reaction. These findings support the view that the phenotypic heterogeneity of sporadic CJD is largely determined by the physicochemical properties of distinct PrPSc conformers.

  8. Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Stephanie M Wittig-Blaich

    2011-07-01

    Full Text Available The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases.

  9. Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

    Science.gov (United States)

    Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

    2011-01-01

    The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

  10. Type-dependent differential expression of neuropeptide Y in chicken hypothalamus (Gallus domesticus)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area(LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions,the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.

  11. IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

    Science.gov (United States)

    Luda, Katarzyna M; Joeris, Thorsten; Persson, Emma K; Rivollier, Aymeric; Demiri, Mimoza; Sitnik, Katarzyna M; Pool, Lieneke; Holm, Jacob B; Melo-Gonzalez, Felipe; Richter, Lisa; Lambrecht, Bart N; Kristiansen, Karsten; Travis, Mark A; Svensson-Frej, Marcus; Kotarsky, Knut; Agace, William W

    2016-04-19

    The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αβ(+) and CD4(+)CD8αα(+) T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

  12. PLASMA-LIPOPROTEIN ABNORMALITIES IN TYPE-1 (INSULIN-DEPENDENT) DIABETES-MELLITUS

    NARCIS (Netherlands)

    DULLAART, RPF

    1995-01-01

    Lipoprotein abnormalities may well contribute to the increased risk of coronary heart disease, cerebrovascular disease and peripheral vascular disease observed in type 1 (insulin-dependent) diabetes mellitus. The spectrum of diabetes-associated changes in lipoprotein metabolism is discussed. The pla

  13. Increased transcapillary escape rate of albumin in type 1 (insulin-dependent) diabetic patients with microalbuminuria

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B

    1986-01-01

    The transcapillary escape rate, intravascular mass and outflux of albumin were measured in 75 Type 1 (insulin-dependent) diabetic patients. The groups were defined as: group 1: normal urinary albumin excretion, less than 30 mg/24 h (n = 21); group 2: microalbuminuria, 30-300 mg/24 h (n = 36); group...

  14. Prion type-dependent deposition of PRNP allelic products in heterozygous sheep

    NARCIS (Netherlands)

    Langeveld, J.P.M.; Jacobs, J.G.; Hunter, N.; Keulen, van L.J.M.; Lantier, F.; Zijderveld, van F.G.; Bossers, A.

    2016-01-01

    Susceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triple

  15. Evaluation of Single Vehicle Data in Dependence of the Vehicle-Type, Lane, and Site

    OpenAIRE

    Tilch, Benno; Helbing, Dirk

    1999-01-01

    In this paper we study dependencies of fundamental diagrams, time gap distributions, and velocity-distance relations on vehicle types, lanes and/or measurement sites. We also propose measurement and aggregation methods that have more favourable statistical properties than conventional methods.

  16. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent

  17. p53-Dependent suppression of genome instability in germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Otozai, Shinji [Department of Otorhinolaryngology and Head and Neck Surgery, Osaka University School of Medicine, Osaka 565-0871 (Japan); Ishikawa-Fujiwara, Tomoko [Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Oda, Shoji [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Kamei, Yasuhiro [Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ryo, Haruko [Nomura Project, National Institute of Biomedical Innovation, Osaka 565-0085 (Japan); Sato, Ayuko [Department of Pathology, Hyogo College of Medicine, Hyogo 663-8501 (Japan); Nomura, Taisei [Nomura Project, National Institute of Biomedical Innovation, Osaka 565-0085 (Japan); Mitani, Hiroshi [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Tsujimura, Tohru [Department of Pathology, Hyogo College of Medicine, Hyogo 663-8501 (Japan); Inohara, Hidenori [Department of Otorhinolaryngology and Head and Neck Surgery, Osaka University School of Medicine, Osaka 565-0871 (Japan); Todo, Takeshi, E-mail: todo@radbio.med.osaka-u.ac.jp [Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2014-02-15

    Highlights: • Radiation-induced microsatellite instability (MSI) was investigated in medaka fish. • msh2{sup −/−} fish had a high frequency of spontaneous MSI. • p53{sup −/−} fish had a high frequency of radiation-induced MSI. • p53 and msh2 suppress MSI by different pathways: mismatch removal and apoptosis. - Abstract: Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2{sup −/−} males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2{sup −/−} and wild-type fish. By contrast, irradiated p53{sup −/−} fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2{sup −/−} fish, but negligible levels in p53{sup −/−} fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

  18. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    Science.gov (United States)

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  19. The development of endometriosis in a murine model is dependent on the presence of dendritic cells.

    Science.gov (United States)

    Pencovich, Niv; Luk, Janelle; Hantisteanu, Shay; Hornstein, Mark D; Fainaru, Ofer

    2014-04-01

    Endometriosis is a common condition associated with pelvic pain and infertility. This study group has previously shown that supplementation of dendritic cells led to enhancement of endometriosis lesion growth and angiogenesis. This study determined whether endometriosis is dependent on the presence of endogenous dendritic cells. Surgical induction of endometriosis was performed in CD11c⁺ DTR/GFP transgenic (Tg) female mice in which dendritic cells were ablated upon injection of diphtheria toxin (DT). Mice were allocated into four groups (n=5 each): group I, wild-type mice treated with vehicle; group II, wild-type mice treated with DT; group III, Tg mice treated with DT; and group IV, Tg mice treated with vehicle. After 10 days, mice were killed and endometriosis lesions were analysed by flow cytometry. DT treatment led to ablation of dendritic cells in spleens and endometriosis lesions in Tg mice while no ablation was observed in controls. Corresponding to dendritic cell ablation, endometriosis lesions in group III were ∼5-fold smaller than in the control groups (ANOVA Pdendritic cells. Therapies designed to inhibit dendritic cell infiltration as possible treatments for endometriosis warrant further study.

  20. Microtubule length dependence of motor traffic in cells.

    Science.gov (United States)

    Zhang, Yunxin

    2012-10-01

    Motor proteins in living cells, such as kinesin and dynein, can move processively along the microtubule (MT), and can also detach from or attach to MT stochastically. Experiments found that the traffic of motors along MT may be jammed; thus various theoretical models were designed to understand this process. However, previous studies mainly focused on motor attachment/detachment rate dependent properties. Leduc et al. recently found that the traffic jam of motor protein Kip3 depends on MT length (Proc. Natl. Acad. Sci. U.S.A. 109, 6100 (2012)). Therefore, this study discusses the MT length-dependent properties of motor traffic. The results showed that MT length has one critical value N(c); a traffic jam occurs only when MT length N > N(c). The jammed MT length increases with total MT length N , whereas the non-jammed MT length may not change monotonically with N . The critical value N(c) increases with motor detachment rate from MT, but decreases with motor attachment rate to MT. Therefore, the traffic of motors will be more likely to be jammed when the MT is long, motor detachment rate is high, and motor detachment rate is low.

  1. Microtubule length dependence of motor traffic in cells

    CERN Document Server

    Zhang, Yunxin

    2012-01-01

    In living cells, motor proteins, such as kinesin and dynein can move processively along microtubule (MT), and also detach from or attach to MT stochastically. Experiments have found that, the traffic of motor might be jammed, and various theoretical models have been designed to understand this traffic jam phenomenon. But previous studies mainly focus on motor attachment/detachment rate dependent properties. Recent experiment of Leduc {\\it et al.} found that the traffic jam formation of motor protein kinesin depends also on the length of MT [Proc. Natl. Acad. Sci. U.S.A. {\\bf 109}, 6100-6105 (2012)]. In this study, the MT length dependent properties of motor traffic will be analyzed. We found that MT length has one {\\it critical value} $N_c$, traffic jam occurs only when MT length $N>N_c$. The jammed length of MT increases with total MT length, while the non-jammed MT length might not change monotonically with the total MT length. The critical value $N_c$ increases with motor detachment rate from MT, but decre...

  2. Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature

    Science.gov (United States)

    Kavanagh, David; Jury, Alexa; Williams, Jac; Scolding, Neil; Bellamy, Chris; Gunther, Claudia; Ritchie, Diane; Gale, Daniel P.; Kanwar, Yashpal S.; Challis, Rachel; Buist, Holly; Overell, James; Weller, Belinda; Flossmann, Oliver; Blunden, Mark; Meyer, Eric P.; Krucker, Thomas; Evans, Stephen J. W.; Campbell, Iain L.; Jackson, Andrew P.; Chandran, Siddharthan

    2016-01-01

    Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/β receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation. PMID:27663672

  3. Hypoxia-induced modulation of apoptosis and BCL-2 family proteins in different cancer cell types.

    Directory of Open Access Journals (Sweden)

    Audrey Sermeus

    Full Text Available Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM(EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and -independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.

  4. Ceruloplasmin enhances smooth muscle cell- and endothelial cell-mediated low density lipoprotein oxidation by a superoxide-dependent mechanism

    Science.gov (United States)

    Mukhopadhyay, C. K.; Ehrenwald, E.; Fox, P. L.

    1996-01-01

    Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

  5. Ovarian Small Cell Carcinoma Hypercalcemic Type: A Case Report

    LENUS (Irish Health Repository)

    Rahma, M B.

    2016-09-01

    A 31-year-old female was diagnosed with small cell carcinoma of the ovary hypercalcaemic type (OSCCHT) post left oophorectomy. This is a rare aggressive ovarian tumour of which less than 300 cases were reported.

  6. IL-10-Producing Type 1 Regulatory T Cells and Allergy

    Institute of Scientific and Technical Information of China (English)

    Kui Wu; Yutian Bi; Kun Sun; Changzheng Wang

    2007-01-01

    As an important subset of regulatory T (Treg) cells, IL-10-producing type 1 regulatory T cells (Tr1), have some different features to thymic-derived naturally occurring CD4+CD25+Foxp3+ Treg cells(nTreg cells). Similar to nTreg cells, Tr1 also play important roles in the control of allergic inflammation in several ways. There is a fine balance between Tr1 and Th2 responses in healthy subjects. Skewing of allergic-specific effctor T cells to a Tr1 phenotype appears to be a critical event in successful allergen-specific immunotherapy and glucocorticoids and β2-agonists treatment. Tr1 suppress Th2 cells and effector cells of allergic inflammation, such as eosinophils, mast cells, basophils, through producing IL-10, and perhaps TGF-β. Understanding of Tr1 may be helpful in developing new strategies for treatment of allergic diseases.

  7. Gene pair signatures in cell type transcriptomes reveal lineage control

    Science.gov (United States)

    Heinäniemi, Merja; Nykter, Matti; Kramer, Roger; Wienecke-Baldacchino, Anke; Sinkkonen, Lasse; Zhou, Joseph Xu; Kreisberg, Richard; Kauffman, Stuart A.; Huang, Sui; Shmulevich, Ilya

    2013-01-01

    The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We compiled a resource of curated human expression data comprising 166 cell types and 2,602 transcription regulating genes and developed a data driven method built around the concept of expression reversal defined at the level of gene pairs, such as those participating in toggle switch circuits. This approach allows us to organize the cell types into their ontogenetic lineage-relationships and to reflect regulatory relationships among genes that explain their ability to function as determinants of cell fate. We show that this method identifies genes belonging to regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, thus offering a novel large-scale perspective on lineage specification. PMID:23603899

  8. Time-dependent Effects in Photospheric-Phase Type II Supernova Spectra

    CERN Document Server

    Dessart, Luc

    2007-01-01

    Spectroscopic modeling of Type II supernovae (SNe) generally assumes steady-state. Following the recent suggestion of Utrobin & Chugai, but using the 1D non-LTE line-blanketed model atmosphere code CMFGEN, we investigate the effects of including time-dependent terms that appear in the statistical and radiative equilibrium equations. We base our discussion on the ejecta properties and the spectroscopic signatures obtained from time-dependent simulations, investigating different ejecta configurations, and covering their evolution from one day to six weeks after shock breakout. Compared to equivalent steady-state models, our time-dependent models produce SN ejecta that are systematically over-ionized, affecting helium at one week after explosion, but ultimately affecting all ions after a few weeks. While the continuum remains essentially unchanged, time-dependence effects on observed spectral lines are large. At the recombination epoch, HI lines and NaID are considerably stronger and broader than in equivale...

  9. Systematic Characterization of Cell Cycle Phase-dependent Protein Dynamics and Pathway Activities by High-content Microscopy-assisted Cell Cycle Phenotyping

    Institute of Scientific and Technical Information of China (English)

    Christopher Bruhn; Torsten Kroll; Zhao-Qi Wang

    2014-01-01

    Cell cycle progression is coordinated with metabolism, signaling and other complex cel-lular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically pow-erful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular locali-zation at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3–4-day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.

  10. G Protein-Dependent CCR5 Signaling Is Not Required for Efficient Infection of Primary T Lymphocytes and Macrophages by R5 Human Immunodeficiency Virus Type 1 Isolates

    OpenAIRE

    2003-01-01

    The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4+-transformed cells or pharmacological inhibition of Gαi proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. ...

  11. Differential effects of genistein on prostate cancer cells depend on mutational status of the androgen receptor.

    Directory of Open Access Journals (Sweden)

    Abeer M Mahmoud

    Full Text Available Blocking the androgen receptor (AR activity is the main goal of therapies for advanced prostate cancer (PCa. However, relapse with a more aggressive, hormone refractory PCa arises, which harbors restored AR activity. One mechanism of such reactivation occurs through acquisition of AR mutations that enable its activation by various steroidal and non-steroidal structures. Thus, natural and chemical compounds that contribute to inappropriate (androgen-independent activation of the AR become an area of intensive research. Here, we demonstrate that genistein, a soy phytoestrogen binds to both the wild and the Thr877Ala (T877A mutant types of AR competitively with androgen, nevertheless, it exerts a pleiotropic effect on PCa cell proliferation and AR activity depending on the mutational status of the AR. Genistein inhibited, in a dose-dependent way, cell proliferation and AR nuclear localization and expression in LAPC-4 cells that have wild AR. However, in LNCaP cells that express the T877A mutant AR, genistein induced a biphasic effect where physiological doses (0.5-5 µmol/L stimulated cell growth and increased AR expression and transcriptional activity, and higher doses induced inhibitory effects. Similar biphasic results were achieved in PC-3 cells transfected with AR mutants; T877A, W741C and H874Y. These findings suggest that genistein, at physiological concentrations, potentially act as an agonist and activate the mutant AR that can be present in advanced PCa after androgen ablation therapy.

  12. Differential effects of genistein on prostate cancer cells depend on mutational status of the androgen receptor.

    Science.gov (United States)

    Mahmoud, Abeer M; Zhu, Tian; Parray, Aijaz; Siddique, Hifzur R; Yang, Wancai; Saleem, Mohammad; Bosland, Maarten C

    2013-01-01

    Blocking the androgen receptor (AR) activity is the main goal of therapies for advanced prostate cancer (PCa). However, relapse with a more aggressive, hormone refractory PCa arises, which harbors restored AR activity. One mechanism of such reactivation occurs through acquisition of AR mutations that enable its activation by various steroidal and non-steroidal structures. Thus, natural and chemical compounds that contribute to inappropriate (androgen-independent) activation of the AR become an area of intensive research. Here, we demonstrate that genistein, a soy phytoestrogen binds to both the wild and the Thr877Ala (T877A) mutant types of AR competitively with androgen, nevertheless, it exerts a pleiotropic effect on PCa cell proliferation and AR activity depending on the mutational status of the AR. Genistein inhibited, in a dose-dependent way, cell proliferation and AR nuclear localization and expression in LAPC-4 cells that have wild AR. However, in LNCaP cells that express the T877A mutant AR, genistein induced a biphasic effect where physiological doses (0.5-5 µmol/L) stimulated cell growth and increased AR expression and transcriptional activity, and higher doses induced inhibitory effects. Similar biphasic results were achieved in PC-3 cells transfected with AR mutants; T877A, W741C and H874Y. These findings suggest that genistein, at physiological concentrations, potentially act as an agonist and activate the mutant AR that can be present in advanced PCa after androgen ablation therapy.

