WorldWideScience

Sample records for cell cycle-dependent localization

  1. Gamma-tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms

    Czech Academy of Sciences Publication Activity Database

    Libusová, Lenka; Sulimenko, Tetyana; Sulimenko, Vadym; Hozák, Pavel; Dráber, Pavel

    2004-01-01

    Roč. 295, - (2004), s. 375-386 ISSN 0014-4827 R&D Projects: GA MŠk ME 310; GA MŠk LN00A026 Keywords : gamma-tubulin * cell cycle * Leishmania Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.007, year: 2004

  2. Cell cycle-dependent changes in localization of a 210 k Da microtubule-interacting protein in Leishmania

    Czech Academy of Sciences Publication Activity Database

    Libusová, Lenka; Dráberová, Eduarda; Juliano, C.; Viklický, Vladimír; Fiori, P.; Cappuccinelli, P.; Dráber, Pavel

    2002-01-01

    Roč. 96, č. 4 (2002), s. 226-227 [Sigma-Aldrich konference mladých chemiků, biochemiků a molekulárních biologů. 22.05.2002-25.05.2002, Velké Meziříčí] R&D Projects: GA ČR GA304/00/0553; GA AV ČR IAA5052004 Keywords : Leishmania * cell cycle * microtubule Subject RIV: EB - Genetics ; Molecular Biology

  3. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  4. Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

    Science.gov (United States)

    Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

    2011-01-01

    The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

  5. 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.

  6. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  7. Cell-cycle-dependent repair of heavy-ion damage

    International Nuclear Information System (INIS)

    Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

    1985-01-01

    Synchronized human T-1 cells have been used to investigate the G1-phase age dependence of repair of potentially lethal damage (PLDR). The cells were irradiated with single doses of either 225 kVp X rays or Bragg-peak 425 MeV/μ neon ions at ages between 1.5 and 6.0 hrs after mitotic selection, and then either trypsinized and plated immediately, or held at 37 0 C for 6 hrs in PBS, or PBS containing 60μM of the DNA-polymerase-inhibitor 1-β-D-arabinofurano-syladenine (β-araA) before trypsinization and plating. Delayed plating showed significant PLDR at all ages irradiated with X rays, with the increase of survival varying between 2- to 8-fold. At equivalent survival levels, there was a reduced capacity for PLDT at each cell age irradiated with neon ions. In early G1 after neon-ion exposures, delayed plating actually enhanced cell killing; whereas, in late G1 the survival increased about 2-fold. β-araA almost completely eliminated the PLDR after X rays, reducing the survival to that measured with immediate plating. β-araA slightly enhanced neon-ion cell killing at all cell ages

  8. Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

    Science.gov (United States)

    Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

    2013-01-01

    The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

  9. Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

    Directory of Open Access Journals (Sweden)

    Yoshinori Kagawa

    Full Text Available The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP, was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

  10. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  11. Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

    Science.gov (United States)

    Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

    2017-03-01

    Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1

    DEFF Research Database (Denmark)

    Walmod, Peter S.; Hartmann-Petersen, Rasmus; Prag, S.

    2004-01-01

    comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner...... for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility...... was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels...

  13. Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

    Science.gov (United States)

    Grolmusz, Vince Kornél; Karászi, Katalin; Micsik, Tamás; Tóth, Eszter Angéla; Mészáros, Katalin; Karvaly, Gellért; Barna, Gábor; Szabó, Péter Márton; Baghy, Kornélia; Matkó, János; Kovalszky, Ilona; Tóth, Miklós; Rácz, Károly; Igaz, Péter; Patócs, Attila

    2016-01-01

    Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

  14. Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

    Science.gov (United States)

    Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

    2013-07-01

    Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

  15. Local time, seasonal, and solar cycle dependency of longitudinal variations of TEC along the crest of EIA over India

    Science.gov (United States)

    Sunda, Surendra; Vyas, B. M.

    2013-10-01

    global wave number 4 structure in the Indian longitudinal region spanning from ~70 to 95°E forming the upward slope of the peak in the total electron content (TEC) are reported along the crest of equatorial ionization anomaly (EIA). The continuous and simultaneous measurements from five GPS stations of GPS Aided Geo Augmented Navigation (GAGAN) network are used in this study. The long-term database (2004-2012) is utilized for examining the local time, seasonal, and solar cycle dependency on the longitudinal variations of TEC. Our results confirm the existence of longitudinal variations of TEC in accordance with wave number 4 longitudinal structure including its strength. The results suggest that these variations, in general, start to develop at ~09 LT, achieve maximum strength at 12-15 LT, and decay thereafter, the decay rate depending on the season. They are more pronounced in equinoctial season followed by summer and winter. The longitudinal variations persist beyond midnight in equinox seasons, whereas in winter, they are conspicuously absent. Interestingly, they also exhibit significant solar cycle dependence in the solstices, whereas in the equinoxes, they are independent of solar activity. The comparison of crest-to-trough ratio (CTR) in the eastern (92°E) and western (72°E) extreme longitudes reveals higher CTR on the eastern side than over the western extreme, suggesting the role of nonmigrating tides in modulating the ExB vertical drift and the consequential EIA crest formation.

  16. CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

    Science.gov (United States)

    Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

    2006-05-01

    We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

  17. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

    International Nuclear Information System (INIS)

    Stamato, T.D.; Weinstein, R.; Giaccia, A.; Mackenzie, L.

    1983-01-01

    A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage

  18. Backup pathways of NHEJ in cells of higher eukaryotes: Cell cycle dependence

    International Nuclear Information System (INIS)

    Iliakis, George

    2009-01-01

    DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs, DNA ligase IV, XRCC4 and XLF/Cernunnos (D-NHEJ) as central components. Work carried out in our laboratory and elsewhere shows that when this pathway is chemically or genetically compromised, cells do not shunt DSBs to homologous recombination repair (HRR) but instead use another form of NHEJ operating as a backup (B-NHEJ). Here I review our efforts to characterize this repair pathway and discuss its dependence on the cell cycle as well as on the growth conditions. I present evidence that B-NHEJ utilizes ligase III, PARP-1 and histone H1. When B-NHEJ is examined throughout the cell cycle, significantly higher activity is observed in G2 phase that cannot be attributed to HRR. Furthermore, the activity of B-NHEJ is compromised when cells enter the plateau phase of growth. Together, these observations uncover a repair pathway with unexpected biochemical constitution and interesting cell cycle and growth factor regulation. They generate a framework for investigating the mechanistic basis of HRR contribution to DSB repair.

  19. Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation.

    Science.gov (United States)

    Erhardt, Sylvia; Mellone, Barbara G; Betts, Craig M; Zhang, Weiguo; Karpen, Gary H; Straight, Aaron F

    2008-12-01

    Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

  20. Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

    Science.gov (United States)

    Delporte, Annelies; De Zaeytijd, Jeroen; De Storme, Nico; Azmi, Abdelkrim; Geelen, Danny; Smagghe, Guy; Guisez, Yves; Van Damme, Els J M

    2014-10-01

    The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Cell cycle-dependent regulation of kainate-induced inward currents in microglia

    International Nuclear Information System (INIS)