  13. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    Science.gov (United States)

    Danto, S I; Zabski, S M; Crandall, E D

    1992-03-01

    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  14. Invasiveness of Hepatocellular Carcinoma Cell Lines: Contribution of Membrane-Type 1 Matrix Metalloproteinase

    Directory of Open Access Journals (Sweden)

    Koji Murakami

    1999-11-01

    Full Text Available Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC. Matrix metalloproteoinases (MMPs and urokinase-type plasminogen activator (u-PA/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MTi-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

  15. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

  16. Towards Optimal Diagnosis of Type II Germ Cell Tumors

    NARCIS (Netherlands)

    J.A. Stoop (Hans)

    2011-01-01

    textabstractThe aim of the work described in this thesis is to improve the understanding of the pathobiology of testicular cancer (type II Germ Cell Tumors) to create possibilities for optimalization of diagnosis for this type of malignancy in routine pathology laboratories. The different studies pr

  17. Influenza A virus inhibits type I IFN signaling via NF-kappaB-dependent induction of SOCS-3 expression.

    Directory of Open Access Journals (Sweden)

    Eva-K Pauli

    2008-11-01

    Full Text Available The type I interferon (IFN system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1. Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3 protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1 was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

  18. Pharmacology of the human cell voltage-dependent cation channel. Part II: inactivation and blocking

    DEFF Research Database (Denmark)

    Bennekou, Poul; Barksmann, Trine L.; Kristensen, Berit I.

    2004-01-01

    Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents......Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents...

  19. Prevalence of hypertension in type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Nørgaard, K; Feldt-Rasmussen, B; Borch-Johnsen, K;

    1990-01-01

    The prevalence of hypertension in a representative sample (n = 10202) of the Danish general population aged 16-59 years was assessed to 4.4% based on three blood pressure readings. In Type 1 (insulin-dependent) diabetic patients of similar age (n = 1703) the prevalence was determined in a similar...... to that observed in the general population. The patients with Type 1 diabetes and essential hypertension had higher systolic (146 +/- 19 vs 133 +/- 18 mm Hg, p less than 0.00001) and diastolic blood pressure (87 +/- 12 vs 79 +/- 7 mm Hg, p less than 0.00001), but less changes in the eye background than patients...... antihypertensive treatment (if any). They were followed-up for a 58 (6-234) month period. We confirmed that hypertension is more common among Type 1 diabetic patients than in the general population and found the prevalence of essential hypertension similar in Type 1 diabetic patients to the non-diabetic population...

  20. Factor XII binding to endothelial cells depends on caveolae

    DEFF Research Database (Denmark)

    Schousboe, Inger; Thomsen, Peter; van Deurs, Bo

    2004-01-01

    to human umbilical vein endothelial cells (HUVEC) has never been shown to be localized to these specialized membrane structures. Using microscopical techniques, we here report that FXII binds to specific patches of the HUVEC plasma membrane with a high density of caveolae. Further investigations of FXII...... binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in noncaveolae...... the structural elements within caveolae. Thus, FXII binding to HUVEC depends on intact caveolae on the cellular surface....

  1. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  2. Adhesion-Dependent Wave Generation in Crawling Cells.

    Science.gov (United States)

    Barnhart, Erin L; Allard, Jun; Lou, Sunny S; Theriot, Julie A; Mogilner, Alex

    2017-01-09

    Dynamic actin networks are excitable. In migrating cells, feedback loops can amplify stochastic fluctuations in actin dynamics, often resulting in traveling waves of protrusion. The precise contributions of various molecular and mechanical interactions to wave generation have been difficult to disentangle, in part due to complex cellular morphodynamics. Here we used a relatively simple cell type-the fish epithelial keratocyte-to define a set of mechanochemical feedback loops underlying actin network excitability and wave generation. Although keratocytes are normally characterized by the persistent protrusion of a broad leading edge, increasing cell-substrate adhesion strength results in waving protrusion of a short leading edge. We show that protrusion waves are due to fluctuations in actin polymerization rates and that overexpression of VASP, an actin anti-capping protein that promotes actin polymerization, switches highly adherent keratocytes from waving to persistent protrusion. Moreover, VASP localizes both to adhesion complexes and to the leading edge. Based on these results, we developed a mathematical model for protrusion waves in which local depletion of VASP from the leading edge by adhesions-along with lateral propagation of protrusion due to the branched architecture of the actin network and negative mechanical feedback from the cell membrane-results in regular protrusion waves. Consistent with our model simulations, we show that VASP localization at the leading edge oscillates, with VASP leading-edge enrichment greatest just prior to protrusion initiation. We propose that the mechanochemical feedbacks underlying wave generation in keratocytes may constitute a general module for establishing excitable actin dynamics in other cellular contexts.

  3. Anti-coreceptor therapy drives selective T cell egress by suppressing inflammation-dependent chemotactic cues

    Science.gov (United States)

    Martin, Aaron J.; Clark, Matthew; Gojanovich, Gregory; Manzoor, Fatima; Miller, Keith; Kline, Douglas E.; Morillon, Y. Maurice; Wang, Bo

    2016-01-01

    There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti–human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell–produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell–mediated pathology by purging T cells in an inflammation-dependent manner.

  4. Reversibility of epithelial-mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling.

    Science.gov (United States)

    Jo, Minji; Lester, Robin D; Montel, Valerie; Eastman, Boryana; Takimoto, Shinako; Gonias, Steven L

    2009-08-21

    Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT. In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O(2), uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O(2) decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O(2)). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer.

  5. Behind the lines–actions of bacterial type III effector proteins in plant cells

    Science.gov (United States)

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:27526699

  6. Plasma somatostatin increases during hypoglycaemia in insulin-dependent patients with and without B-cell function

    DEFF Research Database (Denmark)

    Madsbad, S; Hilsted, J; Krarup, T;

    1983-01-01

    Responses of somatostatin-like immunoreactivity (SLI) to hypoglycaemia were investigated in seven type 1 (insulin-dependent) patients with residual B-cell function, eight patients without B-cell function, and six healthy controls. A higher basal level of SLI was found in the group with B-cell...... function when compared with the group without B-cell function. The basal level in the normal subjects was in between the two diabetic groups. All the diabetics had a somatostatin response to hypoglycaemia which was independent of residual B-cell function and no different from that of normal subjects....

  7. FUNCTIONAL GENOMICS IDENTIFIES TIS21-DEPENDENT MECHANISMS AND PUTATIVE CANCER DRUG TARGETS UNDERLYING MEDULLOBLASTOMA SHH-TYPE DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Giulia Gentile

    2016-11-01

    Full Text Available We have recently generated a novel medulloblastoma (MB mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+-Tis21KO.ts main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs. By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1+-is21 wild-type versus Ptch1+-Tis21 knockout, among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets.The data analysis using bioinformatic tools revealed: i a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; ii a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype the neural cell type involved in group 3 MB; iii the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs.

  8. Generation of diverse neural cell types through direct conversion

    Institute of Scientific and Technical Information of China (English)

    Gayle; F; Petersen; Padraig; M; Strappe

    2016-01-01

    A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace,thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost.The process of neural direct conversion,in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency,shows great potential,with evidence of the generation of a range of functional neural cell types both in vitro and in vivo,through viral and non-viral delivery of exogenous factors,as well as chemical induction methods.Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells,with prospective roles in the investigation of neurological disorders,including neurodegenerative disease modelling,drug screening,and cellular replacement for regenerative medicine applications,however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option.In this review,we describe the generation of diverse neural cell types via direct conversion of somatic cells,with comparison against stem cell-based approaches,as well as discussion of their potential research and clinical applications.

  9. Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

    Science.gov (United States)

    Ludmerer, S W; Benincasa, D; Mark, G E

    1996-01-01

    Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism. PMID:8676509

  10. Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1

    DEFF Research Database (Denmark)

    Petersen, B O; Wagener, C; Marinoni, F;

    2000-01-01

    CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6...... is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6...... in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent...

  11. The Dependence of Physical Mechanical Properties of Concrete Pavement Blocks on Coarse Aggregate Type

    Directory of Open Access Journals (Sweden)

    Malaiškienė Jurgita

    2014-12-01

    Full Text Available The aim of this research is to determine the dependences of the physical mechanical properties of vibropressed concrete (pavement blocks on the type of coarse aggregate used in the main layer. Sustainability of concrete pavement blocks is a really important matter. Five different batches of pavement blocks were produced, changing the consistence ratio of coarse aggregate in the main layer. There are two types of course aggregate: crushed gravel and granite. The consistence of a facing layer was not changed. All tests: density, tensile split strength, water absorption for vibro-pressed concrete units were made according to EN 1338:2003+AC2006.

  12. Abuse and dependence liability of benzodiazepine-type drugs: GABA(A) receptor modulation and beyond.

    Science.gov (United States)

    Licata, Stephanie C; Rowlett, James K

    2008-07-01

    Over the past several decades, benzodiazepines and the newer non-benzodiazepines have become the anxiolytic/hypnotics of choice over the more readily abused barbiturates. While all drugs from this class act at the GABA(A) receptor, benzodiazepine-type drugs offer the clear advantage of being safer and better tolerated. However, there is still potential for these drugs to be abused, and significant evidence exists to suggest that this is a growing problem. This review examines the behavioral determinants of the abuse and dependence liability of benzodiazepine-type drugs. Moreover, the pharmacological and putative biochemical basis of the abuse-related behavior is discussed.

  13. Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling.

    Directory of Open Access Journals (Sweden)

    Dong-Min Shin

    Full Text Available The "enhanced intracellular survival" (eis gene of Mycobacterium tuberculosis (Mtb is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

  14. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D;

    2001-01-01

    .2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed......The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1...

  15. Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes

    DEFF Research Database (Denmark)

    Nielsen, Lars Bo; Ploug, K.B.; Swift, P.;

    2007-01-01

    -dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact...

  16. Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

    Science.gov (United States)

    Cazade, Magali; Bidaud, Isabelle; Lory, Philippe; Chemin, Jean

    2017-01-21

    Voltage-gated Ca(2+) channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca(2+) ion itself. This is well exemplified by the Ca(2+)-dependent inactivation of L-type Ca(2+) channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca(2+) channels, a long-held view is that they are not regulated by intracellular Ca(2+). Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca(2+) channels. We demonstrate that a rise in submembrane Ca(2+) induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca(2+)-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca(2+) entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca(2+) channels to their physiological roles.

  17. Dependence of the characteristics of bubbles on types of sonochemical reactors.

    Science.gov (United States)

    Yasui, Kyuichi; Tuziuti, Toru; Iida, Yasuo

    2005-01-01

    Computer simulations of bubble oscillations in liquid water irradiated by an ultrasonic wave have revealed that the characteristic of bubbles depends on types of sonochemical reactors: a horn-type reactor and a standing-wave type reactor. When the acoustic amplitude is large at 20 kHz, the bubble content is mostly water vapor even at the end of the bubble collapse and the temperature inside a bubble at the collapse is relatively low. On the other hand, when the acoustic amplitude is relatively low, the bubble content is mostly noncondensable gas at the end of the bubble collapse and the bubble temperature is relatively high. In a horn-type sonochemical reactor, the former type of bubbles are dominant because many bubbles exist near the horn-tip where the acoustic amplitude is large, while in a standing-wave type reactor the latter type of bubbles are dominant because the Bjerknes force gathers bubbles at a region where acoustic amplitude is relatively low.

  18. Villous B cells of the small intestine are specialized for invariant NK T cell dependence.

    Science.gov (United States)

    Velázquez, Peter; Wei, Bo; McPherson, Michael; Mendoza, Lesley Marie A; Nguyen, Sandra L; Turovskaya, Olga; Kronenberg, Mitchell; Huang, Tiffany T; Schrage, Matthew; Lobato, Lynn N; Fujiwara, Daisuke; Brewer, Sarah; Arditi, Moshe; Cheng, Genhong; Sartor, R Balfour; Newberry, Rodney D; Braun, Jonathan

    2008-04-01

    B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM(+) IgD(+) cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinase(xid), Galphai2(-/-)), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jalpha18(-/-) or CD1d(-/-) mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.

  19. Velocity dependence of rotational loss in Evershed-type superconducting bearings

    Science.gov (United States)

    Hull, John R.; Mulcahy, Thomas M.; Labataille, Joseph F.

    1997-02-01

    Results of free spin down in vacuum are reported for an Evershed-type superconducting bearing in which a permanent magnet (PM) ring is levitated over an array of high-temperature superconductors (HTSs) and under a similar PM ring in magnetic attraction. The velocity dependence of the rotational loss strongly suggests that the observed velocity-dependent losses are primarily due to eddy currents induced in the PM by inhomogeneity of the field produced by the magnetized HTS array. The results show that the Evershed-type bearing is capable of reducing these eddy-current losses to an extremely low level, so that at a maximum magnet rim velocity of 28 m/s, the fractional kinetic-energy loss per hour was 2.4×10-4. Significant levitation heights are also possible, and at a 23 mm height, we measured a low-speed coefficient of friction of 3×10-8.

  20. IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis

    DEFF Research Database (Denmark)

    Luda, Katarzyna M.; Joeris, Thorsten; Persson, Emma K.;

    2016-01-01

    The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence...... of SI CD8αβ+ and CD4+CD8αα+ T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103+CD11b- DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI......-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8...

  1. Selective loss of TGFbeta Smad-dependent signalling prevents cell cycle arrest and promotes invasion in oesophageal adenocarcinoma cell lines.

    Directory of Open Access Journals (Sweden)

    Benjamin A Onwuegbusi

    Full Text Available In cancer, Transforming Growth Factor beta (TGFbeta increases proliferation and promotes invasion via selective loss of signalling pathways. Oesophageal adenocarcinoma arises from Barrett's oesophagus, progresses rapidly and is usually fatal. The contribution of perturbed TGFbeta signalling in the promotion of metastasis in this disease has not been elucidated. We therefore investigated the role of TGFbeta in Barrett's associated oesophageal adenocarcinoma using a panel of cell lines (OE33, TE7, SEG, BIC, FLO. 4/5 adenocarcinoma cell lines failed to cell cycle arrest, down-regulate c-Myc or induce p21 in response to TGFbeta, and modulation of a Smad3/4 specific promoter was inhibited. These hyperproliferative adenocarcinoma cell lines displayed a TGFbeta induced increase in the expression of the extracellular matrix degrading proteinases, urokinase-type plasminogen activator (uPA and plasminogen activator inhibitor 1 (PAI-1, which correlated with an invasive cell phenotype as measured by in vitro migration, invasion and cell scattering assays. Inhibiting ERK and JNK pathways significantly reduced PAI and uPA induction and inhibited the invasive cell phenotype. These results suggest that TGFbeta Smad-dependent signalling is perturbed in Barrett's carcinogenesis, resulting in failure of growth-arrest. However, TGFbeta can promote PAI and uPA expression and invasion through MAPK pathways. These data would support a dual role for TGFbeta in oesophageal adenocarcinoma.

  2. Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    CHENJian-su; CHENRui; XUJin-tang; DINGYong; ZHAOSong-bin; LISui-lian

    2004-01-01

    Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.

  3. A general principle governs vision-dependent dendritic patterning of retinal ganglion cells.

    Science.gov (United States)

    Xu, Hong-Ping; Sun, Jin Hao; Tian, Ning

    2014-10-15

    Dendritic arbors of retinal ganglion cells (RGCs) collect information over a certain area of the visual scene. The coverage territory and the arbor density of dendrites determine what fraction of the visual field is sampled by a single cell and at what resolution. However, it is not clear whether visual stimulation is required for the establishment of branching patterns of RGCs, and whether a general principle directs the dendritic patterning of diverse RGCs. By analyzing the geometric structures of RGC dendrites, we found that dendritic arbors of RGCs underwent a substantial spatial rearrangement after eye-opening. Light deprivation blocked both the dendritic growth and the branch patterning, suggesting that visual stimulation is required for the acquisition of specific branching patterns of RGCs. We further showed that vision-dependent dendritic growth and arbor refinement occurred mainly in the middle portion of the dendritic tree. This nonproportional growth and selective refinement suggest that the late-stage dendritic development of RGCs is not a passive stretching with the growth of eyes, but rather an active process of selective growth/elimination of dendritic arbors of RGCs driven by visual activity. Finally, our data showed that there was a power law relationship between the coverage territory and dendritic arbor density of RGCs on a cell-by-cell basis. RGCs were systematically less dense when they cover larger territories regardless of their cell type, retinal location, or developmental stage. These results suggest that a general structural design principle directs the vision-dependent patterning of RGC dendrites.

  4. Parameter-dependent Pseudodifferential Operators of Toeplitz Type on Closed Manifolds

    CERN Document Server

    Seiler, Jörg

    2012-01-01

    We present a calculus of zero-order parameter-dependent pseudodifferential operators on a closed manifold $M$ that contains both usual parameter-dependent operators - where the parameter enters as an additional covariable - as well as operators independent of the parameter. Parameter-ellipticity is characterized by the invertibility of three associated principal symbols. In case of ellipticity we can construct a parametrix that is an inverse for large values of the parameter. We then extend this parametrix-construction to operators of Toeplitz type, in particular, to operators of the form $P_1A(\\tau)P_0$ where both $P_0$ and $P_1$ are zero-order projections and $A(\\tau)$ is a usual parameter-dependent operator of arbitrary order or $A(\\tau)=\\tau^{\\mu}-A$ with a pseudodifferential operator $A$ of positiv order $\\mu\\in\

  5. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

    Science.gov (United States)

    Fanger, N A; Wardwell, K; Shen, L; Tedder, T F; Guyre, P M

    1996-07-15

    Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

  6. Transition to chaos in random networks with cell-type-specific connectivity

    Science.gov (United States)

    Aljadeff, Johnatan; Stern, Merav; Sharpee, Tatyana

    2015-01-01

    In neural circuits, statistical connectivity rules strongly depend on cell-type identity. We study dynamics of neural networks with cell-type specific connectivity by extending the dynamic mean field method, and find that these networks exhibit a phase transition between silent and chaotic activity. By analyzing the locus of this transition, we derive a new result in random matrix theory: the spectral radius of a random connectivity matrix with block-structured variances. We apply our results to show how a small group of hyper-excitable neurons within the network can significantly increase the network’s computational capacity by bringing it into the chaotic regime. PMID:25768781

  7. Effect of disodium cromoglycate on mast cell-mediated immediate-type allergic reactions.

    Science.gov (United States)

    Shin, Hye-Young; Kim, Jung-Sook; An, Nyeon-Hyoung; Park, Rae-Kil; Kim, Hyung-Min

    2004-04-23

    We investigated the effect of disodium cromoglycate (DSCG) on mast cell-mediated immediate-type hypersensitivity. DSCG inhibited systemic allergic reaction induced by compound 48/80 dose-dependently. Passive cutaneous anaphylaxis was inhibited by 71.6% by oral administration of DSCG (1 g/kg). When DSCG was pretreated at concentration rang from 0.01-1000 g/kg, the serum histamine levels were reduced in a dose dependent manner. DSCG also significantly inhibited histamine release from rat peritoneal mast cell (RPMC) by compound 48/80. We confirmed that DSCG inhibited compound 48/80-induced degranulation of RPMC by alcian blue/nuclear fast red staining. In addition, DSCG showed a significant inhibitory effect on anti-dinitrophenyl IgE-mediated tumor necrosis factor-alpha production. These results indicate that DSCG inhibits mast cell-mediated immediate-type allergic reaction.

  8. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  9. Surgical treatment of unicentric plasma cell histological type Castleman's disease

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    Marić Nebojša

    2011-01-01

    Full Text Available Introduction. Castleman’s disease or angiofollicular lymph hyperplasia is a rare disease with two identified clinical forms. Unicentric or localized form is characterized by isolated growth of lymph nodes, most often in mediastinum, and multicentric form is expressed as systemic disease with spread lymphadenopathy, organomegaly and presence of general symptoms of the disease. Histological types are hyalovascular, plasma-cell and transitive (mixed cell. Case report. This case report shows a woman, 59 years old, with unicentric form of plasma-cell type of Castleman’s disease. Unicentric form is usually shown as hyalovascular histological type, extremely rare as plasma-cell type, and transitive (mixed cell type was never described in literature as localized clinical form. The disease was manifested with chest pain, loss of body weight, exhaustion and weakness of legs. Further diagnostic procedures found the presence of enlarged lymph nodes paratracheally right, in a close contact with vena cava superior. The disease was confirmed by histopathological analysis of bioptated mediastinal lymph node after mediastinoscopy. Surgical treatment included extirpation of enlarged lymph nodes. After the regular postoperative condition, a full therapy effect was confirmed. Conclusion. Unicentric form of Castleman’s disease is expressed with enlarged lymph nodes on predilected places, usually in mediastinum. Surgical treatment is best method for the management of the disease and brings a full recovery of patient.

  10. METHOD FOR AUTOMATIC ANALYSIS OF WHEAT STRAW PULP CELL TYPES

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    Mikko Karjalainen,

    2012-01-01

    Full Text Available Agricultural residues are receiving increasing interest when studying renewable raw materials for industrial use. Residues, generally referred to as nonwood materials, are usually complex materials. Wheat straw is one of the most abundant agricultural residues around the world and is therefore available for extensive industrial use. However, more information of its cell types is needed to utilize wheat straw efficiently in pulp and papermaking. The pulp cell types and particle dimensions of wheat straw were studied, using an optical microscope and an automatic optical fibre analyzer. The role of various cell types in wheat straw pulp and papermaking is discussed. Wheat straw pulp components were categorized according to particle morphology and categorization with an automatic optical analyzer was used to determine wheat straw pulp cell types. The results from automatic optical analysis were compared to those with microscopic analysis and a good correlation was found. Automatic optical analysis was found to be a promising tool for the in-depth analysis of wheat straw pulp cell types.

  11. High blood ketone body concentration in type 2 non-insulin dependent diabetic patients.

    Science.gov (United States)

    Avogaro, A; Crepaldi, C; Miola, M; Maran, A; Pengo, V; Tiengo, A; Del Prato, S

    1996-02-01

    To assess the metabolic disturbances, and, in particular, the occurrence of high blood ketone body concentration in post-absorptive Type 2 (non-insulin-dependent) diabetic patients as compared to a matched normal population, a study was carried out in a group of 78 Type 2 diabetic outpatients matched for age and sex and in 78 normal individuals. In all subjects we measured HbA1c, and fasting levels of glucose, FFA, lactate, pyruvate, glycerol, alanine, 3-hydroxybutyrate, acetoacetate, uric acid, total cholesterol, triglycerides, creatinine, growth hormone, cortisol, glucagon, free insulin, and C-peptide. Multistix strips were used for urine ketone determination. As expected HbA1c, and plasma glucose were higher in Type 2 diabetics. This was associated with multiple metabolic disturbances as shown by higher circulating concentrations of FFA, glycerol and gluconeogenic precursors. Similarly, blood levels of ketones (351 +/- 29 vs 159 +/- 15 umol/l; P growth hormone. Plasma glucagon levels were higher in Type 2 diabetics. Blood ketone body levels were directly correlated with both plasma glucose and FFA concentrations. These observations clearly show that Type 2 diabetes is a pathologic condition characterised by multiple metabolic disturbances which are fully apparent in the basal state. Furthermore, we emphasise that Type 2 diabetic patients, though not insulin deficient, may present a significant increase in their fasting levels of ketone bodies.

  12. Alveolar epithelial type II cell: defender of the alveolus revisited

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    Fehrenbach Heinz

    2001-01-01

    Full Text Available Abstract In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2 cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today.

  13. Metabolic respiration induces AMPK- and Ire1p-dependent activation of the p38-Type HOG MAPK pathway.

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    Hema Adhikari

    2014-10-01

    Full Text Available Evolutionarily conserved mitogen activated protein kinase (MAPK pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA cycle. The unfolded protein response (UPR kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.

  14. Connectivity between the OFF bipolar type DB3a and six types of ganglion cell in the marmoset retina.

    Science.gov (United States)

    Masri, Rania A; Percival, Kumiko A; Koizumi, Amane; Martin, Paul R; Grünert, Ulrike

    2016-06-15

    Parallel visual pathways originate at the first synapse in the retina, where cones make connections with cone bipolar cells that in turn contact ganglion cells. There are more ganglion cell types than bipolar types, suggesting that there must be divergence from bipolar to ganglion cells. Here we analyze the contacts between an OFF bipolar type (DB3a) and six ganglion cell types in the retina of the marmoset monkey (Callithrix jacchus). Ganglion cells were transfected via particle-mediated gene transfer of an expression plasmid for the postsynaptic density 95-green fluorescent protein (PSD95-GFP), and DB3a cells were labeled via immunohistochemistry. Ganglion cell types that fully or partially costratified with DB3a cells included OFF parasol, OFF midget, broad thorny, recursive bistratified, small bistratified, and large bistratified cells. On average, the number of DB3a contacts to parasol cells (18 contacts per axon terminal) is higher than that to other ganglion cell types (between four and seven contacts). We estimate that the DB3a output to OFF parasol cells accounts for at least 30% of the total DB3a output. Furthermore, we found that OFF parasol cells receive approximately 20% of their total bipolar input from DB3a cells, suggesting that other diffuse bipolar types also provide input to OFF parasol cells. We conclude that DB3a cells preferentially contact OFF parasol cells but also provide input to other ganglion cell types.

  15. Is Transforming Stem Cells to Pancreatic Beta Cells Still the Holy Grail for Type 2 Diabetes?

    Science.gov (United States)

    Kahraman, Sevim; Okawa, Erin R; Kulkarni, Rohit N

    2016-08-01

    Diabetes is a progressive disease affecting millions of people worldwide. There are several medications and treatment options to improve the life quality of people with diabetes. One of the strategies for the treatment of diabetes could be the use of human pluripotent stem cells or induced pluripotent stem cells. The recent advances in differentiation of stem cells into insulin-secreting beta-like cells in vitro make the transplantation of the stem cell-derived beta-like cells an attractive approach for treatment of type 1 and type 2 diabetes. While stem cell-derived beta-like cells provide an unlimited cell source for beta cell replacement therapies, these cells can also be used as a platform for drug screening or modeling diseases.

  16. Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

    Science.gov (United States)

    Lund, A W; Stegemann, J P; Plopper, G E

    2009-01-01

    The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

  17. Spatiotemporally restricted arenavirus replication induces immune surveillance and type I interferon-dependent tumour regression

    Science.gov (United States)

    Kalkavan, Halime; Sharma, Piyush; Kasper, Stefan; Helfrich, Iris; Pandyra, Aleksandra A.; Gassa, Asmae; Virchow, Isabel; Flatz, Lukas; Brandenburg, Tim; Namineni, Sukumar; Heikenwalder, Mathias; Höchst, Bastian; Knolle, Percy A.; Wollmann, Guido; von Laer, Dorothee; Drexler, Ingo; Rathbun, Jessica; Cannon, Paula M.; Scheu, Stefanie; Bauer, Jens; Chauhan, Jagat; Häussinger, Dieter; Willimsky, Gerald; Löhning, Max; Schadendorf, Dirk; Brandau, Sven; Schuler, Martin; Lang, Philipp A.; Lang, Karl S.

    2017-01-01

    Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses. PMID:28248314

  18. Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence.

    Directory of Open Access Journals (Sweden)

    Cameron R Cunningham

    2016-01-01

    Full Text Available Persistent viral infections are simultaneously associated with chronic inflammation and highly potent immunosuppressive programs mediated by IL-10 and PDL1 that attenuate antiviral T cell responses. Inhibiting these suppressive signals enhances T cell function to control persistent infection; yet, the underlying signals and mechanisms that program immunosuppressive cell fates and functions are not well understood. Herein, we use lymphocytic choriomeningitis virus infection (LCMV to demonstrate that the induction and functional programming of immunosuppressive dendritic cells (DCs during viral persistence are separable mechanisms programmed by factors primarily considered pro-inflammatory. IFNγ first induces the de novo development of naive monocytes into DCs with immunosuppressive potential. Type I interferon (IFN-I then directly targets these newly generated DCs to program their potent T cell immunosuppressive functions while simultaneously inhibiting conventional DCs with T cell stimulating capacity. These mechanisms of monocyte conversion are constant throughout persistent infection, establishing a system to continuously interpret and shape the immunologic environment. MyD88 signaling was required for the differentiation of suppressive DCs, whereas inhibition of stimulatory DCs was dependent on MAVS signaling, demonstrating a bifurcation in the pathogen recognition pathways that promote distinct elements of IFN-I mediated immunosuppression. Further, a similar suppressive DC origin and differentiation was also observed in Mycobacterium tuberculosis infection, HIV infection and cancer. Ultimately, targeting the underlying mechanisms that induce immunosuppression could simultaneously prevent multiple suppressive signals to further restore T cell function and control persistent infections.

  19. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.

    Science.gov (United States)

    Christianson, Helena C; Svensson, Katrin J; van Kuppevelt, Toin H; Li, Jin-Ping; Belting, Mattias

    2013-10-22

    Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  20. Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types

    Science.gov (United States)

    Ferreira, Lauren; Macaulay, Iain C.; Stubbington, Michael J.T.

    2017-01-01

    The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell–specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates. PMID:28087841

  1. Effect of epithelial cell type on in vitro invasion of non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Singh, Neeraj Kumar; Kunde, Dale A; Tristram, Stephen G

    2016-10-01

    Non-typeable Haemophilus influenzae (NTHi) have been shown to have variable ability for in vitro invasion with a range of epithelial cells, and increased invasion of BEAS-2B cells has been associated with altered penicillin binding protein3 (PBP3), which is concerning as these strains are increasing worldwide. The aim of the study was to investigate the effect of respiratory cell type and the presence of altered PBP3 on the in vitro invasion of NTHi. A collection of 16 clinical NTHi isolates was established, 7 had normal PBP3, and 9 had altered PBP3 as defined by an N526K substitution. The isolates were tested for invasion of BEAS-2B, NHBE, A549 and NCI-H292 respiratory epithelial cells in vitro using a gentamicin survival assay, with invasion measured as the percentage of intracellular organisms relative to the initial inoculum. The overall median invasion for the 16 NTHi isolates for cell types BEAS-2B, NHBE, A549 and NCI-H292 cells were 3.17, 2.31, 0.11 and 1.52 respectively. The differences were statistically significant for BEAS-2B compared to A549 (P=0.015) and A549 compared to NCI-H292 (P=0.015), and there were also very marked differences in invasion for some individual isolates depending on the cell type used. There was a consistent bias for invasion of isolates with normal versus abnormal PBP3: and this was statistically significant for BEAS-2B (0.07 to 9.90, P=0.031) and A549 cells (0.02 to 1.68, P=0.037). These results show that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the cell line used, and that the association between altered PBP3 and increased invasion is conserved across multiple cell lines.

  2. Using typed dependencies to study and recognise conceptualisation zones in biomedical literature.

    Science.gov (United States)

    Groza, Tudor

    2013-01-01

    In the biomedical domain, authors publish their experiments and findings using a quasi-standard coarse-grained discourse structure, which starts with an introduction that sets up the motivation, continues with a description of the materials and methods, and concludes with results and discussions. Over the course of the years, there has been a fair amount of research done in the area of scientific discourse analysis, with a focus on performing automatic recognition of scientific artefacts/conceptualisation zones from the raw content of scientific publications. Most of the existing approaches use Machine Learning techniques to perform classification based on features that rely on the shallow structure of the sentence tokens, or sentences as a whole, in addition to corpus-driven statistics. In this article, we investigate the role carried by the deep (dependency) structure of the sentences in describing their rhetorical nature. Using association rule mining techniques, we study the presence of dependency structure patterns in the context of a given rhetorical type, the use of these patterns in exploring differences in structure between the rhetorical types, and their ability to discriminate between the different rhetorical types. Our final goal is to provide a series of insights that can be used to complement existing classification approaches. Experimental results show that, in particular in the context of a fine-grained multi-class classification context, the association rules emerged from the dependency structure are not able to produce uniform classification results. However, they can be used to derive discriminative pair-wise classification mechanisms, in particular for some of the most ambiguous types.

  3. SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors.

    Science.gov (United States)

    De Calisto, Jaime; Wang, Ninghai; Wang, Guoxing; Yigit, Burcu; Engel, Pablo; Terhorst, Cox

    2014-01-01

    Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF) of receptors, whose expression is essential for T, NK, and B-cell responses. Additionally, the expression of SAP in double-positive thymocytes is mandatory for natural killer T (NKT) cells and, in mouse, for innate CD8(+) T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1 + 6](-/-) and Slamf[1 + 5 + 6](-/-) B6 mice have ~70% reduction of NKT cells compared to wild-type B6 mice. Unexpectedly, the proportion of innate CD8(+) T cells slightly increased in the Slamf[1 + 5 + 6](-/-) , but not in the Slamf[1 + 6](-/-) strain, suggesting that Slamf5 may function as a negative regulator of innate CD8(+) T cell development. Accordingly, Slamf5(-/-) B6 mice showed an exclusive expansion of innate CD8(+) T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7(-/-) strain showed an expansion of both splenic innate CD8(+) T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3(-/-) BALB/c mice, the proportions of thymic promyelocytic leukemia zinc finger (PLZF(hi)) NKT cells and innate CD8(+) T cells significantly increased in the SAP-independent Slamf8(-/-) BALB/c strain. In summary, these results show that NKT and innate CD8(+) T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8(+) T cells.