    Yamada, Jun; Sawada, Makoto; Nakanishi, Hiroshi

    2006-01-01

    Microglia are reported to have α-amino-hydroxy-5-methyl-isoxazole-4-propionate/kainate (KA) types. However, only small population of primary cultured rat microglia (approximately 20%) responded to KA. In the present study, we have attempted to elucidate the regulatory mechanism of responsiveness to KA in GMIR1 rat microglial cell line. When the GMIR1 cells were plated at a low density in the presence of granulocyte macrophage colony-stimulating factor, the proliferation rate increased and reached the peak after 2 days in culture and then gradually decreased because of density-dependent inhibition. At cell proliferation stage, approximately 80% of the GMIR1 cells exhibited glutamate (Glu)- and KA-induced inward currents at cell proliferation stage, whereas only 22.5% of the cells showed responsiveness to Glu and KA at cell quiescent stage. Furthermore, the mean amplitudes of inward currents induced by Glu and KA at cell proliferation stage (13.8 ± 3.0 and 8.4 ± 0.6 pA) were significantly larger than those obtained at cell quiescent stage (4.7 ± 0.8 and 6.2 ± 1.2 pA). In the GMIR1 cells, KA-induced inward currents were markedly inhibited by (RS)-3-(2-carboxybenzyl) willardiine (UBP296), a selective antagonist for KA receptors. The KA-responsive cells also responded to (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist for GluR5, in both GMIR1 cells and primary cultured rat microglia. Furthermore, mRNA levels of the KA receptor subunits, GluR5 and GluR6, at the cell proliferation stage were significantly higher than those at the cell quiescent stage. Furthermore, the immunoreactivity for GluR6/7 was found to increase in activated microglia in the post-ischemic hippocampus. These results strongly suggest that microglia have functional KA receptors mainly consisting of GluR5 and GluR6, and the expression levels of these subunits are closely regulated by the cell cycle mechanism

  2. Cell cycle dependence of boron uptake in various boron compounds used for neutron capture therapy

    International Nuclear Information System (INIS)

    Yoshida, F.; Matsumura, A.; Shibata, Y.; Yamamoto, T.; Nose, T.; Okumura, M.

    2000-01-01

    In neutron capture therapy, it is important that the tumor take boron in selectively. Furthermore, it is ideal when the uptake is equal in each tumor cell. Some indirect proof of differences in boron uptake among neoplastic cell cycles has been documented. However, no investigation has yet measured boron uptake directly. Using flow cytometry, in the present study cells were sorted by G0/G1 phase and G2/M phase, and the boron concentration of each fraction was measured with inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The results were that BSH (sodiumborocaptate) and BPA (p-boronophenylalanine) had higher rates of boron uptake in the G2/M group than in the G0/G1 group. However, in BPA the difference was more prominent, which revealed a 2.2-3.3 times higher uptake of boron in the G2/M group than in the G0/G1 group. (author)

  3. Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.

    Directory of Open Access Journals (Sweden)

    Ronan Broderick

    Full Text Available Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.

  4. Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas

    DEFF Research Database (Denmark)

    Kim, Yung Hae; Larsen, Hjalte List; Rué, Paul

    2015-01-01

    Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine...

  5. Cell Cycle Dependent Expression of Plk1 in Synchronized Porcine Fetal Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Anger, Martin; Kues, W. A.; Klíma, Jiří; Mielenz, M.; Kubelka, Michal; Motlík, Jan; Ešner, M.; Dvořák, P.; Carnwath, J. W.; Niemann, H.

    2003-01-01

    Roč. 65, č. 3 (2003), s. 245-253 ISSN 1040-452X R&D Projects: GA MŠk LN00A065 Grant - others:FIRCA(XX) R03-TW-05530-01 Institutional research plan: CEZ:AV0Z5045916 Keywords : Plk1 * serum deprivation * cell cycle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.543, year: 2003

  6. p53 represses autophagy in a cell cycle-dependent fashion.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, Maria Chiara; Orhon, Idil; Kepp, Oliver; Morselli, Eugenia; Criollo, Alfredo; Kroemer, Guido

    2008-10-01

    Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-alpha), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-X(L) with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.

  7. Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1

    International Nuclear Information System (INIS)

    Kang, Du-Seock; Hong, Kyeong-Man; Park, Joobae; Bae, Chang-Dae

    2012-01-01

    Highlights: ► We identified a GC box and a CHR element in human CKAP2 minimal promoter. ► The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. ► The GC box was essential for the basic promoter activity of human CKAP2. ► The GC box was also essential for the cyclic expression of human CKAP2. ► The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (−110 to −104 bp) and a GC box (−41 to −32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

  8. Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

    1980-01-01

    A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

  9. Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Bergdahl, Andreas; Christophersen, Palle

    2007-01-01

    Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored...... during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current......+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle...

  10. The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

    Science.gov (United States)

    Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Bögler, Oliver; Duchrow, Michael

    2003-01-01

    The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase. Copyright 2002 John Wiley & Sons, Ltd.

  11. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Siem van der Laan

    Full Text Available Pluripotency of embryonic stem cells (ESC is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb. Esrrb contributes to the relaxation of the G1 to S-phase (G1/S checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

  12. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

    Science.gov (United States)

    van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

    2014-01-01

    Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

  13. FasL and FADD delivery by a glioma-specific and cell cycle-dependent HSV-1 amplicon virus enhanced apoptosis in primary human brain tumors

    Directory of Open Access Journals (Sweden)

    Lam Paula Y

    2010-10-01

    Full Text Available Abstract Background Glioblastoma multiforme is the most malignant cancer of the brain and is notoriously difficult to treat due to the highly proliferative and infiltrative nature of the cells. Herein, we explored the combination treatment of pre-established human glioma xenograft using multiple therapeutic genes whereby the gene expression is regulated by both cell-type and cell cycle-dependent transcriptional regulatory mechanism conferred by recombinant HSV-1 amplicon vectors. Results We demonstrated for the first time that Ki67-positive proliferating primary human glioma cells cultured from biopsy samples were effectively induced into cell death by the dual-specific function of the pG8-FasL amplicon vectors. These vectors were relatively stable and exhibited minimal cytotoxicity in vivo. Intracranial implantation of pre-transduced glioma cells resulted in better survival outcome when compared with viral vectors inoculated one week post-implantation of tumor cells, indicating that therapeutic efficacy is dependent on the viral spread and mode of viral vectors administration. We further showed that pG8-FasL amplicon vectors are functional in the presence of commonly used treatment regimens for human brain cancer. In fact, the combined therapies of pG8-FasL and pG8-FADD in the presence of temozolomide significantly improved the survival of mice bearing intracranial high-grade gliomas. Conclusion Taken together, our results showed that the glioma-specific and cell cycle-dependent HSV-1 amplicon vector is potentially useful as an adjuvant therapy to complement the current gene therapy strategy for gliomas.

  14. Genes adopt non-optimal codon usage to generate cell cycle-dependent oscillations in protein levels

    DEFF Research Database (Denmark)

    Frenkel-Morgenstern, Milana; Danon, Tamar; Christian, Thomas

    2012-01-01

    The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle a...

  15. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Ken Kikuchi

    2014-01-01

    Full Text Available Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

  16. Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective or radiosensitizing effects as elucidated by the PCC and G2-assay

    Energy Technology Data Exchange (ETDEWEB)

    Sebastià, N., E-mail: natividad.sebastia@uv.es [Radiation Protection Service, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Montoro, A. [Radiation Protection Service, Universitary and Politechnic Hospital La Fe, Valencia (Spain); Grupo de Investigación Biomédica en Imagen GIBI230, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética Clínica, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Hervás, D. [Biostatistics Unit, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Pantelias, G.; Hatzi, V.I. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National Centre for Scientific Research “Demokritos”, Aghia Paraskevi, Athens (Greece); Soriano, J.M. [Grupo de Investigación Biomédica en Imagen GIBI230, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética Clínica, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Department of Preventive Medicine and Public Health, Faculty of Pharmacy, University of Valencia, Burjassot, Valencia (Spain); Villaescusa, J.I. [Radiation Protection Service, Universitary and Politechnic Hospital La Fe, Valencia (Spain); and others

    2014-08-15

    Highlights: • Curcumin and trans-resveratrol can exert radioprotective or radiosensitizing effects. • The mechanisms underlying such dual action were elucidated using the PCC and G2-assay. • Radioprotection occurs in non-cycling cells exposed to curcumin and resveratrol. • Radiosensitization occurs in cycling cells exposed to the chemicals. • G2-checkpoint abrogation by the chemicals underlies the radiosensitizing mechanism. - Abstract: Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140 μM curcumin and 2.2 to 220 μM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity

  17. Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective or radiosensitizing effects as elucidated by the PCC and G2-assay

    International Nuclear Information System (INIS)

    Sebastià, N.; Montoro, A.; Hervás, D.; Pantelias, G.; Hatzi, V.I.; Soriano, J.M.; Villaescusa, J.I.