  4. SAP-independent and -dependent regulation of innate T cell development involving SLAMF receptors

    Directory of Open Access Journals (Sweden)

    Jaime eDe Calisto

    2014-04-01

    Full Text Available Signaling lymphocytic activation molecule (SLAM-associated protein (SAP plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF of receptors, whose expression is essential for T, NK, and B cell responses. Additionally, the expression of SAP in double-positive (DP thymocytes is mandatory for natural killer T (NKT cells and, in mouse, for innate CD8+ T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1+6]-/- and Slamf[1+5+6]-/- B6 mice have an approximately 70% reduction of NKT cells compared to wild-type (WT B6 mice. Unexpectedly, the proportion of innate CD8+ T cells slightly increased in the Slamf[1+5+6]-/-, but not in the Slamf[1+6]-/- strain, suggesting that Slamf5 may function as a negative regulator of innate CD8+ T cell development. Accordingly, Slamf5-/- B6 mice showed an exclusive expansion of innate CD8+ T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7-/- strain showed an expansion of both splenic innate CD8+ T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3-/- BALB/c mice, the proportions of thymic PLZFhi NKT cells and innate CD8+ T cells significatively increased in the SAP-independent Slamf8-/- BALB/c strain. In summary, these results show that NKT and innate CD8+ T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8+ T cells.

  5. Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells

    Science.gov (United States)

    Monti, Marcello; Iommelli, Francesca; De Rosa, Viviana; Carriero, Maria Vincenza; Miceli, Roberta; Camerlingo, Rosa; Di Minno, Giovanni; Del Vecchio, Silvana

    2017-01-01

    Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells. PMID:28166238

  6. Social network types and functional dependency in older adults in Mexico

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    Espinosa-Alarcón Patricia

    2010-02-01

    Full Text Available Abstract Background Social networks play a key role in caring for older adults. A better understanding of the characteristics of different social networks types (TSNs in a given community provides useful information for designing policies to care for this age group. Therefore this study has three objectives: 1 To derive the TSNs among older adults affiliated with the Mexican Institute of Social Security; 2 To describe the main characteristics of the older adults in each TSN, including the instrumental and economic support they receive and their satisfaction with the network; 3 To determine the association between functional dependency and the type of social network. Methods Secondary data analysis of the 2006 Survey of Autonomy and Dependency (N = 3,348. The TSNs were identified using the structural approach and cluster analysis. The association between functional dependency and the TSNs was evaluated with Poisson regression with robust variance analysis in which socio-demographic characteristics, lifestyle and medical history covariates were included. Results We identified five TSNs: diverse with community participation (12.1%, diverse without community participation (44.3%; widowed (32.0%; nonfriends-restricted (7.6%; nonfamily-restricted (4.0%. Older adults belonging to widowed and restricted networks showed a higher proportion of dependency, negative self-rated health and depression. Older adults with functional dependency more likely belonged to a widowed network (adjusted prevalence ratio 1.5; 95%CI: 1.1-2.1. Conclusion The derived TSNs were similar to those described in developed countries. However, we identified the existence of a diverse network without community participation and a widowed network that have not been previously described. These TSNs and restricted networks represent a potential unmet need of social security affiliates.

  7. Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms

    Institute of Scientific and Technical Information of China (English)

    Ying-jia ZHONG; Yong QIN; Lin-chuan; LIAO Xia WANG; Fang SHI; Xue-lian ZHENG; Qiong WANG; Lan YANG; Hong SUN; Fan HE; Lin ZHANG; Yong LIN

    2011-01-01

    To investigate the anticancer effect of crocetin,a major ingredient in saffron,and its underlying mechanisms.Methods:Cervical cancer cell line HeLa,non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine.Cell proliferation was examined using MTT assay.Cell cycle distribution and sub-G1 fraction were analyzed using flow cytometric analysis after propidium iodide staining.Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry.Cell death was measured based on the release of lactate dehydrogenase (LDH).The expression levels of p53 and p21wAF1/Cip1 as well as caspase activation were examined using Western blot analysis.Results:Treatment of the 3 types of cancer cells with crocetin (60-240 μmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner.Crocetin (240 μmol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21WAF1/Cip1 induction.Crocetin (120-240 μmoVL) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner.In the 3 types of cancer cells,crocetin (60 μmol/L) significantly enhanced the cytotoxicity induced by vincristine (1 μmol/L).Furthermore,this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR.Conclusion:Ccrocetin is a potential anticancer agent,which may be used as a chemotherapeutic drug or as a chemosensitizer for vin-cristine.

  8. Cell-type specific four-component hydrogel.

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    Timo Aberle

    Full Text Available In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin to generate a blend (technical term: quattroGel, an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

  9. Intestinal CX3C chemokine receptor 1(high) (CX3CR1(high)) myeloid cells prevent T-cell-dependent colitis.

    Science.gov (United States)

    Kayama, Hisako; Ueda, Yoshiyasu; Sawa, Yukihisa; Jeon, Seong Gyu; Ma, Ji Su; Okumura, Ryu; Kubo, Atsuko; Ishii, Masaru; Okazaki, Taku; Murakami, Masaaki; Yamamoto, Masahiro; Yagita, Hideo; Takeda, Kiyoshi

    2012-03-27

    Adequate activation of CD4(+) T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4(+) T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX(3)C chemokine receptor 1(high) (CX(3)CR1(high)) CD11b(+) CD11c(+) cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4(+) T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX(3)CR1(high) CD11b(+) CD11c(+) cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX(3)CR1(high) CD11b(+) CD11c(+) cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis.

  10. Intestinal CX3C chemokine receptor 1high (CX3CR1high) myeloid cells prevent T-cell-dependent colitis

    Science.gov (United States)

    Kayama, Hisako; Ueda, Yoshiyasu; Sawa, Yukihisa; Jeon, Seong Gyu; Ma, Ji Su; Okumura, Ryu; Kubo, Atsuko; Ishii, Masaru; Okazaki, Taku; Murakami, Masaaki; Yamamoto, Masahiro; Yagita, Hideo; Takeda, Kiyoshi

    2012-01-01

    Adequate activation of CD4+ T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4+ T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX3C chemokine receptor 1high (CX3CR1high) CD11b+ CD11c+ cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4+ T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX3CR1high CD11b+ CD11c+ cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX3CR1high CD11b+ CD11c+ cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis. PMID:22403066

  11. CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways

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    Blay Jean-Yves

    2006-03-01

    Full Text Available Abstract Background CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. Methods The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. Results Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89% the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. Conclusion These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively.

  12. A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Schiefelbein, John

    2008-12-23

    Cellular pattern formation in the root epidermis of Arabidopsis occurs in a position-dependent manner, generating root-hair (H) cells contacting two underlying cortical cells and nonhair (N) cells contacting one cortical cell. SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase (LRR-RLK), mediates this process through its effect on a downstream transcription factor regulatory network. After perception of a positional cue, the SCM signaling pathway is proposed to preferentially repress WEREWOLF (WER) transcription factor expression in H cells and thereby bias the outcome of mutual lateral inhibition acting between H and N cells. However, the molecular mechanism responsible for this preferential SCM signaling is unknown. Here, we analyze the distribution of the SCM receptor and the biological effect of altering its accumulation pattern. We find that SCM expression and accumulation in the epidermal cell layer is necessary and sufficient to direct the cell-type pattern. Further, SCM preferentially accumulates in H cells, and this accumulation pattern is dependent on the downstream transcription factors. Thus, SCM participates in an autoregulatory feedback loop, enabling cells engaged in SCM signaling to maintain high levels of SCM receptor, which provides a simple mechanism for reinforcing a bias in receptor-mediated signaling to ensure robust pattern formation.

  13. Local intra-arterial thrombolysis in the carotid territory: does recanalization depend on the thromboembolus type?

    Energy Technology Data Exchange (ETDEWEB)

    Urbach, H.; Wilhelm, K.; Flacke, S.; Schild, H.H. [Department of Radiology/Neuroradiology, University of Bonn, Sigmund Freud Strasse 25, 53105 Bonn (Germany); Hartmann, A.; Pohl, C.; Klockgether, T. [Department of Neurology, University of Bonn, Sigmund Freud Strasse 25, 53105 Bonn (Germany); Omran, H. [Department of Cardiology, University of Bonn, Sigmund Freud Strasse 25, 53105 Bonn (Germany)

    2002-08-01

    Little is known about whether recanalization of carotid territory occlusions by local intra-arterial thrombolysis (LIT) depends on the type of the occluding thromboembolus. We retrospectively analysed the records of 62 patients with thromboembolic occlusions of the intracranial internal carotid artery (ICA) bifurcation or the middle cerebral artery who were undergoing LIT with urokinase within 6 h of symptom onset. We determined the influence of thromboembolus type (according to the TOAST criteria), thromboembolus location, leptomeningeal collaterals, time interval from onset of symptoms to onset of thrombolysis, and patient's age on recanalization. The thromboembolus type was atherosclerotic in six patients, cardioembolic in 29, of other determined etiology in four, and of undetermined etiology in 23 patients. Thirty-three (53%) thromboembolic occlusions were recanalized. The thromboembolus location but not the TOAST stroke type nor other parameters affected recanalization. In the TOAST group of patients with cardioembolic occlusions recanalization occurred significantly less frequently when transoesophageal echocardiography showed cardiac thrombus. The present study underlines the thromboembolus location as being the most important parameter affecting recanalization. The fact that thromboembolic occlusions originating from cardiac thrombi had a lower likelihood of being resolved by thrombolysis indicates the thromboembolus type as another parameter affecting recanalization. (orig.)

  14. Role of T Cells in Type 2 Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Chia-Chao Wu

    2011-01-01

    Full Text Available Type 2 diabetic nephropathy (DN is the most common cause of end-stage renal disease and is increasingly considered as an inflammatory disease characterized by leukocyte infiltration at every stage of renal involvement. Inflammation and activation of the immune system are closely involved in the pathogenesis of diabetes and its microvascular complications. Macrophage has been well recognized to play an important role in type 2 DN, leukocyte infiltration, and participated in process of DN, as was proposed recently. Th1, Th2, Th17, T reg, and cytotoxic T cells are involved in the development and progression of DN. The purpose of this review is to assemble current information concerning the role of T cells in the development and progression of type 2 DN. Specific emphasis is placed on the potential interaction and contribution of the T cells to renal damage. The therapeutic strategies involving T cells in the treatment of type 2 DN are also reviewed. Improving knowledge of the recognition of T cells as significant pathogenic mediators in DN reinforces the possibility of new potential therapeutic targets translated into future clinical treatments.

  15. Towards Optimal Diagnosis of Type II Germ Cell Tumors

    OpenAIRE

    Stoop, Hans

    2011-01-01

    textabstractThe aim of the work described in this thesis is to improve the understanding of the pathobiology of testicular cancer (type II Germ Cell Tumors) to create possibilities for optimalization of diagnosis for this type of malignancy in routine pathology laboratories. The different studies presented here show valuable additional information on the microscopic diagnostics in daily practice. This enables proper and complete diagnosis of this relative rare variant of cancer ensuring the b...

  16. History-Dependent Excitability as a Single-Cell Substrate of Transient Memory for Information Discrimination

    Science.gov (United States)

    Baroni, Fabiano; Torres, Joaquín J.; Varona, Pablo

    2010-01-01

    Neurons react differently to incoming stimuli depending upon their previous history of stimulation. This property can be considered as a single-cell substrate for transient memory, or context-dependent information processing: depending upon the current context that the neuron “sees” through the subset of the network impinging on it in the immediate past, the same synaptic event can evoke a postsynaptic spike or just a subthreshold depolarization. We propose a formal definition of History-Dependent Excitability (HDE) as a measure of the propensity to firing in any moment in time, linking the subthreshold history-dependent dynamics with spike generation. This definition allows the quantitative assessment of the intrinsic memory for different single-neuron dynamics and input statistics. We illustrate the concept of HDE by considering two general dynamical mechanisms: the passive behavior of an Integrate and Fire (IF) neuron, and the inductive behavior of a Generalized Integrate and Fire (GIF) neuron with subthreshold damped oscillations. This framework allows us to characterize the sensitivity of different model neurons to the detailed temporal structure of incoming stimuli. While a neuron with intrinsic oscillations discriminates equally well between input trains with the same or different frequency, a passive neuron discriminates better between inputs with different frequencies. This suggests that passive neurons are better suited to rate-based computation, while neurons with subthreshold oscillations are advantageous in a temporal coding scheme. We also address the influence of intrinsic properties in single-cell processing as a function of input statistics, and show that intrinsic oscillations enhance discrimination sensitivity at high input rates. Finally, we discuss how the recognition of these cell-specific discrimination properties might further our understanding of neuronal network computations and their relationships to the distribution and functional

  17. History-dependent excitability as a single-cell substrate of transient memory for information discrimination.

    Directory of Open Access Journals (Sweden)

    Fabiano Baroni

    Full Text Available Neurons react differently to incoming stimuli depending upon their previous history of stimulation. This property can be considered as a single-cell substrate for transient memory, or context-dependent information processing: depending upon the current context that the neuron "sees" through the subset of the network impinging on it in the immediate past, the same synaptic event can evoke a postsynaptic spike or just a subthreshold depolarization. We propose a formal definition of History-Dependent Excitability (HDE as a measure of the propensity to firing in any moment in time, linking the subthreshold history-dependent dynamics with spike generation. This definition allows the quantitative assessment of the intrinsic memory for different single-neuron dynamics and input statistics. We illustrate the concept of HDE by considering two general dynamical mechanisms: the passive behavior of an Integrate and Fire (IF neuron, and the inductive behavior of a Generalized Integrate and Fire (GIF neuron with subthreshold damped oscillations. This framework allows us to characterize the sensitivity of different model neurons to the detailed temporal structure of incoming stimuli. While a neuron with intrinsic oscillations discriminates equally well between input trains with the same or different frequency, a passive neuron discriminates better between inputs with different frequencies. This suggests that passive neurons are better suited to rate-based computation, while neurons with subthreshold oscillations are advantageous in a temporal coding scheme. We also address the influence of intrinsic properties in single-cell processing as a function of input statistics, and show that intrinsic oscillations enhance discrimination sensitivity at high input rates. Finally, we discuss how the recognition of these cell-specific discrimination properties might further our understanding of neuronal network computations and their relationships to the distribution and

  18. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

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    Xinna Li

    Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

  19. Particle Size-Dependent Antibacterial Activity and Murine Cell Cytotoxicity Induced by Graphene Oxide Nanomaterials

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    Lin Zhao

    2016-01-01

    Full Text Available Recent studies have indicated that graphene and its derivative graphene oxide (GO engage in a wide range of antibacterial activities with limited toxicity to human cells. Here, we systematically evaluate the dependence of GO toxicity on the size of the nanoparticles used in treatments: we compare the cytotoxic effects of graphene quantum dots (GQDs, <15 nm, small GOs (SGOs, 50–200 nm, and large GOs (LGOs, 0.5–3 μm. We synthesize the results of bacterial colony count assays and SEM-based observations of morphological changes to assess the antibacterial properties that these GOs bring into effect against E. coli. We also use Live/Dead assays and morphological analysis to investigate changes to mammalian (Murine macrophage-like Raw 264.7 cells induced by the presence of the various GO particle types. Our results demonstrate that LGOs, SGOs, and GQDs possess antibacterial activities and cause mammalian cell cytotoxicity at descending levels of potency. Placing our observations in the context of previous simulation results, we suggest that both the lateral size and surface area of GO particles contribute to cytotoxic effects. We hope that the size dependence elucidated here provides a useful schematic for tuning GO-cell interactions in biomedical applications.

  20. XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

    DEFF Research Database (Denmark)

    Yabal, Monica; Müller, Nicole; Adler, Heiko;

    2014-01-01

    X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss...... but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1...... of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3...