    2014-01-01

    Highlights: • Curcumin and trans-resveratrol can exert radioprotective or radiosensitizing effects. • The mechanisms underlying such dual action were elucidated using the PCC and G2-assay. • Radioprotection occurs in non-cycling cells exposed to curcumin and resveratrol. • Radiosensitization occurs in cycling cells exposed to the chemicals. • G2-checkpoint abrogation by the chemicals underlies the radiosensitizing mechanism. - Abstract: Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140 μM curcumin and 2.2 to 220 μM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity

  18. Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

    Science.gov (United States)

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2013-01-01

    Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

  19. Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

    Science.gov (United States)

    Bruni, R; Roizman, B

    1998-11-01

    The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the alpha22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparent Mr of 78,000 and two minor bands with apparent Mrs of 62, 000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.

  20. Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging

    Science.gov (United States)

    Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

    2017-01-01

    ABSTRACT We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression. PMID:27715464

  1. The PPARα/p16INK4a Pathway inhibits Vascular Smooth Muscle Cell Proliferation by repressing Cell Cycle-dependent Telomerase Activation

    Science.gov (United States)

    Gizard, Florence; Nomiyama, Takashi; Zhao, Yue; Findeisen, Hannes M.; Heywood, Elizabeth B.; Jones, Karrie L.; Staels, Bart; Bruemmer, Dennis

    2009-01-01

    Peroxisome Proliferator-Activated Receptor (PPAR) α, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARα activation suppresses G1→S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16INK4a (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARα is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARα activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect which was dependent on p16. The inhibition of cell proliferation by PPARα activation was lost in VSMC following TERT overexpression or knock-down, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARα. Finally, we demonstrate that PPARα agonists suppress telomerase activation during the proliferative response following vascular injury indicating that these findings are applicable in vivo. In concert, these results demonstrate that the anti-proliferative effects of PPARα in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade. PMID:18818403

  2. Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

    Science.gov (United States)

    Cáceres-Cortés, J R; Cantú-Garza, F A; Mendoza-Mata, M T; Chavez-González, M A; Ramos-Mandujano, G; Zambrano-Ramírez, I R

    2001-12-01

    Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation. Copyright 2001 John Wiley & Sons, Ltd.

  3. Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of gammaH2AX- and NBS1-positive repair foci

    Czech Academy of Sciences Publication Activity Database

    Suchánková, Jana; Kozubek, Stanislav; Legartová, Soňa; Sehnalová, Petra; Kuntzinger, T.; Bártová, Eva

    2015-01-01

    Roč. 107, č. 12 (2015), s. 440-454 ISSN 0248-4900 R&D Projects: GA ČR(CZ) GBP302/12/G157; GA ČR(CZ) GA13-07822S Institutional support: RVO:68081707 Keywords : Cell cycle * DNA repair * Interphase Subject RIV: BO - Biophysics Impact factor: 2.552, year: 2015

  4. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

    Science.gov (United States)

    Escribano-Díaz, Cristina; Orthwein, Alexandre; Fradet-Turcotte, Amélie; Xing, Mengtan; Young, Jordan T F; Tkáč, Ján; Cook, Michael A; Rosebrock, Adam P; Munro, Meagan; Canny, Marella D; Xu, Dongyi; Durocher, Daniel

    2013-03-07

    DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

    Science.gov (United States)

    Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

    2003-11-15

    Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

  6. Cell cycle-dependent changes in localization of a 210-kDa microtubule-interacting protein in .I.Leishmania./I..

    Czech Academy of Sciences Publication Activity Database

    Libusová, Lenka; Dráberová, Eduarda; Juliano, C.; Viklický, Vladimír; Fiori, P. L.; Cappuccinelli, P.; Dráber, Petr

    2001-01-01

    Roč. 266, č. 2 (2001), s. 270-278 ISSN 0014-4827 R&D Projects: GA ČR GA304/00/0553; GA AV ČR IAA5052004 Keywords : microtubule-associated proteins * Leishmania-antibody Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.096, year: 2001

  7. A new analysis of radiation-induced cytogenetic damage in human lymphocytes using the PCC technique, and its implications for biological dosimetry and the understanding of cell-cycle-dependent radiosensitivity fluctuations

    International Nuclear Information System (INIS)

    Zannos, A.; Pantelias, G.E.

    1993-01-01

    The objectives of the project are: to develop a sensitive biological dosemeter, based on the analysis of C-banded peripheral blood lymphocyte prematurely condensed chromosomes (PCCs), for the early assessment of radiation injury and the establishment of absorbed dose estimates in accidental overexposures; and to elucidate the mechanisms of radiation action at the molecular, chromosomal and cellular levels by the study of the effects of DNA repair inhibitors on the repair of radiation damage, effects of BrdUrd incorporation on radiation damage, effects of hyperthermia on the induction and repair of radiation-induced damage, and induction and repair of radiation damage in an X-ray sensitive CHO mutant cell line. (authors) 16 refs., 1 fig

  8. Local advanced transitional cell cancer and squamous cell cancer of ...

    African Journals Online (AJOL)

    Case report: A 51-year-old man presented with a locally advanced squamous cell cancer of the periurethral tissues as well as an underlying isolated transitional cell cancer of the urethra. Chemotherapy with Gemcitabin and Cisplatinum together with local radiation to the pelvis and the perineum was given. There was ...

  9. Local cell metrics: a novel method for analysis of cell-cell interactions.

    Science.gov (United States)

    Su, Jing; Zapata, Pedro J; Chen, Chien-Chiang; Meredith, J Carson

    2009-10-23

    The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs), which addresses this shortcoming. The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical role in determining cell fate, e.g., cancer, developmental

  10. Business Cycle Dependent Unemployment Benefits with Wealth Heterogeneity and Precautionary Savings

    DEFF Research Database (Denmark)

    Kristoffersen, Mark Strøm

    In the wake of the financial and economic crisis the discussion about social insurance and optimal stabilization policies has re-blossomed. This paper adds to the literature by studying the effects of a business cycle dependent level of unemployment benefits in a model with labor market matching......, wealth heterogeneity, precautionary savings, and aggregate fluctuations in productivity. The results are ambiguous: both procyclical and countercyclical unemployment benefits can increase welfare relative to business cycle invariant benefits. Procyclical benefits are beneficial due to countercyclicality...

  11. Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

    Science.gov (United States)

    Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago; Kulesa, Paul M.

    2013-06-01

    Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

  12. Giant cell angiofibroma or localized periorbital lymphedema?

    Science.gov (United States)

    Lynch, Michael C; Chung, Catherine G; Specht, Charles S; Wilkinson, Michael; Clarke, Loren E

    2013-12-01

    Giant cell angiofibroma represents a rare soft tissue neoplasm with a predilection for the orbit. We recently encountered a mass removed from the lower eyelid of a 56-year-old female that histopathologically resembled giant cell angiofibroma. The process consisted of haphazardly arranged CD34-positive spindled and multinucleated cells within an edematous, densely vascular stroma. However, the patient had recently undergone laryngectomy and radiotherapy for a laryngeal squamous cell carcinoma. A similar mass had arisen on the contralateral eyelid, and both had developed several months post-therapy. Lymphedema of the orbit can present as tumor-like nodules and in some cases may share histopathologic features purported to be characteristic of giant cell angiofibroma. A relationship between giant cell angiofibroma and lymphedema has not been established, but our case suggests there may be one. The potential overlap of these two conditions should be recognized, as should other entities that may enter the differential diagnosis. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. MUSIC for localization of thunderstorm cells

    Energy Technology Data Exchange (ETDEWEB)

    Mosher, J.C.; Lewis, P.S. [Los Alamos National Lab., NM (United States); Rynne, T.M. [Scientific Applications and Research Associates, Inc., Huntington Beach, CA (United States)

    1993-12-31

    Lightning represents an event detectable optically, electrically, and acoustically, and several systems are already in place to monitor such activity. Unfortunately, such detection of lightning can occur too late, since operations need to be protected in advance of the first lightning strike. Additionally, the bolt itself can traverse several kilometers before striking the ground, leaving a large region of uncertainty as to the center of the storm and its possible strike regions. NASA Kennedy Space Center has in place an array of electric field mills that monitor the (effectively) DC electric field. Prior to the first lightning strike, the surface electric fields rise as the storm generator within a thundercloud begins charging. Extending methods we developed for an analogous source localization problem in mangnetoencephalography, we present Cramer-Rao lower bounds and MUSIC scans for fitting a point-charge source model to the electric field mill data. Such techniques can allow for the identification and localization of charge centers in cloud structures.

  14. Local cell metrics: a novel method for analysis of cell-cell interactions

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chiang

    2009-10-01

    Full Text Available Abstract Background The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs, which addresses this shortcoming. Results The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. Conclusion LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical

  15. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  16. Effect of antigen on localization of immunologically specific B cells

    International Nuclear Information System (INIS)

    Ponzio, N.M.; Chapman, J.M.; Thorbecke, G.J.

    1976-01-01

    Studies were conducted to demonstrate homing of memory B cells to sites of antigen localization in lymph nodes, using functional criteria to detect local presence of memory cells at varying intervals after intravenous injection. Cell suspensions were prepared from spleens of donor mice injected with complete Freund's adjuvant. Recipient mice were injected with Escherichia coli endotoxin and immune or normal spleen cells and were gamma-irradiated. Results indicated that passively transferred unilateral B cell memory was established. The development over a period of several days of this difference between left and right lymph nodes suggests that recirculating memory B cells are being progressively selected by antigen in the lymph node, rather than that this difference is due to a specific exit of cells from the circulation towards the antigen

  17. In vivo localization of cloned IL-2-dependent T cells

    International Nuclear Information System (INIS)

    Carroll, A.M.; Palladino, M.A.; Oettgen, H.; De Sousa, M.

    1983-01-01

    The quantitative organ distribution and tissue microenvironment positioning of radioisotopically labeled cloned T cells were characterized. Intravenous (iv) injection of 51chromium ( 51 Cr)-labeled, long-term cultured cloned T-helper cells and cells from several cloned cytolytic T-lymphocyte lines (CTLL) resulted in poor localization of these cells in recipient lymphoid tissues, similar to results reported for activated lymphoblastoid cells. Simultaneous administration of interleukin 2 (IL-2) with labeled cells resulted in enhanced recovery from recipient spleen. By the intraperitoneal (ip) injection route, overall percentage recovery of injected radioactivity was lower than by the iv route, but significant localization to lymph nodes occurred. Examination of autoradiographs of tissue sections from recipients of [ 3 H]adenosine-labeled cells showed most label associated with intact, isolated cells in the liver, lungs, spleen, and small intestine. By 24 hr after iv injection, labeled cells in spleen sections were distributed to both nonlymphoid and T- and B-lymphoid areas. These findings suggest that poor localization of these cells to recipient lymphoid tissue is due both to intrinsic characteristics of cultured lymphocytes and to the possible reduced viability of IL-2-dependent cells in vivo

  18. Studying biomolecule localization by engineering bacterial cell wall curvature.

    Directory of Open Access Journals (Sweden)

    Lars D Renner

    Full Text Available In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

  19. Regulation of NKT Cell Localization in Homeostasis and Infection

    Science.gov (United States)

    Slauenwhite, Drew; Johnston, Brent

    2015-01-01

    Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection. PMID:26074921

  20. Regulation of NKT Cell Localization in Homeostasis and Infection.

    Science.gov (United States)

    Slauenwhite, Drew; Johnston, Brent

    2015-01-01

    Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection.

  1. Cell cycle dependent expression of Plk 1 in synchronized porcine fetal fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Anger, M.; Kues, W. A.; Klíma, J.; Mielenz, M.; Kubelka, M.; Motlík, J.; Ešner, M.; Dvořák, Petr; Carnwath, J. W.; Niemann, H.

    2003-01-01

    Roč. 65, - (2003), s. 245-253 ISSN 1040-452X R&D Projects: GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5039906 Keywords : serum deprivation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.543, year: 2003

  2. Localization of functional memory B cells at sites of antigen localization and its relationship to local aspects of immunological memory

    International Nuclear Information System (INIS)

    Ponzio, N.M.; Baine, Y.; Thorbecke, G.J.

    1980-01-01

    Experiments are described which have been designed to test whether antigen in a draining lymph node can mediate local accumulation of passively transferred antigen-specific memory B cells, using recipients whose own immune response is inhibited via γ-irradiation or by injection of cyclophosphamide. (Auth.)

  3. Long-period variations of wind parameters in the mesopause region and the solar cycle dependence

    International Nuclear Information System (INIS)

    Greisiger, K.M.; Schminder, R.; Kuerschner, D.

    1987-01-01

    The solar cycle dependence of wind parameters below 100 km on the basis of long term continuous ionospheric drift measurements in the low frequency range is discussed. For the meridional prevailing wind no significant variation was found. The same comparison as for winter was done for summer where the previous investigations gave no correlation. Now the radar meteor wind measurement values, too, showed a significant negative correlation of the zonal prevailing wind with solar activity for the years 1976 to 1983. The ionospheric drift measurement results of Collm have the same tendency but a larger dispersion due to the lower accuracy of the harmonic analysis because of the shorter daily measuring interval in summer. Continuous wind observations in the upper mesopause region over more than 20 years revealed distinct long term variations, the origin of which cannot be explained with the present knowledge

  4. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

    2010-01-01

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  5. Giant Cell Angiofibroma in Unusual Localization: A Case Report

    Directory of Open Access Journals (Sweden)

    Emel Ebru Pala

    2012-01-01

    Full Text Available Giant cell angiofibroma (GCA was initially described as a potentially recurrent tumor in the orbit of adults. However, it is now recognized that it can also present in other locations. The morphological hallmark is a richly vascularized patternless spindle cell proliferation containing pseudovascular spaces and floret like multinucleate giant cells. Our case was a 32-years-old female complaining of painless solitary nodule arising on the occipital region of the scalp, which was diagnosed as giant cell angiofibroma. We report the case because of its extremely rare localization.

  6. Effect of localized polycrystalline silicon properties on solar cell performance

    Science.gov (United States)

    Leung, D.; Iles, P. A.; Hyland, S.; Kachare, A.