  1. Estrogen Receptor-Dependent Regulation of Dendritic Cell Development and Function

    Science.gov (United States)

    Laffont, Sophie; Seillet, Cyril; Guéry, Jean-Charles

    2017-01-01

    Autoimmunity, infectious diseases and cancer affect women and men differently. Because they tend to develop more vigorous adaptive immune responses than men, women are less susceptible to some infectious diseases but also at higher risk of autoimmunity. The regulation of immune responses by sex-dependent factors probably involves several non-redundant mechanisms. A privileged area of study, however, concerns the role of sex steroid hormones in the biology of innate immune cells, especially dendritic cells (DCs). In recent years, our understanding of the lineage origin of DC populations has expanded, and the lineage-committing transcription factors shaping peripheral DC subsets have been identified. Both progenitor cells and mature DC subsets express estrogen receptors (ERs), which are ligand-dependent transcription factors. This suggests that estrogens may contribute to the reported sex differences in immunity by regulating DC biology. Here, we review the recent literature and highlight evidence that estrogen-dependent activation of ERα regulates the development or the functional responses of particular DC subsets. The in vitro model of GM-CSF-induced DC differentiation shows that CD11c+ CD11bint Ly6cneg cells depend on ERα activation by estrogen for their development, and for the acquisition of competence to activate naive CD4+ T lymphocytes and mount a robust pro-inflammatory cytokine response to CD40 stimulation. In this model, estrogen signaling in conjunction with GM-CSF is necessary to promote early interferon regulatory factor (Irf)-4 expression in macrophage-DC progenitors and their subsequent differentiation into IRF-4hi CD11c+ CD11bint Ly6cneg cells, closely related to the cDC2 subset. The Flt3L-induced model of DC differentiation in turn shows that ERα signaling promotes the development of conventional DC (cDC) and plasmacytoid DC (pDC) with higher capability of pro-inflammatory cytokine production in response to TLR stimulation. Likewise, cell

  2. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  3. Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji, E-mail: kzsuzuki@nagasaki-u.ac.jp [Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Kodama, Seiji [Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-machi, Sakai 599-8570 (Japan); Watanabe, Masami [Kyoto University Research Reactor Institute, Kumatori-cho Sennan-gun, Osaka 590-0494 (Japan)

    2010-01-05

    Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.

  4. Entry of human papillomavirus type 16 by actin-dependent, clathrin- and lipid raft-independent endocytosis.

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    Mario Schelhaas

    Full Text Available Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16, the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na⁺/H⁺ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

  5. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    Science.gov (United States)

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-08-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells.

  6. Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Tadafumi Yokoyama

    Full Text Available The Wiskott-Aldrich syndrome (WAS is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg obtained from Was gene knockout (WKO mice and found that their numbers were significantly lower in these mice compared to wild type (WT controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

  7. Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

    Science.gov (United States)

    Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

    2016-09-01

    Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

  8. Bonzo/CXCR6 expression defines type 1–polarized T-cell subsets with extralymphoid tissue homing potential

    OpenAIRE

    2001-01-01

    Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell–dependent upregulation, while IL-4 inhibits it. In bloo...

  9. Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

    NARCIS (Netherlands)

    di Pietro, Alessandra; Koster, Roelof; Boersma-van Eck, Wytske; Dam, Wendy A.; Mulder, Nanno H.; Gietema, Jourik A.; de Vries, Elisabeth G. E.; de Jong, Steven

    2012-01-01

    In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivi

  10. Proton and Fe Ion-Induced Early and Late Chromosome Aberrations in Different Cell Types

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Yeshitla, Samrawit; Bowler, Deborah; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

    2016-01-01

    Genomic instability, induced by various metabolic, genetic, and environmental factors, is the driving force of tumorigenesis. Radiation exposure from different types of radiation sources induces different types of DNA damages, increases mutation and chromosome aberration rates, and increases cellular transformation in vitro and in vivo experiments. The cell survival rates and frequency of chromosome aberrations depend on the genetic background and radiation sources. To further understand genomic instability induced by charged particles, we exposed human lymphocytes ex vivo, human fibroblast cells, human mammary epithelial cells, and bone marrow cells isolated from CBA/CaH and C57BL/6 mice to high energy protons and Fe ions, and collected chromosomes at different generations after exposure. Chromosome aberrations were analyzed with fluorescent in situ hybridization with whole chromosome specific probes.

  11. Chemotransduction in the Carotid Body: K+ Current Modulated by Po2 in Type I Chemoreceptor Cells

    Science.gov (United States)

    Lopez-Barneo, Jose; Lopez-Lopez, Jose R.; Urena, Juan; Gonzalez, Constancio

    1988-07-01

    The ionic currents of carotid body type I cells and their possible involvement in the detection of oxygen tension (Po2) in arterial blood are unknown. The electrical properties of these cells were studied with the whole-cell patch clamp technique, and the hypothesis that ionic conductances can be altered by changes in Po2 was tested. The results show that type I cells have voltage-dependent sodium, calcium, and potassium channels. Sodium and calcium currents were unaffected by a decrease in Po2 from 150 to 10 millimeters of mercury, whereas, with the same experimental protocol, potassium currents were reversibly reduced by 25 to 50 percent. The effect of hypoxia was independent of internal adenosine triphosphate and calcium. Thus, ionic conductances, and particularly the O2-sensitive potassium current, play a key role in the transduction mechanism of arterial chemoreceptors.

  12. Endoplasmic Reticulum Stress in the β-Cell Pathogenesis of Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Sung Hoon Back

    2012-01-01

    Full Text Available Type 2 diabetes is a complex metabolic disorder characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency by β-cell failure. Even if the mechanisms underlying the pathogenesis of β-cell failure are still under investigation, recent increasing genetic, experimental, and clinical evidence indicate that hyperactivation of the unfolded protein response (UPR to counteract metabolic stresses is closely related to β-cell dysfunction and apoptosis. Signaling pathways of the UPR are “a double-edged sword” that can promote adaptation or apoptosis depending on the nature of the ER stress condition. In this paper, we summarized our current understanding of the mechanisms and components related to ER stress in the β-cell pathogenesis of type 2 diabetes.

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

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

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

  14. Time-Dependent Toroidal Compactification Proposals and the Bianchi Type I Model: Classical and Quantum Solutions

    Directory of Open Access Journals (Sweden)

    L. Toledo Sesma

    2016-01-01

    Full Text Available We construct an effective four-dimensional model by compactifying a ten-dimensional theory of gravity coupled with a real scalar dilaton field on a time-dependent torus. This approach is applied to anisotropic cosmological Bianchi type I model for which we study the classical coupling of the anisotropic scale factors with the two real scalar moduli produced by the compactification process. Under this approach, we present an isotropization mechanism for the Bianchi I cosmological model through the analysis of the ratio between the anisotropic parameters and the volume of the Universe which in general keeps constant or runs into zero for late times. We also find that the presence of extra dimensions in this model can accelerate the isotropization process depending on the momenta moduli values. Finally, we present some solutions to the corresponding Wheeler-DeWitt (WDW equation in the context of standard quantum cosmology.

  15. The Tool Life of Ball Nose end Mill Depending on the Different Types of Ramping

    Directory of Open Access Journals (Sweden)

    Vopát Tomáš

    2014-12-01

    Full Text Available The article deals with the cutting tool wear measurement process and tool life of ball nose end mill depending on upward ramping and downward ramping. The aim was to determine and compare the wear (tool life of ball nose end mill for different types of copy milling operations, as well as to specify particular steps of the measurement process. In addition, we examined and observed cutter contact areas of ball nose end mill with machined material. For tool life test, DMG DMU 85 monoBLOCK 5-axis CNC milling machine was used. In the experiment, cutting speed, feed rate, axial depth of cut and radial depth of cut were not changed. The cutting tool wear was measured on Zoller Genius 3s universal measuring machine. The results show different tool life of ball nose end mills depending on the copy milling strategy.

  16. On a Camassa-Holm type equation with two dependent variables

    Energy Technology Data Exchange (ETDEWEB)

    Falqui, Gregorio [SISSA, Via Beirut 2/4, I-34014 Trieste (Italy)

    2006-01-13

    We consider a generalization of the Camassa-Holm (CH) equation with two dependent variables, called CH2, introduced in a paper by Liu and Zhang (Liu S-Q and Zhang Y 2005 J. Geom. Phys. 54 427-53). We briefly provide an alternative derivation of it based on the theory of Hamiltonian structures on (the dual of) a Lie algebra. The Lie algebra involved here is the same algebra as underlies the NLS hierarchy. We study the structural properties of the hierarchy defined by the CH2 equation within the bi-Hamiltonian theory of integrable PDEs, and provide its Lax representation. Then we explicitly discuss how to construct classes of solutions, both of peakon and of algebro-geometrical type. Finally we sketch the construction of a class of singular solutions, defined by setting to zero one of the two dependent variables.

  17. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  18. Time-dependent regulation of yeast glycolysis upon nitrogen starvation depends on cell history

    NARCIS (Netherlands)

    van Eunen, K.; Dool, P.; Canelas, A. B.; Kiewiet, J.; Bouwman, J.; van Gulik, W. M.; Westerhoff, H. V.; Bakker, B. M.

    2010-01-01

    In this study, the authors investigated how the glycolytic flux was regulated in time upon nitrogen starvation of cells with different growth histories. We have compared cells grown in glucose-limited chemostat cultures under respiratory conditions (low dilution rate of 0.1/h) to cells grown under r

  19. 17beta-hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone-dependent breast cancer.

    Science.gov (United States)

    Day, Joanna M; Foster, Paul A; Tutill, Helena J; Parsons, Michael F C; Newman, Simon P; Chander, Surinder K; Allan, Gillian M; Lawrence, Harshani R; Vicker, Nigel; Potter, Barry V L; Reed, Michael J; Purohit, Atul

    2008-05-01

    Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

  20. Engineering controlled mammalian type O-Glycosylation in plant cells

    DEFF Research Database (Denmark)

    Yang, Zhang; Drew, Damian Paul; Jørgensen, Bodil

    2011-01-01

    Human mucins are large heavily O-glycosylated glycoproteins (>200 kDa), which account for the majority of proteins in mucus layers that e.g. hydrate, lubricate and protect cells from proteases as well as from pathogens. O-linked mucin glycans are truncated in many cancers, yielding truncated cancer...... specific glyco-peptide epitopes, such as the Tn epitope (GalNAc sugar attached to either Serine or Threonine), which are antigenic to the immune system. In the present study, we have identified plant cells as the only eukaryotic cells without mammalian type O-glycosylation or competing (for sites) O...

  1. Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner

    Science.gov (United States)

    Arulmoli, Janahan; Pathak, Medha M.; McDonnell, Lisa P.; Nourse, Jamison L.; Tombola, Francesco; Earthman, James C.; Flanagan, Lisa A.

    2015-02-01

    Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.

  2. Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection.

    Directory of Open Access Journals (Sweden)

    Darin L Wiesner

    2015-03-01

    Full Text Available Pulmonary mycoses are often associated with type-2 helper T (Th2 cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

  3. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  4. Cell type mediated resistance of vesicular stomatitis virus and Sendai virus to ribavirin.

    Science.gov (United States)

    Shah, Nirav R; Sunderland, Amanda; Grdzelishvili, Valery Z

    2010-06-22

    Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.

  5. Cell type mediated resistance of vesicular stomatitis virus and Sendai virus to ribavirin.

    Directory of Open Access Journals (Sweden)

    Nirav R Shah

    Full Text Available Ribavirin (RBV is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus and Sendai virus (SeV, a paramyxovirus. Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.

  6. Porosity dependence of positive magnetoconductance in n-type porous silicon

    Energy Technology Data Exchange (ETDEWEB)

    Chouaibi, Bassem; Radaoui, Moufid; Benfredj, Amel; Bouchriha, Habib [Laboratoire Materiaux Avances et Phenomenes Quantiques, Faculte des Sciences de Tunis, Universite El Manar, 2092 Campus universitaire, Tunis (Tunisia); Romdhane, Samir [Laboratoire Materiaux Avances et Phenomenes Quantiques, Faculte des Sciences de Tunis, Universite El Manar, 2092 Campus universitaire, Tunis (Tunisia); Faculte des Sciences de Bizerte, 7021 Zarzouna, Bizerte, Universite de Carthage (Tunisia); Bouaicha, Mongi [Laboratoire de Photovoltaique, Centre de Recherches et des Technologies de l' Energie, BP 95, Hammam-Lif 2050 (Tunisia)

    2012-10-15

    Positive magnetoconductance (MC) on n-type porous silicon (PS) based devices was observed at room temperature for low static magnetic field (under 6000 G). We found that the measured MC decreases when the film porosity is increased. Obtained results were analyzed by means of the quasi-1D weak localization (WL) theory. From the dependence of the MC vs. applied magnetic field, we determine the phase coherence length L{sup {phi}}. Good agreement between theoretical and experimental results was found (copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  7. Bonzo/CXCR6 expression defines type 1–polarized T-cell subsets with extralymphoid tissue homing potential

    Science.gov (United States)

    Kim, Chang H.; Kunkel, Eric J.; Boisvert, Judie; Johnston, Brent; Campbell, James J.; Genovese, Mark C.; Greenberg, Harry B.; Butcher, Eugene C.

    2001-01-01

    Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell–dependent upregulation, while IL-4 inhibits it. In blood, 35–56% of Bonzo+ CD4 T cells are Th1 cells, and 60–65% of Bonzo+ CD8 T cells are Tc1 cells, while few Bonzo+ cells are type 2 T cells. Almost all Bonzo+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Most Bonzo+ T cells lack L-selectin and/or CCR7, homing receptors for lymphoid tissues. Instead, Bonzo+ T cells are dramatically enriched among T cells in tissue sites of inflammation, such as rheumatoid joints and inflamed livers. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases. PMID:11238560

  8. Plasma adrenaline kinetics in type 1 (insulin-dependent) diabetic patients with and without autonomic neuropathy

    DEFF Research Database (Denmark)

    Dejgaard, Anders; Hilsted, J; Henriksen, J H

    1989-01-01

    Plasma adrenaline kinetics (clearance, extraction across the forearm, initial plasma disappearance rate, mean sojourn time, volume of distribution) were studied in sixteen Type 1 (insulin-dependent) diabetic patients during constant i.v. infusion of tritium labelled adrenaline. In patients with (n...... = 8) and without (n = 8) neuropathy forearm venous plasma noradrenaline and adrenaline concentrations as well as plasma clearance of adrenaline based on arterial sampling (1.7 vs 2.1 l/min) were not significantly different. The initial disappearance time (T 1/2) after the infusion of the tritium...... labelled adrenaline had been stopped was significantly prolonged in Type 1 diabetic patients with neuropathy compared to those without (after 20 min infusion 2.7 vs 2.2 min, p less than 0.02, after 75 min infusion 3.7 vs 2.9 min, p less than 0.05). The corresponding values for the mean sojourn time...

  9. Impaired autoregulation of glomerular filtration rate in type 1 (insulin-dependent) diabetic patients with nephropathy

    DEFF Research Database (Denmark)

    Parving, H H; Kastrup, Helge; Smidt, U M

    1984-01-01

    The effect of acute lowering of arterial blood pressure upon kidney function in nephropathy was studied in 13 patients with long-term Type 1 (insulin-dependent) diabetes. Ten normal subjects (six normotensive and four hypertensive) and five short-term Type 1 diabetic patients without nephropathy...... micrograms) or saline (0.154 mmol/l). The arterial blood pressure was similar in the diabetic patients with nephropathy (mean 136 +/- 11 divided by 88 +/- mmHg) and in the non-diabetic control subjects (mean 140 +/- 25 divided by 92 +/- 15 mmHg). The clonidine injection induced similar reductions in mean...... excretion declined from 1707 to 938 micrograms/min (p less than 0.01) in the patients with diabetic nephropathy. Our results suggest that an intrinsic vascular (arteriolar) mechanism underlying the normal autoregulation of glomerular filtration rate, i.e. the relative constancy of glomerular filtration rate...

  10. Transcapillary escape rate of albumin in hypertensive patients with type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Nørgaard, K; Jensen, T; Feldt-Rasmussen, B

    1993-01-01

    . The systemic blood pressure and the transcapillary escape rate of albumin were measured in the following groups after 4 weeks without antihypertensive treatment: Group 1--eleven healthy control subjects. Group 2--ten Type 1 (insulin-dependent) diabetic patients with incipient nephropathy (urinary albumin......Diabetic patients with elevated urinary albumin excretion rate (incipient or clinical nephropathy) also have an increased transcapillary escape rate of albumin. This study was designed to clarify whether this is caused by a general vascular dysfunction or by elevated systemic blood pressure...... excretion rate: 30-300 mg/24 h) and normal blood pressure. Group 3--eleven non-diabetic patients with essential hypertension. Group 4--nine Type 1 diabetic patients with hypertension but normal urinary albumin excretion (diabetic patients with nephropathy (urinary...

  11. Direct evidence of fiber type-dependent GLUT-4 expression in human skeletal muscle

    DEFF Research Database (Denmark)

    Gaster, M; Poulsen, P; Handberg, A

    2000-01-01

    GLUT-4 expression in individual fibers of human skeletal muscles in younger and older adults was studied. Furthermore, the dependency of insulin-stimulated glucose uptake on fiber type distribution was investigated. Fiber type distribution was determined in cryosections of muscle biopsies from 8...... younger (29 yr) and 8 older (64 yr) healthy subjects, and estimates of GLUT-4 expression in individual fibers were obtained by combining immunohistochemistry and stereology. GLUT-4 was more abundantly expressed in slow compared with fast muscle fibers in both younger (P ...) subjects. A 25% reduction of GLUT-4 density in fast fibers (P GLUT-4 density in slow fibers were demonstrated in older compared with younger subjects. Insulin-stimulated glucose uptake rates measured by hyperinsulinemic, euglycemic clamp were not correlated with the fraction...

  12. Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Zeuthen, Louise Hjerrild; Ferlazzo, Guido

    2007-01-01

    , in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells......The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However...... (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and naïve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production...

  13. Metabolism Kinetics of Glucose in Anchorage-dependent Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    孙祥明; 张元兴

    2001-01-01

    The kinetic model of glucose metabolism was established and successfully applied to batchcultures of rCHO and rBHK cells. It was found that a large amount of glucose was utilized for cellmaintenance, and the overwhelming majority of maintenance energy from glucose was by its anaerobicmetabolism in both rBHK and rCHO cell cultures. The overall maintenance coefficients from aerobicmetabolism were 1.9×10-13 mmol/(cell.h) for rCHO cells and 7×10-13 mmol/(cell.h) for rBHK cells. Inaddition, all Go/T and Eo/T gradually increased with the same trend as the cell growth in the culture ofboth rCHO and rBHK cells. The overall molecule yield coefficients of lactate to glucose were 1.61 for rCHO cells and 1.38 for rBHK cells. The yield coefficients of cell to glucose were 4.5×108 cells/mmol for rCHO cells and 1.9 × 108 cells/mmol for rBHK cells, respectively.

  14. Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-I and LC3-II activation-dependent autophagy.

    Science.gov (United States)

    Huang, An-Cheng; Lien, Jin-Cherng; Lin, Meng-Wei; Yang, Jai-Sing; Wu, Ping-Ping; Chang, Shu-Jen; Lai, Tung-Yuan

    2013-08-01

    Numerous studies have demonstrated that autophagy is associated with cancer development. Thus, agents to induce autophagy could be employed in some cases for the treatment of cancer. Our results showed that tetrandrine significantly decreased the viability of SAS cells in a concentration- and time-dependent manner. Tetrandrine induced nuclear condensation, demonstrated by DAPI staining. The early events in apoptosis analysed by Annexin V/PI staining indicated that the percentage of cells staining positive for Annexin V was slightly increased in SAS cells with tetrandrine treatment but was much lower following bafilomycin A1 pre-treatment. Tetrandrine caused AVO and MDC induction in SAS cells in a concentration-dependent manner by fluorescence microscopy. Tetrandrine also caused LC-3 expression in SAS cells in a time-dependent manner. Our results show that tetrandrine treatment induced the levels of cleaved caspase-3 in a concentration- and time-dependent manner. Tetrandrine treatment induced the levels of LC-3 II, Atg-5, beclin-1, p-S6, p-ULK, p-mTOR, p-Akt (S473) and raptor. Tetrandrine decreased cell viability, but bafilomycin A1, 3-MA, chloroquine and NAC protected tetrandrine-treated SAS cells against decrease of cell viability. Atg-5, beclin-1 siRNA decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells and protected tetrandrine-treated SAS cells against decrease in cell viability. Chloroquine, NAC and bafilomycin A1 also decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells. Our results indicate the tetrandrine induces apoptosis and autophagy of SAS human cancer cells via caspase-dependent and LC3-I and LC3-II‑dependent pathways.

  15. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways.

    Science.gov (United States)

    Berkowska, Magdalena A; Driessen, Gertjan J A; Bikos, Vasilis; Grosserichter-Wagener, Christina; Stamatopoulos, Kostas; Cerutti, Andrea; He, Bing; Biermann, Katharina; Lange, Johan F; van der Burg, Mirjam; van Dongen, Jacques J M; van Zelm, Menno C

    2011-08-25

    Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27⁻IgG⁺ and CD27⁺IgM⁺ B cells are derived from primary germinal center reactions, and CD27⁺IgA⁺ and CD27⁺IgG⁺ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27⁻IgA⁺ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27⁻IgA⁺ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

  16. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China); Fujisaki, Hitomi; Hattori, Shunji [Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017 (Japan); Tashiro, Shin-ichi [Institute for Clinical and Biomedical Sciences, Kyoto 603-8072 (Japan); Onodera, Satoshi [Department of Clinical and Pharmaceutical Sciences, Showa Pharmaceutical University, Tokyo 194-8543 (Japan); Ikejima, Takashi, E-mail: ikejimat@vip.sina.com [China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016 (China)

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  17. Soluble interleukin-15 complexes are generated in vivo by type I interferon dependent and independent pathways.

    Directory of Open Access Journals (Sweden)

    Scott M Anthony

    Full Text Available Interleukin (IL-15 associates with IL-15Rα on the cell surface where it can be cleaved into soluble cytokine/receptor complexes that have the potential to stimulate CD8 T cells and NK cells. Unfortunately, little is known about the in vivo production of soluble IL-15Rα/IL-15 complexes (sIL-15 complexes, particularly regarding the circumstances that induce them and the mechanisms responsible. The main objective of this study was to elucidate the signals leading to the generation of sIL-15 complexes. In this study, we show that sIL-15 complexes are increased in the serum of mice in response to Interferon (IFN-α. In bone marrow derived dendritic cells (BMDC, IFN-α increased the activity of ADAM17, a metalloproteinase implicated in cleaving IL-15 complexes from the cell surface. Moreover, knocking out ADAM17 in BMDCs prevented the ability of IFN-α to induce sIL-15 complexes demonstrating ADAM17 as a critical protease mediating cleavage of IL-15 complexes in response to type I IFNs. Type I IFN signaling was required for generating sIL-15 complexes as in vivo induction of sIL-15 complexes by Poly I:C stimulation or total body irradiation (TBI was impaired in IFNAR-/- mice. Interestingly, serum sIL-15 complexes were also induced in mice infected with Vesicular stomatitis virus (VSV or mice treated with agonistic CD40 antibodies; however, sIL-15 complexes were still induced in IFNAR-/- mice after VSV infection or CD40 stimulation indicating pathways other than type I IFNs induce sIL-15 complexes. Overall, this study has shown that type I IFNs, VSV infection, and CD40 stimulation induce sIL-15 complexes suggesting the generation of sIL-15 complexes is a common event associated with immune activation. These findings reveal an unrealized mechanism for enhanced immune responses occurring during infection, vaccination, inflammation, and autoimmunity.

  18. [CHARACTERISTICS OF COMBINED ANESTHESIA WITH EPIDURAL COMPONENTE DEPENDING ON VEGETATIVE NERVOUS SYSTEM TYPE].

    Science.gov (United States)

    Hasanov, F J; Aslanov, A A; Muradov, N F; Namazova, K N

    2016-01-01

    The research objective was to study the characteristics of combined anesthesia with epidural componente (CAEC) depending on vegetative nervous system type (VNS) in patients who underwent large scale traumatic surgical operations on abdominal cavity organs. The scientific research was conducted in Anaesthesiology--Reanimation Department of the Scientific Surgical Centre named after acad. MA. Topchubashev, the Ministry of Health of the Azerbaijan Republic. The research objects were 69 patients who underwent operations in conditions of CAEC due to different serious surgical pathologies of abdominal cavity organs. VNS type was identified based on electroencephalogram, Cerdo Vegetative Index (CVI), Hildebrandt coefficient (HC) and single neurophysiological tests. The patients were divided into three groups depending on VNS type: I--normotonics--17 patients (24.7%), II--sympathatonics--25 patients (36.2%), and III--vagotonics--27 patients (39.1%). Blood adrenocorticotropic hormone (ACTH) and cortisol concentration were studied in 3 stages: I -preoperative, II--operation traumatic stage, III--the 1st postoperative days. The other indicators (heart rate, systolic blood pressure--SBP, dyastolic blood pressure--DBR average blood pressure--BP ave., pulse oximetry SpO₂, ECG, gases in blood and acid-base balance, electrolytes, blood glucose level, myocardium oxygen demand--MOD) were registered after 20 minutes and the 2nd day after operation besides the above stages. The research results indicated that it is possible to define the vegetative nervous system type superiority based on complex of single tests data, EEG, ECG, Cerdo Vegetative Index, Hildebrandt coefficient. CAEC can be considered optimun alternative of general anesthesia ensuring neurohumoral and hemodynamic stability in large scale, traumatic operations on abdominal cavity organs. Clinical course of CAEC is characterized by firmer hemodynamic and humoral stability in patients with functional balance of

  19. Spin-dependent transport and recombination in solar cells studied by pulsed electrically detected magnetic resonance

    Energy Technology Data Exchange (ETDEWEB)

    Behrends, Jan

    2009-11-11

    This thesis deals with spin-dependent transport and recombination of charge carriers in solar cells. A systematic study on the influence of localized paramagnetic states which act as trapping and recombination centres for photogenerated charge carriers, is presented for three different types of solar cells. The central technique used in this thesis is electrically detected magnetic resonance (EDMR). The capabilities of pulsed (p) EDMR were extended with regard to the detection sensitivity. These improvements allowed pEDMR measurements on fully processed devices from cryogenic to room temperature. The instrumental upgrades also set the stage for pEDMR measurements at different resonance frequencies. In high-efficiency solar cells based on the heterojunction between hydrogenated amorphous silicon (a-Si:H) and crystalline silicon (c-Si), recombination via performancelimiting interface states could directly be measured electrically for the first time. The identification of these defects could be achieved by exploiting their orientation with regard to the surface. In thin-film solar cells based on hydrogenated microcrystalline silicon ({mu}-Si:H) the situation is more complex due to the heterogeneous and disordered structure of the material itself. In addition, these cells are multilayer-systems comprising three different silicon layers with different doping levels and microstructures. By combining a systematic alteration of the sample structure with the information extracted from deconvoluting spectrally overlapping signals in the time domain, it was possible to assign the spin-dependent signals to defects in the individual layers of the solar cells. Benefiting from the instrumental improvements, recombination via dangling bond states in silicon-based solar cells could be investigated by pEDMR at room temperature for the first time. In organic bulk heterojunction solar cells based on MEH-PPV and PCBM two different spin-dependent mechanisms coexist. Both processes

  20. Single-cell LEP-type cavity on measurement stand

    CERN Multimedia

    1982-01-01

    A single-cell cavity, made of copper, with tapered connectors for impedance measurements. It was used as a model of LEP-type superconducting cavities, to investigate impedance and higher-order modes and operated at around 600 MHz (the LEP acceleration frequency was 352.2 MHz). See 8202500.

  1. The role of emotions for moral judgments depends on the type of emotion and moral scenario.

    Science.gov (United States)

    Ugazio, Giuseppe; Lamm, Claus; Singer, Tania

    2012-06-01

    Emotions seem to play a critical role in moral judgment. However, the way in which emotions exert their influence on moral judgments is still poorly understood. This study proposes a novel theoretical approach suggesting that emotions influence moral judgments based on their motivational dimension. We tested the effects of two types of induced emotions with equal valence but with different motivational implications (anger and disgust), and four types of moral scenarios (disgust-related, impersonal, personal, and beliefs) on moral judgments. We hypothesized and found that approach motivation associated with anger would make moral judgments more permissible, while disgust, associated with withdrawal motivation, would make them less permissible. Moreover, these effects varied as a function of the type of scenario: the induced emotions only affected moral judgments concerning impersonal and personal scenarios, while we observed no effects for the other scenarios. These findings suggest that emotions can play an important role in moral judgment, but that their specific effects depend upon the type of emotion induced. Furthermore, induced emotion effects were more prevalent for moral decisions in personal and impersonal scenarios, possibly because these require the performance of an action rather than making an abstract judgment. We conclude that the effects of induced emotions on moral judgments can be predicted by taking their motivational dimension into account. This finding has important implications for moral psychology, as it points toward a previously overlooked mechanism linking emotions to moral judgments.

  2. Susceptibility of different leukocyte cell types to Vaccinia virus infection

    Directory of Open Access Journals (Sweden)

    Sánchez-Puig Juana M

    2004-11-01

    Full Text Available Abstract Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP, and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.

  3. Innate immune response to pulmonary contusion: identification of cell type-specific inflammatory responses.

    Science.gov (United States)

    Hoth, J Jason; Wells, Jonathan D; Yoza, Barbara K; McCall, Charles E

    2012-04-01

    Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma, such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety of inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll-like receptors 2 and 4 (TLR2 and TLR4) mediate the inflammatory response to lung injury. In this study, we used chimeric mice generated by adoptive bone marrow transfer between TLR2 or TLR4 and wild-type mice. We found that, in the lung, both bone marrow-derived and nonmyeloid cells contribute to TLR-dependent inflammatory responses after injury in a cell type-specific manner. We also show a novel TLR2-dependent injury mechanism that is associated with enhanced airway epithelial cell apoptosis and increased pulmonary FasL and Fas expression in the lungs from injured mice. Thus, in addition to cardiopulmonary physiological dysfunction, cell type-specific TLR and their differential response to injury may provide novel specific targets for management of patients with pulmonary contusion.

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

    Science.gov (United States)

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

    2011-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Eric Oswald

    2011-03-01

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

  6. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    Science.gov (United States)

    2005-01-01

    sequencing or hybridization array capillary chromatography. After a 6-min-long preliminary technologies.30,31 separation, fractions from the first...characterize single cells. These tools include mass cating cells contain diploid, S-phase and tetraploid frac- spectrometry, electrochemistry and capillary...separation tions; and some advanced tumors contain tetraploid and methods. This review focuses on the use of capillary aneuploid cells [2

  7. Novel 8-hydroxylquinoline analogs induce copper-dependent proteasome inhibition and cell death in human breast cancer cells.

    Science.gov (United States)

    Milacic, Vesna; Jiao, Peifu; Zhang, Bin; Yan, Bing; Dou, Q Ping

    2009-12-01

    An elevated level of copper (Cu), which is necessary for the growth and metastasis of tumor cells, has been found in many types of cancer, including breast, prostate, lung and brain. Although its molecular basis is unclear, this tumor-specific Cu elevation has been proposed to be a novel target for developing selective anti-cancer therapies. We previously reported that 8-hydroxylquinoline (8-OHQ) is able to form a Cu complex that inhibits the proteasome and induces apoptosis in cultured cancer cells. Toward the goal of discovering novel 8-OHQ analogs as potential anti-copper and anti-cancer drugs, in the current study we synthesized several 8-OHQ analogs and their copper complexes and evaluated their biological activities in human breast cancer cells. We report that when substitutions are made on the hydroxyl group of 8-OHQ, their copper mixtures have profound effects on the proteasome-inhibitory and apoptosis-inducing abilities in breast cancer MDA-MB-231 cells. In addition, the proteasome-inhibitory and apoptosis-inducing activities of 8-OHQ analog-copper mixtures are determined by both the polarity and position of the substituents. Finally, a synthetic complex of 8-OHQ analog-copper was able to inhibit the proteasome activity, induce cell death and suppress the growth selectively in breast cancer MDA-MB-231 cells, but not in normal immortalized human breast MCF-10A cells. Our results support the concept that human cancer cells and tissues, which contain an elevated copper level and are highly dependent on proteasome activity for their survival, should be sensitive to treatment with anti-copper drugs such as the novel 8-OHQ analogs described here.

  8. Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Qian Wang

    Full Text Available Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

  9. CD117+ Dendritic and Mast Cells Are Dependent on RasGRP4 to Function as Accessory Cells for Optimal Natural Killer Cell-Mediated Responses to Lipopolysaccharide.

    Directory of Open Access Journals (Sweden)

    Saijun Zhou

    Full Text Available Ras guanine nucleotide-releasing protein-4 (RasGRP4 is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117+ mast cells (MCs, its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117+ dendritic cells (DCs in wild-type (WT C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117+ DCs from WT mice induced natural killer (NK cells to produce much more interferon-γ (IFN-γ than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117+ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.

  10. Context-dependent wiring of Sox2 regulatory networks for self-renewal of embryonic and trophoblast stem cells.

    Science.gov (United States)

    Adachi, Kenjiro; Nikaido, Itoshi; Ohta, Hiroshi; Ohtsuka, Satoshi; Ura, Hiroki; Kadota, Mitsutaka; Wakayama, Teruhiko; Ueda, Hiroki R; Niwa, Hitoshi

    2013-11-07

    Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential role in different types of multipotent stem cells, raising the possibility that Sox2 governs the common stemness phenotype. Here we show that Sox2 is a critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal of trophoblast stem cells (TSCs). Sustained expression of Sox2 together with Esrrb or Tfap2c can replace FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and TSCs combined with inducible knockout systems, we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct sets of genes with unique functions in these two different yet developmentally related types of stem cells. Our findings provide insights into the functional versatility of transcription factors during embryogenesis, during which they can be recursively utilized in a variable manner within discrete network structures.