    1984-01-01

    Several forms of polycrystalline silicon, mostly from cast ingots, (including UCP, SILSO and HEM) were studied. On typical slices, localized properties were studied in two ways. Small area (about 2.5 sq mm) mesa diodes were formed, and localized photovoltaic properties were measured. Also a small area (about .015 sq mm) light spot was scanned across the cells; the light spot response was calibrated to measure local diffusion length directly. Using these methods, the effects of grain boundaries, or of intragrain imperfections were correlated with cell performance. Except for the fine grain portion of SILSO, grain boundaries played only a secondary role in determining cell performance. The major factor was intra-grain material quality and it varied with position in ingots and probably related to solidification procedure.

  7. Localized tenosynovial giant cell tumor in both knee joints

    International Nuclear Information System (INIS)

    Kim, Hyun Su; Kwon, Jong Won; Ahn, Jin Hwan; Chang, Moon Jong; Cho, Eun Yoon

    2010-01-01

    Tenosynovial giant cell tumor, previously called pigmented villonodular synovitis (PVNS), is a rare benign neoplastic process that may involve the synovium of the joint. The disorder is usually monoarticular and only a few cases have been reported on polyarticular involvement. Herein, we present a case of localized intra-articular tenosynovial giant cell tumor in a 29-year-old man involving both knee joints with a description of the MR imaging and histological findings. (orig.)

  8. Localization of urease activity in ureaplasma urealyticum cells

    International Nuclear Information System (INIS)

    Vinther, O.

    1976-01-01

    Measurements of the urease activity of various cell fractions of U. urealyticum showed that this activity was confined to the soluble fraction of the cytoplasm. An attempt was made to devise a method for electron microscopic detection of the sites of urease activity based on precipitation of electron-dense MnO 2 at the alkaline pH created by the hydrolysis of urea. The results obtained supported the previous results indicating a cytoplasmatic localization of the urease activity in the cells. Helical ribosome patterns were observed when glutaraldehyde-fixed cells were treated with cytochemical test solutions. (author)

  9. Localization of urease activity in Ureaplasma urealyticum cells

    Energy Technology Data Exchange (ETDEWEB)

    Vinther, O [Aarhus Univ. (Denmark)

    1976-01-01

    Measurements of the urease activity of various cell fractions of U. urealyticum showed that this activity was confined to the soluble fraction of the cytoplasm. An attempt was made to devise a method for electron microscopic detection of the sites of urease activity based on precipitation of electron-dense MnO/sub 2/ at the alkaline pH created by the hydrolysis of urea. The results obtained supported the previous results indicating a cytoplasmatic localization of the urease activity in the cells. Helical ribosome patterns were observed when glutaraldehyde-fixed cells were treated with cytochemical test solutions.

  10. QUANTIFYING THE ANISOTROPY AND SOLAR CYCLE DEPENDENCE OF '1/f' SOLAR WIND FLUCTUATIONS OBSERVED BY ADVANCED COMPOSITION EXPLORER

    International Nuclear Information System (INIS)

    Nicol, R. M.; Chapman, S. C.; Dendy, R. O.

    2009-01-01

    The power spectrum of the evolving solar wind shows evidence of a spectral break between an inertial range (IR) of turbulent fluctuations at higher frequencies and a '1/f' like region at lower frequencies. In the ecliptic plane at ∼1 AU, this break occurs approximately at timescales of a few hours and is observed in the power spectra of components of velocity and magnetic field. The '1/f' energy range is of more direct coronal origin than the IR, and carries signatures of the complex magnetic field structure of the solar corona, and of footpoint stirring in the solar photosphere. To quantify the scaling properties we use generic statistical methods such as generalized structure functions and probability density functions (PDFs), focusing on solar cycle dependence and on anisotropy with respect to the background magnetic field. We present structure function analysis of magnetic and velocity field fluctuations, using a novel technique to decompose the fluctuations into directions parallel and perpendicular to the mean local background magnetic field. Whilst the magnetic field is close to '1/f', we show that the velocity field is '1/f α ' with α ≠ 1. For the velocity, the value of α varies between parallel and perpendicular fluctuations and with the solar cycle. There is also variation in α with solar wind speed. We have examined the PDFs in the fast, quiet solar wind and intriguingly, whilst parallel and perpendicular are distinct, both the B field and velocity show the same PDF of their perpendicular fluctuations, which is close to gamma or inverse Gumbel. These results point to distinct physical processes in the corona and to their mapping out into the solar wind. The scaling exponents obtained constrain the models for these processes.

  11. Local Actions of Melatonin in Somatic Cells of the Testis.

    Science.gov (United States)

    Frungieri, Mónica Beatriz; Calandra, Ricardo Saúl; Rossi, Soledad Paola

    2017-05-31

    The pineal hormone melatonin regulates testicular function through the hypothalamic-adenohypophyseal axis. In addition, direct actions of melatonin in somatic cells of the testis have been described. Melatonin acts as a local modulator of the endocrine activity in Leydig cells. In Sertoli cells, melatonin influences cellular growth, proliferation, energy metabolism and the oxidation state, and consequently may regulate spermatogenesis. These data pinpoint melatonin as a key player in the regulation of testicular physiology (i.e., steroidogenesis, spermatogenesis) mostly in seasonal breeders. In patients with idiopathic infertility, melatonin exerts anti-proliferative and anti-inflammatory effects on testicular macrophages, and provides protective effects against oxidative stress in testicular mast cells. Consequently, melatonin is also involved in the modulation of inflammatory and oxidant/anti-oxidant states in testicular pathology. Overall, the literature data indicate that melatonin has important effects on testicular function and male reproduction.

  12. Local cell-mediated immune reactions in cancer patients

    International Nuclear Information System (INIS)

    Bilynskij, B.T.; Vasil'ev, N.V.; Volod'ko, N.A.; Akademiya Meditsinskikh Nauk SSSR, Tomsk. Onkologicheskij Nauchnyj Tsentr)

    1988-01-01

    The analysis of 178 cases of stage I-II breast cancer showed morphological features of local cell-mediated immune reactions to be of limited prognostic value. A comparative evaluation of some characteristics of cell surface receptors, such as ability to spontaneous rosette formation with sheep erythrocytes and sensitivty to theophylline, was carried out in lymphocyte samples obtained from tumor tissue and peripheral blood of 76 cancer patients subjected to preoperative radiotherapy. The said parameters were studied in breast cancer patients of rosette-forming cell reaction to theophylline were identified, the incidence of some of them being determined by the presence or absence of regional metastases. The level and functional activity of surface receptors of tumor mononuclear cells proved to influence prognosis

  13. Antigen localization controls T cell-mediated tumor immunity.

    Science.gov (United States)

    Zeelenberg, Ingrid S; van Maren, Wendy W C; Boissonnas, Alexandre; Van Hout-Kuijer, Maaike A; Den Brok, Martijn H M G M; Wagenaars, Jori A L; van der Schaaf, Alie; Jansen, Eric J R; Amigorena, Sebastian; Théry, Clotilde; Figdor, Carl G; Adema, Gosse J

    2011-08-01

    Effective antitumor immunotherapy requires the identification of suitable target Ags. Interestingly, many of the tumor Ags used in clinical trials are present in preparations of secreted tumor vesicles (exosomes). In this study, we compared T cell responses elicited by murine MCA101 fibrosarcoma tumors expressing a model Ag at different localizations within the tumor cell in association with secreted vesicles (exosomes), as a nonsecreted cell-associated protein, or as secreted soluble protein. Remarkably, we demonstrated that only the tumor-secreting vesicle-bound Ag elicited a strong Ag-specific CD8(+) T cell response, CD4(+) T cell help, Ag-specific Abs, and a decrease in the percentage of immunosuppressive regulatory T cells in the tumor. Moreover, in a therapeutic tumor model of cryoablation, only in tumors secreting vesicle-bound Ag could Ag-specific CD8(+) T cells still be detected up to 16 d after therapy. We concluded that the localization of an Ag within the tumor codetermines whether a robust immunostimulatory response is elicited. In vivo, vesicle-bound Ag clearly skews toward a more immunogenic phenotype, whereas soluble or cell-associated Ag expression cannot prevent or even delay outgrowth and results in tumor tolerance. This may explain why particular immunotherapies based on these vesicle-bound tumor Ags are potentially successful. Therefore, we conclude that this study may have significant implications in the discovery of new tumor Ags suitable for immunotherapy and that their location should be taken into account to ensure a strong antitumor immune response.