  11. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    Science.gov (United States)

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-04

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  12. Galangin induces human colon cancer cell death via the mitochondrial dysfunction and caspase-dependent pathway.

    Science.gov (United States)

    Ha, Tae Kwun; Kim, Mi Eun; Yoon, Ju Hwa; Bae, Sung Jin; Yeom, Jihye; Lee, Jun Sik

    2013-09-01

    Galangin is a member of flavonols and found in Alpinia officinarum, galangal root, and propolis. Previous studies have demonstrated that galangin has anti-cancer effects on several cancers, including melanoma, hepatoma, and leukaemia cells. However, anti-cancer activity of galangin on human colon cancer has not been established yet. In this study, we investigated the anti-cancer effects of galangin on two types of human colon cancer cells (HCT-15 and HT-29). We found that galangin induced apoptosis and DNA condensation of human colon cancer cells in a dose-dependent manner. We also determined that galangin increased the activation of caspase-3 and -9, and release of apoptosis inducing factor from the mitochondria into the cytoplasm by Western blot analysis. In addition, galangin induced human colon cancer cell death through the alteration of mitochondria membrane potential and dysfunction. These results suggest that galangin induces apoptosis of HCT-15 and HT-29 human colon cancer cells and may prove useful in the development of therapeutic agents for human colon cancer.

  13. Widespread FRA1-dependent control of mesenchymal transdifferentiation programs in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Jeannine Diesch

    Full Text Available Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs, and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.

  14. Trefoil factor 3 peptide regulates migration via a Twist-dependent pathway in gastric cell.

    Science.gov (United States)

    Zheng, Qianqian; Gao, Jian; Li, Honglin; Guo, Wendong; Mao, Qi; Gao, Enhui; Zhu, Ya-qin

    2013-08-16

    Trefoil factor 3 (TFF3) is a member of the TFF-domain peptide family and essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract. However, the role of TFF3 and its downstream regulating mechanisms in cancer cell migration remain unclear. We previously reported that TFF3 prolonged the up-regulation of Twist protein to modulate IL-8 secretion in intestinal epithelial cells. In this study, we investigated the role of Twist protein in TFF3-induced migration of SGC7901 cells. While Twist was activated by TFF3, siRNA-mediated knockdown of Twist abolished TFF3-induced cell migration. Furthermore, the migration related marker CK-8 as well as ZO-1 and MMP-9 was also regulated by TFF3 via a Twist-dependent mechanism. Our study suggests that Twist, as an important potential downstream effector, plays a key role in TFF3-modulated metastasis in gastric cancer and can be a promising therapeutic target against intestinal-type gastric cancer.

  15. The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells.

    Science.gov (United States)

    Wolff, Svenja; Groseth, Allison; Meyer, Bjoern; Jackson, David; Strecker, Thomas; Kaufmann, Andreas; Becker, Stephan

    2016-04-01

    The Arenaviridae is a diverse and growing family of viruses that already includes more than 25 distinct species. While some of these viruses have a significant impact on public health, others appear to be non-pathogenic. At present little is known about the host cell responses to infection with different arenaviruses, particularly those found in the New World; however, apoptosis is known to play an important role in controlling infection of many viruses. Here we show that infection with Tacaribe virus (TCRV), which is widely considered the prototype for non-pathogenic arenaviruses, leads to stronger induction of apoptosis than does infection with its human-pathogenic relative Junín virus. TCRV-induced apoptosis occurred in several cell types during late stages of infection and was shown to be caspase-dependent, involving the activation of caspases 3, 7, 8 and 9. Further, UV-inactivated TCRV did not induce apoptosis, indicating that the activation of this process is dependent on active viral replication/transcription. Interestingly, when apoptosis was inhibited, growth of TCRV was not enhanced, indicating that apoptosis does not have a direct negative effect on TCRV infection in vitro. Taken together, our data identify and characterize an important virus-host cell interaction of the prototypic, non-pathogenic arenavirus TCRV, which provides important insight into the growing field of arenavirus research aimed at better understanding the diversity in responses to different arenavirus infections and their functional consequences.

  16. The Dependence of Type Ia Supernova Luminosities on their Host Galaxies

    CERN Document Server

    Sullivan, M; Howell, D A; Neill, J D; Astier, P; Balland, C; Basa, S; Carlberg, R G; Fouchez, D; Guy, J; Hardin, D; Hook, I M; Pain, R; Palanque-Delabrouille, N; Perrett, K M; Pritchet, C J; Regnault, N; Rich, J; Ruhlmann-Kleider, V; Baumont, S; Hsiao, E; Kronborg, T; Lidman, C; Perlmutter, S; Walker, E S

    2010-01-01

    (Abridged) Precision cosmology with Type Ia supernovae (SNe Ia) makes use of the fact that SN Ia luminosities depend on their light-curve shapes and colours. Using Supernova Legacy Survey (SNLS) and other data, we show that there is an additional dependence on the global characteristics of their host galaxies: events of the same light-curve shape and colour are, on average, 0.08mag (~4.0sigma) brighter in massive host galaxies (presumably metal-rich) and galaxies with low specific star-formation rates (sSFR). SNe Ia in galaxies with a low sSFR also have a smaller slope ("beta") between their luminosities and colours with ~2.7sigma significance, and a smaller scatter on SN Ia Hubble diagrams (at 95% confidence), though the significance of these effects is dependent on the reddest SNe. SN Ia colours are similar between low-mass and high-mass hosts, leading us to interpret their luminosity differences as an intrinsic property of the SNe and not of some external factor such as dust. If the host stellar mass is in...

  17. Rapid diagnosis of vitamin D-dependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes.

    Science.gov (United States)

    Takeda, E; Kuroda, Y; Saijo, T; Toshima, K; Naito, E; Kobashi, H; Iwakuni, Y; Miyao, M

    1986-03-28

    The interactions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with phytohemagglutinin (PHA)-stimulated lymphocytes from normal subjects and three patients with vitamin D-dependent rickets (DDR) type II were investigated. Impaired nuclear uptake and normal cytosol binding of [3H]1,25-(OH)2D3 were observed with PHA-stimulated lymphocytes of these patients as with their cultured skin fibroblasts. Furthermore, the incorporation of [14C]thymidine into PHA-stimulated lymphocytes of the patients was not reduced by 1,25-(OH)2D3, which is known to inhibit proliferation of various cells. These findings suggest that 1,25-(OH)2D3 receptors are reduced or absent in patients with DDR type II. Thus, the capacities of cytosol binding and nuclear uptake of 1,25-(OH)2D3 in PHA-stimulated lymphocytes seem to reflect those of endo-organs such as the intestine and bone. These findings show that a test of the effect of 1,25-(OH)2D3 on thymidine incorporation into PHA-stimulated lymphocytes is useful for rapid diagnosis of DDR type II.

  18. Single-cell RNA sequencing identifies distinct mouse medial ganglionic eminence cell types

    Science.gov (United States)

    Chen, Ying-Jiun J.; Friedman, Brad A.; Ha, Connie; Durinck, Steffen; Liu, Jinfeng; Rubenstein, John L.; Seshagiri, Somasekar; Modrusan, Zora

    2017-01-01

    Many subtypes of cortical interneurons (CINs) are found in adult mouse cortices, but the mechanism generating their diversity remains elusive. We performed single-cell RNA sequencing on the mouse embryonic medial ganglionic eminence (MGE), the major birthplace for CINs, and on MGE-like cells differentiated from embryonic stem cells. Two distinct cell types were identified as proliferating neural progenitors and immature neurons, both of which comprised sub-populations. Although lineage development of MGE progenitors was reconstructed and immature neurons were characterized as GABAergic, cells that might correspond to precursors of different CINs were not identified. A few non-neuronal cell types were detected, including microglia. In vitro MGE-like cells resembled bona fide MGE cells but expressed lower levels of Foxg1 and Epha4. Together, our data provide detailed understanding of the embryonic MGE developmental program and suggest how CINs are specified. PMID:28361918

  19. Substrate-dependent cell elasticity measured by optical tweezers indentation

    Science.gov (United States)

    Yousafzai, Muhammad S.; Ndoye, Fatou; Coceano, Giovanna; Niemela, Joseph; Bonin, Serena; Scoles, Giacinto; Cojoc, Dan

    2016-01-01

    In the last decade, cell elasticity has been widely investigated as a potential label free indicator for cellular alteration in different diseases, cancer included. Cell elasticity can be locally measured by pulling membrane tethers, stretching or indenting the cell using optical tweezers. In this paper, we propose a simple approach to perform cell indentation at pN forces by axially moving the cell against a trapped microbead. The elastic modulus is calculated using the Hertz-model. Besides the axial component, the setup also allows us to examine the lateral cell-bead interaction. This technique has been applied to measure the local elasticity of HBL-100 cells, an immortalized human cell line, originally derived from the milk of a woman with no evidence of breast cancer lesions. In addition, we have studied the influence of substrate stiffness on cell elasticity by performing experiments on cells cultured on two substrates, bare and collagen-coated, having different stiffness. The mean value of the cell elastic modulus measured during indentation was 26±9 Pa for the bare substrate, while for the collagen-coated substrate it diminished to 19±7 Pa. The same trend was obtained for the elastic modulus measured during the retraction of the cell: 23±10 Pa and 13±7 Pa, respectively. These results show the cells adapt their stiffness to that of the substrate and demonstrate the potential of this setup for low-force probing of modifications to cell mechanics induced by the surrounding environment (e.g. extracellular matrix or other cells).

  20. The role of mast cells and fibre type in ischaemia reperfusion injury of murine skeletal muscles

    Directory of Open Access Journals (Sweden)

    Bortolotto Susan K

    2004-09-01

    Full Text Available Abstract Background Ischaemia reperfusion (IR injury of skeletal muscle, is a significant cause of morbidity following trauma and surgical procedures, in which muscle fibre types exhibit different susceptibilities. The relative degree of mast cell mediated injury, within different muscle types, is not known. Methods In this study we compared susceptibility of the fast-twitch, extensor digitorum longus (EDL, mixed fast/slow-twitch gastrocnemius and the predominately slow-twitch soleus, muscles to ischemia reperfusion (IR injury in four groups of mice that harbour different mast cell densities; C57/DBA mast cell depleted (Wf/Wf, their heterozygous (Wf/+ and normal littermates (+/+ and control C57BL/6 mice. We determined whether susceptibility to IR injury is associated with mast cell content and/or fibre type and/or mouse strain. In experimental groups, the hind limbs of mice were subjected to 70 minutes warm tourniquet ischemia, followed by 24 h reperfusion, and the muscle viability was assessed on fresh whole-mount slices by the nitroblue tetrazolium (NBT histochemical assay. Results Viability was remarkably higher in the Wf/Wf strain irrespective of muscle type. With respect to muscle type, the predominately slow-twitch soleus muscle was significantly more resistant to IR injury than gastrocnemius and the EDL muscles in all groups. Mast cell density was inversely correlated to muscle viability in all types of muscle. Conclusion These results show that in skeletal muscle, IR injury is dependent upon both the presence of mast cells and on fibre type and suggest that a combination of preventative therapies may need to be implemented to optimally protect muscles from IR injury.

  1. Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.

    Directory of Open Access Journals (Sweden)

    Elena Rahn

    Full Text Available Herpes simplex virus type 1 (HSV-1 can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s. Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.

  2. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    Science.gov (United States)

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  3. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Directory of Open Access Journals (Sweden)

    Teruhito Yamashita

    Full Text Available Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA, a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the

  4. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  5. Carrier concentration dependence of band gap shift in n-type ZnO:Al films

    Science.gov (United States)

    Lu, J. G.; Fujita, S.; Kawaharamura, T.; Nishinaka, H.; Kamada, Y.; Ohshima, T.; Ye, Z. Z.; Zeng, Y. J.; Zhang, Y. Z.; Zhu, L. P.; He, H. P.; Zhao, B. H.

    2007-04-01

    Al-doped ZnO (AZO) thin films have been prepared by mist chemical vapor deposition and magnetron sputtering. The band gap shift as a function of carrier concentration in n-type zinc oxide (ZnO) was systematically studied considering the available theoretical models. The shift in energy gap, evaluated from optical absorption spectra, did not depend on sample preparations; it was mainly related to the carrier concentrations and so intrinsic to AZO. The optical gap increased with the electron concentration approximately as ne2/3 for ne≤4.2×1019 cm-3, which could be fully interpreted by a modified Burstein-Moss (BM) shift with the nonparabolicity of the conduction band. A sudden decrease in energy gap occurred at 5.4-8.4×1019 cm-3, consistent with the Mott criterion for a semiconductor-metal transition. Above the critical values, the band gap increased again at a different rate, which was presumably due to the competing BM band-filling and band gap renormalization effects, the former inducing a band gap widening and the latter an offsetting narrowing. The band gap narrowing (ΔEBGN) derived from the band gap renormalization effect did not show a good ne1/3 dependence predicated by a weakly interacting electron-gas model, but it was in excellent agreement with a perturbation theory considering different many-body effects. Based on this theory a simple expression, ΔEBGN=Ane1/3+Bne1/4+Cne1/2, was deduced for n-type ZnO, as well as p-type ZnO, with detailed values of A, B, and C coefficients. An empirical relation once proposed for heavily doped Si could also be used to describe well this gap narrowing in AZO.

  6. Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses

    Directory of Open Access Journals (Sweden)

    Pryor Kellyann

    2009-04-01

    Full Text Available Abstract Background Current literature suggests that dipeptidyl peptidase IV (DPP-IV; CD26 plays an essential role in T-dependent immune responses, a role that could have important clinical consequences. To rigorously define the role of DPP-IV in the immune system, we evaluated genetic and pharmacological inhibition of the enzyme on T-dependent immune responses in vivo. Results The DPP-IV null animals mounted robust primary and secondary antibody responses to the T dependent antigens, 4-hydroxy-3-nitrophenylacetyl-ovalbumin (NP-Ova and 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin (NP-CGG, which were comparable to wild type mice. Serum levels of antigen specific IgM, IgG1, IgG2a, IgG2b and IgG3 were similar between the two groups of animals. DPP-IV null animals mounted an efficient germinal center reaction by day 10 after antigen stimulation that was comparable to wild type mice. Moreover, the antibodies produced by DPP-IV null animals after repeated antigenic challenge were affinity matured. Similar observations were made using wild type animals treated with a highly selective DPP-IV inhibitor during the entire course of the experiments. T cell recall responses to ovalbumin and MOG peptide, evaluated by measuring proliferation and IL-2 release from cells isolated from draining lymph nodes, were equivalent in DPP-IV null and wild type animals. Furthermore, mice treated with DPP-IV inhibitor had intact T-cell recall responses to MOG peptide. In addition, female DPP-IV null and wild type mice treated with DPP-IV inhibitor exhibited normal and robust in vivo cytotoxic T cell responses after challenge with cells expressing the male H-Y minor histocompatibility antigen. Conclusion These data indicate Selective inhibition of DPP-IV does not impair T dependent immune responses to antigenic challenge.

  7. Evolution of nonclassical MHC-dependent invariant T cells.

    Science.gov (United States)

    Edholm, Eva-Stina; Grayfer, Leon; Robert, Jacques

    2014-12-01

    TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.

  8. Clonal progression during the T cell-dependent B cell antibody response depends on the immunoglobulin DH gene segment repertoire.

    Directory of Open Access Journals (Sweden)

    Ahmad eTrad

    2014-08-01

    Full Text Available The diversity of the third complementarity determining region of the Ig H chain is constrained by natural selection of immunoglobulin diversity (DH sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA. We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered D-DFS and D-iD strains were significantly reduced. An increased prevalence of IgM producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion as well as class switch recombination indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

  9. Genes affecting β-cell function in type 1 diabetes

    DEFF Research Database (Denmark)

    Fløyel, Tina; Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Type 1 diabetes (T1D) is a multifactorial disease resulting from an immune-mediated destruction of the insulin-producing pancreatic β cells. Several environmental and genetic risk factors predispose to the disease. Genome-wide association studies (GWAS) have identified around 50 genetic regions...... that affect the risk of developing T1D, but the disease-causing variants and genes are still largely unknown. In this review, we discuss the current status of T1D susceptibility loci and candidate genes with focus on the β cell. At least 40 % of the genes in the T1D susceptibility loci are expressed in human...... islets and β cells, where they according to recent studies modulate the β-cell response to the immune system. As most of the risk variants map to noncoding regions of the genome, i.e., promoters, enhancers, intergenic regions, and noncoding genes, their possible involvement in T1D pathogenesis as gene...

  10. Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-08-08

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

  11. Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-01-01

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

  12. Changes in brain activation in breast cancer patients depend on cognitive domain and treatment type

    Science.gov (United States)

    Menning, Sanne; de Ruiter, Michiel B.; Veltman, Dick J.; Boogerd, Willem; Oldenburg, Hester S. A.; Reneman, Liesbeth

    2017-01-01

    integrity after systemic treatment. Overall these results suggest different neurobehavioral trajectories in breast cancer patients depending on treatment type. PMID:28267750

  13. Soil type-depending effect of paddy management: composition and distribution of soil organic matter

    Science.gov (United States)

    Urbanski, Livia; Kölbl, Angelika; Lehndorff, Eva; Houtermans, Miriam; Schad, Peter; Zhang, Gang-Lin; Rahayu Utami, Sri; Kögel-Knabner, Ingrid

    2016-04-01

    Paddy soil management is assumed to promote soil organic matter accumulation and specifically lignin caused by the resistance of the aromatic lignin structure against biodegradation under anaerobic conditions during inundation of paddy fields. The present study investigates the effect of paddy soil management on soil organic matter composition compared to agricultural soils which are not used for rice production (non-paddy soils). A variety of major soil types, were chosen in Indonesia (Java), including Alisol, Andosol and Vertisol sites (humid tropical climate of Java, Indonesia) and in China Alisol sites (humid subtropical climate, Nanjing). This soils are typically used for rice cultivation and represent a large range of soil properties to be expected in Asian paddy fields. All topsoils were analysed for their soil organic matter composition by solid-state 13C nuclear magnetic resonance spectroscopy and lignin-derived phenols by CuO oxidation method. The soil organic matter composition, revealed by solid-state 13C nuclear magnetic resonance, was similar for the above named different parent soil types (non-paddy soils) and was also not affected by the specific paddy soil management. The contribution of lignin-related carbon groups to total SOM was similar in the investigated paddy and non-paddy soils. A significant proportion of the total aromatic carbon in some paddy and non-paddy soils was attributed to the application of charcoal as a common management practise. The extraction of lignin-derived phenols revealed low VSC (vanillyl, syringyl, cinnamyl) values for all investigated soils, being typical for agricultural soils. An inherent accumulation of lignin-derived phenols due to paddy management was not found. Lignin-derived phenols seem to be soil type-dependent, shown by different VSC concentrations between the parent soil types. The specific paddy management only affects the lignin-derived phenols in Andosol-derived paddy soils which are characterized by

  14. Angiogenesis and collagen type IV expression in different endothelial cell culture systems.

    Science.gov (United States)

    Bahramsoltani, M; Slosarek, I; De Spiegelaere, W; Plendl, J

    2014-04-01

    In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro- and anti-angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1-6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non-angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-11

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

  16. Generation of cloned calves from different types of somatic cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Six types of bovine somatic cell lines,including a granulosa cell line of Chinese red-breed yellow cattle(YGR),a granulosa cell line of Holstein cow(HGR),two skin fibroblast cell lines of two adult Holstein cows respectively(AFB1 and AFB2),a skin fibroblast cell line(FFB)and an oviduct epithelial cell line(FOV)of a Holstein fetus,were established.Somatic cell nuclear transfer(SCNT)was carried out using these cells as nuclei donor,and a total of 12 healthy calves were cloned.The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated.(i)There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR(33.2% and 35.1%,respectively).Pregnancy rates of them were 33.3% and 30.2%,respectively; and birth rates were 16.7%and 11.6%,respectively.(ii)Development rates to the blastocyst stage for SCNT embryos from diffetent individuals(AFB1 and AFB2)differed significantly(27.9% and 39.4%,respectively,P <0.05).Pregnancy rates of them were 36.2% and 36.4%,respectively; and birth rates were 14.9% and 27.3%,respectively.(iii)There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus(37.9% and 41.5%,respectively,P < 0.05).Pregnancy rates of them were 45.7% and 24.1%,respectively; and birth rates were 22.9 % and 10.3%,respectively.Finally,developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows(HGR,AFB,FFB and FOV)were compared.For in vitro development stage,development rates to the blastocyst stage for SCNT embryos from HGR,AFB,FFB and FOV were 35.1%A,29.4%B,37.9%A and 41.5%C,respectively(pABC<0.05); for in vivo development stage,pregnancy rates of them were 30.2%,36.2%,45.7%and 24.1%,respectively; and birth rates of them were 11.6%,17.2%,22.9% and 10.3% respectively.

  17. Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling*

    Science.gov (United States)

    Mistry, Rajesh K.; Murray, Thomas V. A.; Prysyazhna, Oleksandra; Martin, Daniel; Burgoyne, Joseph R.; Santos, Celio; Eaton, Philip; Shah, Ajay M.; Brewer, Alison C.

    2016-01-01

    The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production. PMID:26620565

  18. Dopaminergic neurons write and update memories with cell-type-specific rules.

    Science.gov (United States)

    Aso, Yoshinori; Rubin, Gerald M

    2016-07-21

    Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. Previously we described the anatomy of the adult MB and defined 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments (Aso et al., 2014a, 2014b). Here we compare the properties of memories formed by optogenetic activation of individual DAN cell types. We found extensive differences in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. Our results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences.

  19. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo); W.C. Poller; B.C. Tilly (Bernard); A. Smolenski; S. Schneider-Rasp; A.G. Bot (Alice); M.J. Edixhoven (Marcel); B.J. Scholte (Bob); T. Jarchau; U. Walter

    1997-01-01

    textabstractIn order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was ana

  20. Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba.

    Science.gov (United States)

    Polit, Justyna Teresa; Kaźmierczak, Andrzej; Walczak-Drzewiecka, Aurelia

    2012-01-01

    The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition.

  1. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-09-15

    When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

  2. Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

    Directory of Open Access Journals (Sweden)

    Becker Walter

    2011-09-01

    Full Text Available Abstract Background Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells. We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. Results The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM. UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS. This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin and BDNF (brain derived neurotrophic factor. Conclusion The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein

  3. The STATs in cell stress-type responses

    Directory of Open Access Journals (Sweden)

    Best James

    2004-08-01

    Full Text Available Abstract In the early 1990's, a new cell signaling pathway was described. This new paradigm, now known as the JAK/STAT pathway, has been extensively investigated in immune-type cells in response to interferons and interleukins. However, recent evidence suggests that the JAK/STAT pathway also mediates diverse cellular responses to various forms of biological stress including hypoxia/reperfusion, endotoxin, ultraviolet light, and hyperosmolarity. The current literature describing the JAK/STAT pathway's role in cellular stress responses has been reviewed herein, but it is clear that our knowledge in this area is far from complete.

  4. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  5. Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

    Directory of Open Access Journals (Sweden)

    Crandall Edward D

    2005-04-01

    Full Text Available Abstract Background Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. Methods Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. Results This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. Conclusion This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.

  6. Age-dependent change in biological characteristics of stem cells in radiation-induced mammary carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Yoshiya; Nishimura, Mayumi; Kakinuma, Shizuko; Imaoka, Tatsuhiko [National Institute of Radiological Sciences, Anagawa, Chiba (Japan); Yasukawa-Barnes, Jane; Gould, Michael N.; Clifton, Kelly H. [Univ. of Wisconsin, Department of Human Oncology, Madison, WI (United States)

    2003-07-01

    If you ask what types of cells are the targets for carcinogenesis, a popular answer would be that cancer arises from stem cells. Stem cells are cells that are capable of both self-renewal and generation of differentiated progenies. If the hypothesis of 'cancer as stem cell disease' is correct, the risk of carcinogenesis should be a function of the number of stem cells and their responsiveness of carcinogen-induced damage. In the present study, we addressed the feasibility of this hypothesis using the rat mammary carcinogenesis model. One of the important conclusions emerging from studies on atomic bomb survivors concerns age-related changes in the susceptibility to breast cancer. The relative risk of breast cancer is very high among women exposed to ionizing radiation before or during puberty, and it decreases thereafter. Little information is available, however, on age-related changes in the radiobiological nature of mammary stem cells. We examined age-associated changes in the number of mammary stem-like cells (clonogens) and their susceptibility to radiation in terms of cell death and carcinogenic initiation frequency. The results were as follows. (1) During the prepubertal period, the total number of mammary clonogens per rat increased exponentially with a population doubling time of {approx}4 days. After puberty, the doubling time lengthened to {approx}30 days. The total number of clonogens in abdominal and inguinal mammary glands was {approx}200 in 2-week-old rats, while it was {approx}5600 in 8-week-old rats. (2) The survival curves of clonogenic cells after irradiation indicated that radiation sensitivity of the cells before and during puberty was much higher than after puberty. (3) The initiation frequency of the clonogens from prepubertal rats after 5 Gy irradiation was four times higher than that of the clonogens from post-pubertal rats. These results suggest that changes in the number of stem cells and their radiobiological characteristics

  7. d,l-Sulforaphane Induces ROS-Dependent Apoptosis in Human Gliomablastoma Cells by Inactivating STAT3 Signaling Pathway

    Science.gov (United States)

    Miao, Ziwei; Yu, Fei; Ren, Yahao; Yang, Jun

    2017-01-01

    d,l-Sulforaphane (SFN), a synthetic analogue of broccoli-derived isomer l-SFN, exerts cytotoxic effects on multiple tumor cell types through different mechanisms and is more potent than the l-isomer at inhibiting cancer growth. However, the means by which SFN impairs glioblastoma (GBM) cells remains poorly understood. In this study, we investigated the anti-cancer effect of SFN in GBM cells and determined the underlying molecular mechanisms. Cell viability assays, flow cytometry, immunofluorescence, and Western blot results revealed that SFN could induced apoptosis of GBM cells in a dose- and time-dependent manner, via up-regulation of caspase-3 and Bax, and down-regulation of Bcl-2. Mechanistically, SFN treatment led to increase the intracellular reactive oxygen species (ROS) level in GBM cells. Meanwhile, SFN also suppressed both constitutive and IL-6-induced phosphorylation of STAT3, and the activation of upstream JAK2 and Src tyrosine kinases, dose- and time-dependently. Moreover, blockage of ROS production by using the ROS inhibitor N-acetyl-l-cysteine totally reversed SFN-mediated down-regulation of JAK2/Src-STAT3 signaling activation and the subsequent effects on apoptosis by blocking the induction of apoptosis-related genes in GBM cells. Taken together, our data suggests that SFN induces apoptosis in GBM cells via ROS-dependent inactivation of STAT3 phosphorylation. These findings motivate further evaluation of SFN as a cancer chemopreventive agent in GBM treatment. PMID:28054986

  8. d,l-Sulforaphane Induces ROS-Dependent Apoptosis in Human Gliomablastoma Cells by Inactivating STAT3 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ziwei Miao

    2017-01-01

    Full Text Available d,l-Sulforaphane (SFN, a synthetic analogue of broccoli-derived isomer l-SFN, exerts cytotoxic effects on multiple tumor cell types through different mechanisms and is more potent than the l-isomer at inhibiting cancer growth. However, the means by which SFN impairs glioblastoma (GBM cells remains poorly understood. In this study, we investigated the anti-cancer effect of SFN in GBM cells and determined the underlying molecular mechanisms. Cell viability assays, flow cytometry, immunofluorescence, and Western blot results revealed that SFN could induced apoptosis of GBM cells in a dose- and time-dependent manner, via up-regulation of caspase-3 and Bax, and down-regulation of Bcl-2. Mechanistically, SFN treatment led to increase the intracellular reactive oxygen species (ROS level in GBM cells. Meanwhile, SFN also suppressed both constitutive and IL-6-induced phosphorylation of STAT3, and the activation of upstream JAK2 and Src tyrosine kinases, dose- and time-dependently. Moreover, blockage of ROS production by using the ROS inhibitor N-acetyl-l-cysteine totally reversed SFN-mediated down-regulation of JAK2/Src-STAT3 signaling activation and the subsequent effects on apoptosis by blocking the induction of apoptosis-related genes in GBM cells. Taken together, our data suggests that SFN induces apoptosis in GBM cells via ROS-dependent inactivation of STAT3 phosphorylation. These findings motivate further evaluation of SFN as a cancer chemopreventive agent in GBM treatment.

  9. Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells.

    Science.gov (United States)

    Strock, Christopher J; Park, Jong-In; Nakakura, Eric K; Bova, G Steven; Isaacs, John T; Ball, Douglas W; Nelkin, Barry D

    2006-08-01

    We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.

  10. Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

    Science.gov (United States)

    Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

    2014-01-01

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

  11. Preimplantation HLA typing for stem cell transplantation treatment of hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    Anver Kuliev

    2014-09-01

    Full Text Available Preimplantation genetic diagnosis (PGD for HLA typing is steadily becoming an option for at risk couples with thalassemic children, requiring HLA matched bone marrow transplantation treatment. The paper presents the world’s largest PGD experience of 475 cases for over 2 dozens thalassemia mutations, resulting in birth of 132 unaffected children. A total of 146 cases were performed together with preimplantation HLA typing, resulting in detection and transfer of HLA matched unaffected embryos in 83 of them, yielding the birth of 16 HLA matched children, potential donors for their affected siblings. The presented experience of HLA matched stem cell transplantation for thalassemia, following PGD demonstrated a successful hematopoietic reconstitution both for younger and older patients. The data show that PGD is an efficient approach for HLA matched stem cell transplantation treatment for thalassemia.

  12. Voltage-Dependent Intrinsic Bursting in Olfactory Bulb Golgi Cells

    Science.gov (United States)

    Pressler, R. Todd; Rozman, Peter A.; Strowbridge, Ben W.

    2013-01-01

    In the mammalian olfactory bulb (OB), local synaptic circuits modulate the evolving pattern of activity in mitral and tufted cells following olfactory sensory stimulation. GABAergic granule cells, the most numerous interneuron subtype in this brain region, have been extensively studied. However, classic studies using Golgi staining methods…

  13. On the metallicity dependence of the [Y/Mg]-age relation for solar-type stars

    Science.gov (United States)

    Feltzing, Sofia; Howes, Louise M.; McMillan, Paul J.; Stonkutė, Edita

    2017-02-01

    Several recent studies of solar twins in the solar neighbourhood have shown a tight correlation between various elemental abundances and age, in particular [Y/Mg]. If this relation is real and valid for other types of stars as well as elsewhere in the Galaxy, it would provide a very powerful tool to derive ages of stars without the need to resort to determining their masses (evolutionary stage) very precisely. The method would also likely work if the stellar parameters have relatively large errors. The studies presented in the recent literature span a narrow range of [Fe/H]. By studying a larger sample of solar neighbourhood dwarfs with a much larger range of [Fe/H], we find that the relation between [Y/Mg] and age depends on the [Fe/H] of the stars. Hence, it appears that the [Y/Mg]-age relation is unique to solar analogues.

  14. Nocturnal electroencephalogram registrations in type 1 (insulin-dependent) diabetic patients with hypoglycaemia

    DEFF Research Database (Denmark)

    Bendtson, I; Gade, J; Rosenfalck, A M;

    1991-01-01

    Eight Type 1 (insulin-dependent) diabetic patients with no diabetic complications were studied overnight for two consecutive and one subsequent night with continuous monitoring of electroencephalogram and serial hormone measurements. The aims were: 1) to evaluate the influence of spontaneous...... and insulin-induced hypoglycaemia on nocturnal electroencephalogram sleep-patterns and, 2) to evaluate counter-regulatory hormone responses. Spontaneous hypoglycaemia occurred on six nights (38%) with blood glucose concentrations less than 3.0 mmol/l and on four nights less than 2.0 mmol/l. All the patients...... be correlated to the rank of individual electroencephalogram-patterns during the whole night, and specific hypoglycaemic amplitude-frequency patterns could be assigned. Three of the eight patients showed electroencephalogram changes at blood glucose levels below 2.0 (1.6-2.0) mmol/l. The electroencephalogram...

  15. On the metallicity dependance of the [Y/Mg] - age relation for solar type stars

    CERN Document Server

    Feltzing, S; McMillan, P J; Stonkute, E

    2016-01-01

    Several recent studies of Solar twins in the Solar neighbourhood have shown a tight correlation between various elemental abundances and age, in particular [Y/Mg]. If th