  14. Feeding cycle-dependent circulating insulin fluctuation is not a dominant Zeitgeber for mouse peripheral clocks except in the liver: Differences between endogenous and exogenous insulin effects.

    Science.gov (United States)

    Oishi, Katsutaka; Yasumoto, Yuki; Higo-Yamamoto, Sayaka; Yamamoto, Saori; Ohkura, Naoki

    2017-01-29

    The master clock in the suprachiasmatic nucleus synchronizes peripheral clocks via humoral and neural signals in mammals. Insulin is thought to be a critical Zeitgeber (synchronizer) for peripheral clocks because it induces transient clock gene expression in cultured cells. However, the extent to which fluctuations in feeding-dependent endogenous insulin affect the temporal expression of clock genes remains unclear. We therefore investigated the temporal expression profiles of clock genes in the peripheral tissues of mice fed for 8 h during either the daytime (DF) or the nighttime (NF) for one week to determine the involvement of feeding cycle-dependent endogenous insulin rhythms in the circadian regulation of peripheral clocks. The phase of circulating insulin fluctuations was reversed in DF compared with NF mice, although those of circulating corticosterone fluctuations and nocturnal locomotor activity were identical between these mice. The reversed feeding cycle affected the circadian phases of Per1 and Per2 gene expression in the liver and not in heart, lung, white adipose and skeletal muscle tissues. On the other hand, injected exogenous insulin significantly induced Akt phosphorylation in the heart and skeletal muscle as well as the liver, and significantly induced Per1 and Per2 gene expression in all examined tissues. These findings suggest that feeding cycles and feeding cycle-dependent endogenous insulin fluctuations are not dominant entrainment signals for peripheral clocks other than the liver, although exogenous insulin might reset peripheral oscillators in mammals. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Quantification and localization of mast cells in periapical lesions.

    Science.gov (United States)

    Mahita, V N; Manjunatha, B S; Shah, R; Astekar, M; Purohit, S; Kovvuru, S

    2015-01-01

    Periapical lesions occur in response to chronic irritation in periapical tissue, generally resulting from an infected root canal. Specific etiological agents of induction, participating cell population and growth factors associated with maintenance and resolution of periapical lesions are incompletely understood. Among the cells found in periapical lesions, mast cells have been implicated in the inflammatory mechanism. Quantifications and the possible role played by mast cells in the periapical granuloma and radicular cyst. Hence, this study is to emphasize the presence (localization) and quantification of mast cells in periapical granuloma and radicular cyst. A total of 30 cases and out of which 15 of periapical granuloma and 15 radicular cyst, each along with the case details from the previously diagnosed cases in the department of oral pathology were selected for the study. The gender distribution showed male 8 (53.3%) and females 7 (46.7%) in periapical granuloma cases and male 10 (66.7%) and females 5 (33.3%) in radicular cyst cases. The statistical analysis used was unpaired t-test. Mean mast cell count in periapical granuloma subepithelial and deeper connective tissue, was 12.40 (0.99%) and 7.13 (0.83%), respectively. The mean mast cell counts in subepithelial and deeper connective tissue of radicular cyst were 17.64 (1.59%) and 12.06 (1.33%) respectively, which was statistically significant. No statistical significant difference was noted among males and females. Mast cells were more in number in radicular cyst. Based on the concept that mast cells play a critical role in the induction of inflammation, it is logical to use therapeutic agents to alter mast cell function and secretion, to thwart inflammation at its earliest phases. These findings may suggest the possible role of mast cells in the pathogenesis of periapical lesions.

  16. Secondary Metabolite Localization by Autofluorescence in Living Plant Cells

    Directory of Open Access Journals (Sweden)

    Pascale Talamond

    2015-03-01

    Full Text Available Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla. This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

  17. Local and Regional Spread of Primary Conjunctival Squamous Cell Carcinoma.

    Science.gov (United States)

    Desai, Shilpa J; Pruzan, Noelle L; Geske, Michael J; Jeng, Bennie H; Bloomer, Michele M; Vagefi, M Reza

    2016-04-06

    Two cases of biopsy-proven conjunctival squamous cell carcinoma (SCC) that developed local and regional spread are described. The cases involved a 65-year-old woman and a 79-year-old man who were initially treated at outside institutions for SCC of the conjunctiva. The patients did not have a history of immune compromise. The female patient presented with direct extension into the lacrimal gland but deferred recommended exenteration. Despite eventual exenteration, she developed metastasis to a neck node 6 months later, which was treated with radiotherapy. The male patient presented with local recurrence and a parotid node metastasis treated with exenteration, parotidectomy, selective neck dissection, and postoperative radiotherapy. Review of the outside pathology of both cases revealed positive tumor margins at the time of original resection. Local control of conjunctival SCC is of critical importance to reduce the risk of orbital extension and regional spread.

  18. Salvage surgery for local failure of oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Omura, Ken; Harada, Hiroyuki; Shimamoto, Hiroaki

    2003-01-01

    Local failure rates following treatment for squamous cell carcinoma of the oral cavity have been reported to be in the range of 25%-48%. This study investigated the pattern of failure for squamous cell carcinoma (SCC) of the oral cavity and evaluated salvage surgery for local recurrent disease. The tumor files of 549 patients undergoing treatment for SCC of the oral cavity between 1980 and 2000 at Chiba Cancer Center Hospital were reviewed. Of 549 patients, 289 were initially treated with radiation therapy for their primary tumor, and 260 with surgery, either with or without radiation therapy. One hundred and sixty-one patients developed failures, yielding a failure rate of 29.3%. The site of recurrent tumor was local in 72 (13.1%) patients, regional in 89 (16.2%) patients, and distant in 5 (0.9%) patients. Local failure developed in 49 (17.0%) patients with radiation therapy and in 23 (8.8%) patients with surgery. Out of these 72 patients with local failure, 41 (56.9%) patients had salvage surgery for their disease. Twenty-five (73.5%) of the 34 patients initially treated with radiation therapy were salvaged with surgery, and 4 (57.1%) of the 7 patients initially treated with surgery were cured with further surgery. For the treatment of patients with local failure, the disease should be diagnosed more carefully than untreated disease. The disease that has recurred after radiation therapy may be more likely to be salvaged by surgery, however, the disease that has developed after surgery may have a limited indication for re-surgery. (author)

  19. Follow-the-leader cell migration requires biased cell–cell contact and local microenvironmental signals

    International Nuclear Information System (INIS)

    Wynn, Michelle L; Rupp, Paul; Trainor, Paul A; Kulesa, Paul M; Schnell, Santiago

    2013-01-01

    Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell–cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns. (paper)

  20. The local origin of decidual cells in pregnant mice

    International Nuclear Information System (INIS)

    Zorn, T.M.T.; Abrahamsohn, P.A.; Mariano, M.

    1986-01-01

    In order to evaluate the participation of extrauterine cells in the formation of mouse antimesometrial decidua, [ 3 H]-thymidine was administered ip on days 1, 5 and 6 of pregnancy and the animals were killed 1 h afterwards. A second group of mice received four ip injections of [ 3 H]-thymidine at 6-h intervals on the 1st day of pregnancy and were killed on the 2nd, 5th or 6th day of pregnancy. A third group of virgin mice in estrus received [ 3 H]-thymidine ip four times at 6-h intervals and was killed 96 h after the first injection. Radioautographs of the uteri showed that few endometrial stomal cells were labelled on the 1st and 2nd day of pregnancy. Although many decidual cells incorporated thymidine on the 5th and 6th day of pregnancy in pulse-labelled animals, only few labelled decidual cells were found on the 5th and 6th day of pregnancy in animals that received several injections of thymidine on the 1st and 2nd day of pregnancy. These results indicate that the antimesometrial decidual cells that develop at the beginning of pregnancy are mostly of local origin. The short-term migration of extraneous cells into the uterus to participate in decidualization is not supported by these data. (author) [pt

  1. Internalization and localization of basal insulin peglispro in cells.

    Science.gov (United States)

    Moyers, Julie S; Volk, Catherine B; Cao, Julia X C; Zhang, Chen; Ding, Liyun; Kiselyov, Vladislav V; Michael, M Dodson

    2017-10-15

    Basal insulin peglispro (BIL) is a novel, PEGylated insulin lispro that has a large hydrodynamic size compared with insulin lispro. It has a prolonged duration of action, which is related to a delay in insulin absorption and a reduction in clearance. Given the different physical properties of BIL compared with native insulin and insulin lispro, it is important to assess the cellular internalization characteristics of the molecule. Using immunofluorescent confocal imaging, we compared the cellular internalization and localization patterns of BIL, biosynthetic human insulin, and insulin lispro. We assessed the effects of BIL on internalization of the insulin receptor (IR) and studied cellular clearance of BIL. Co-localization studies using antibodies to either insulin or PEG, and the early endosomal marker EEA1 showed that the overall internalization and subcellular localization pattern of BIL was similar to that of human insulin and insulin lispro; all were rapidly internalized and co-localized with EEA1. During ligand washout for 4 h, concomitant loss of insulin, PEG methoxy group, and PEG backbone immunostaining was observed for BIL, similar to the loss of insulin immunostaining observed for insulin lispro and human insulin. Co-localization studies using an antibody to the lysosomal marker LAMP1 did not reveal evidence of lysosomal localization for insulin lispro, human insulin, BIL, or PEG using either insulin or PEG immunostaining reagents. BIL and human insulin both induced rapid phosphorylation and internalization of human IR. Our findings show that treatment of cells with BIL stimulates internalization and localization of IR to early endosomes. Both the insulin and PEG moieties of BIL undergo a dynamic cellular process of rapid internalization and transport to early endosomes followed by loss of cellular immunostaining in a manner similar to that of insulin lispro and human insulin. The rate of clearance for the insulin lispro portion of BIL was slower than

  2. Localization of mitochondria in living cells with rhodamine 123.

    Science.gov (United States)

    Johnson, L V; Walsh, M L; Chen, L B

    1980-01-01

    The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

  3. Radio(chemotherapy in locally advanced nonsmall cell lung cancer

    Directory of Open Access Journals (Sweden)

    Markus Glatzer

    2016-03-01

    Full Text Available Definitive radiochemotherapy is the standard treatment for many patients with locally advanced nonsmall cell lung cancer (NSCLC. Treatment outcomes have improved over the last decades. Several treatment regimens have been shown effective and safe. This review summarises the results of significant studies between 1996 and 2015 on concomitant and sequential radiochemotherapy regimens and radiation dose per fraction. Beside therapy regimens, optimised radiotherapy planning is indispensable to improve outcome and minimise radiation-induced toxicity. An insight into the rationale of radiotherapy planning for stage III NSCLC is also provided.

  4. Local probing and stimulation of neuronal cells by optical manipulation

    Science.gov (United States)

    Cojoc, Dan

    2014-09-01

    During development and in the adult brain, neurons continuously explore the environment searching for guidance cues, leading to the appropriate connections. Elucidating these mechanisms represents a gold goal in neurobiology. Here, I discuss our recent achievements developing new approaches to locally probe the growth cones and stimulate neuronal cell compartments with high spatial and temporal resolution. Optical tweezers force spectroscopy applied in conjunction with metabolic inhibitors reveals new properties of the cytoskeleton dynamics. On the other hand, using optically manipulated microvectors as functionalized beads or filled liposomes, we demonstrate focal stimulation of neurons by small number of signaling molecules.

  5. A space-jump derivation for non-local models of cell-cell adhesion and non-local chemotaxis.

    Science.gov (United States)

    Buttenschön, Andreas; Hillen, Thomas; Gerisch, Alf; Painter, Kevin J

    2018-01-01

    Cellular adhesion provides one of the fundamental forms of biological interaction between cells and their surroundings, yet the continuum modelling of cellular adhesion has remained mathematically challenging. In 2006, Armstrong et al. proposed a mathematical model in the form of an integro-partial differential equation. Although successful in applications, a derivation from an underlying stochastic random walk has remained elusive. In this work we develop a framework by which non-local models can be derived from a space-jump process. We show how the notions of motility and a cell polarization vector can be naturally included. With this derivation we are able to include microscopic biological properties into the model. We show that particular choices yield the original Armstrong model, while others lead to more general models, including a doubly non-local adhesion model and non-local chemotaxis models. Finally, we use random walk simulations to confirm that the corresponding continuum model represents the mean field behaviour of the stochastic random walk.

  6. A model for cell type localization in the migrating slug of ...

    Indian Academy of Sciences (India)

    PRAKASH

    . Localization of the three major cell types within the migrating slug stage is a dynamic process (Sternfeld 1992;. A model for cell type localization in the migrating slug of Dictyostelium discoideum based on differential chemotactic sensitivity to ...

  7. Axin localizes to mitotic spindles and centrosomes in mitotic cells

    International Nuclear Information System (INIS)

    Kim, Shi-Mun; Choi, Eun-Jin; Song, Ki-Joon; Kim, Sewoon; Seo, Eunjeong; Jho, Eek-Hoon; Kee, Sun-Ho

    2009-01-01

    Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor

  8. Update on contemporary management of clinically localized renal cell carcinoma.

    Science.gov (United States)

    Jorns, J J; Thiel, D D; Castle, E P

    2012-12-01

    Renal cell carcinoma (RCC) continues to increase in incidence with the largest increase manifesting in small, organ-confined tumors. This review outlines the epidemiology and current data pertaining to the management of clinically-localized RCC. In this manuscript, the current data outlining the benefit of nephron sparing to the overall survival of the patient is described. The data pertaining to minimally invasive nephron sparing is also explained in detail. From laparoscopic and robotic partial nephrectomy to watchful waiting and percutaneous ablation, the urologist is continually assaulted with new data for the management of clinically-localized RCC. The data can be confusing, and much of it is conflicting. The addition of new scoring systems or nomograms may aid in predicting which therapy would be most beneficial in certain patient groups. New scoring systems may also predict the difficulty of surgical resection and predict surgical complications. The limitations of the data pertaining to the management of clinically-localized RCC are also outlined.

  9. Local therapy for small cell carcinoma of the cervix

    International Nuclear Information System (INIS)

    Nakamura, C.; Chen, Y.; DuBeshter, B.; Angel, C.; Dawson, A.; Casey, W.

    1996-01-01

    Objective: Small cell carcinoma of the uterine cervix is a rare and aggressive tumor. This tumor is similar to small cell carcinoma of the lung with a tendency to metastasize early. While there has been an increasing interest in the use of chemotherapy regimens similar to those used for small cell carcinoma of the lung, the optimum local therapy for small cell carcinoma of the cervix remains unknown. We reviewed the treatment outcome of patients with small cell carcinoma of the cervix diagnosed in our cancer center with an emphasis on the local/regional disease control. Material and Methods: Between 1983 and 1993, medical records of patients diagnosed with carcinoma of the uterine cervix were reviewed. There were 281 patients with carcinoma of the uterine cervix referred to our department for radiation treatment. Seven patients had pathologic diagnosis of either small cell or neuroendocrine histology. Details of the treatments and follow-up information of these patients were reviewed with a medium follow-up period of three years (range - 1 to 4 years). Results: Five patients had pure small cell histology. Two patients had mixed histology: one with mixed small cell anaplastic neuroendocrine cells and a small foci of adenocarcinoma, the other had mixed small cell and squamous cell histology. Four patients had clinical stage IB disease. The others had IIA, IIB, and IIIB disease, respectively. All patients received either irradiation (XRT) alone or as part of the local therapy. Three patients received XRT alone, one received surgery followed by XRT, one received XRT followed by surgery, and the remaining two had triple modality treatment (chemotherapy, surgery, and XRT). Three patients were alive without evidence of disease recurrence at the last follow-up. Two of these received adjuvant chemotherapy in addition to local therapy. The third patient, whose tumor was smaller than one cm at the time of diagnosis, received XRT alone. Four patients died with disease

  10. Cancer cell uptake behavior of Au nanoring and its localized surface plasmon resonance induced cell inactivation

    International Nuclear Information System (INIS)

    Chu, Che-Kuan; Tu, Yi-Chou; Chang, Yu-Wei; Chu, Chih-Ken; Chen, Shih-Yang; Chi, Ting-Ta; Kiang, Yean-Woei; Yang, Chih-Chung

    2015-01-01

    Au nanorings (NRIs), which have the localized surface plasmon resonance (LSPR) wavelength around 1058 nm, either with or without linked antibodies, are applied to SAS oral cancer cells for cell inactivation through the LSPR-induced photothermal effect when they are illuminated by a laser of 1065 nm in wavelength. Different incubation times of cells with Au NRIs are considered for observing the variations of cell uptake efficiency of Au NRI and the threshold laser intensity for cell inactivation. In each case of incubation time, the cell sample is washed for evaluating the total Au NRI number per cell adsorbed and internalized by the cells based on inductively coupled plasma mass spectrometry measurement. Also, the Au NRIs remaining on cell membrane are etched with KI/I 2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I 2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center with various laser power levels. By using Au NRIs with antibodies, the internalized Au NRI number per cell increases monotonically with incubation time up to 24 h. However, the number of Au NRI remaining on cell membrane reaches a maximum at 12 h in incubation time. The cell uptake behavior of an Au NRI without antibodies is similar to that with antibodies except that the uptake NRI number is significantly smaller and the incubation time for the maximum NRI number remaining on cell membrane is delayed to 20 h. By comparing the threshold laser intensities before and after KI/I 2 etching, it is found that the Au NRIs remaining on cell membrane cause more effective cancer cell inactivation, when compared with the internalized Au NRIs. (paper)

  11. Localization of ORC1 During the Cell Cycle in Human Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Frederick D. Coffman

    2011-01-01

    Full Text Available The interaction of the origin recognition complex (ORC with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.

  12. Local stem cell depletion model for normal tissue damage

    International Nuclear Information System (INIS)

    Yaes, R.J.; Keland, A.

    1987-01-01

    The hypothesis that radiation causes normal tissue damage by completely depleting local regions of tissue of viable stem cells leads to a simple mathematical model for such damage. In organs like skin and spinal cord where destruction of a small volume of tissue leads to a clinically apparent complication, the complication probability is expressed as a function of dose, volume and stem cell number by a simple triple negative exponential function analogous to the double exponential function of Munro and Gilbert for tumor control. The steep dose response curves for radiation myelitis that are obtained with our model are compared with the experimental data for radiation myelitis in laboratory rats. The model can be generalized to include other types or organs, high LET radiation, fractionated courses of radiation, and cases where an organ with a heterogeneous stem cell population receives an inhomogeneous dose of radiation. In principle it would thus be possible to determine the probability of tumor control and of damage to any organ within the radiation field if the dose distribution in three dimensional space within a patient is known

  13. Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells

    Directory of Open Access Journals (Sweden)

    Mazel-Sanchez Beryl

    2010-03-01

    Full Text Available Abstract In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

  14. Development of a Fatigue Model for Low Alloy Steels Using a Cycle-Dependent Cohesive Zone Law

    Directory of Open Access Journals (Sweden)

    Kyungmok Kim

    2014-03-01

    Full Text Available A fatigue model for SAE 4130 steels is developed using a cycle-dependent cohesive zone law. Reduction of fracture energy and degradation of stiffness are considered to describe failure resistance after certain number of cycles. The reduction rate of fracture energy is determined with experimental stress (S- number of cycles to failure (N scatter found in the literature. Three-dimensional finite element models containing a cohesive zone are generated with commercial software (ABAQUS. Calculated fatigue lives at different stress ratios are in good agreement with experimental ones. In addition, fatigue behavior of hardened SAE 4130 steels is predicted with that of normalized material.

  15. Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

    Science.gov (United States)

    Sotiriou, P; Giannoutsou, E; Panteris, E; Galatis, B; Apostolakos, P

    2018-03-01

    The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

  16. Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes.

    Directory of Open Access Journals (Sweden)

    Hui Shi

    Full Text Available Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM. We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

  17. Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes.

    Science.gov (United States)

    Shi, Hui; Liu, Zhuo; Li, Ang; Yin, Jing; Chong, Alvin G L; Tan, Kevin S W; Zhang, Yong; Lim, Chwee Teck

    2013-01-01

    Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

  18. Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture.

    Science.gov (United States)

    Chhabra, Aastha; Jaiswal, Astha; Malhotra, Umang; Kohli, Shrey; Rani, Vibha

    2012-09-01

    In situ zymography is a unique technique for detection and localization of enzyme-substrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the 'in position' activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.

  19. Influence of local porosity and local permeability on the performances of a polymer electrolyte membrane fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Akiki, Tilda [Universite Saint Esprit Kaslik (Lebanon); Universite de Technologie de Belfort-Montbeliard, FCLAB Institute for Research on Fuel Cell Systems, 90010 Belfort (France); Charon, Willy; Iltchev, Marie-Christine; Kouta, Raed [Universite de Technologie de Belfort-Montbeliard, FCLAB Institute for Research on Fuel Cell Systems, 90010 Belfort (France); Accary, Gilbert [Universite Saint Esprit Kaslik (Lebanon)

    2010-08-15

    In the literature, many models and studies focused on the steady-state aspect of fuel cell systems while their dynamic transient behavior is still a wide area of research. In the present paper, we study the effects of mechanical solicitations on the performance of a proton exchange membrane fuel cell as well as the coupling between the physico-chemical phenomena and the mechanical behavior. We first develop a finite element method to analyze the local porosity distribution and the local permeability distribution inside the gas diffusion layer induced by different pressures applied on deformable graphite or steel bipolar plates. Then, a multi-physical approach is carried out, taking into account the chemical phenomena and the effects of the mechanical compression of the fuel cell, more precisely the deformation of the gas diffusion layer, the changes in the physical properties and the mass transfer in the gas diffusion layer. The effects of this varying porosity and permeability fields on the polarization and on the power density curves are reported, and the local current density is also investigated. Unlike other studies, our model accounts for a porosity field that varies locally in order to correctly simulate the effect of an inhomogeneous compression in the cell. (author)

  20. Localization of thymosin ß10 in breast cancer cells

    DEFF Research Database (Denmark)

    Mælan, A.ase Elisabeth; Rasmussen, Trine Kring; Larsson, Lars-Inge

    2007-01-01

    as in cell motility and spreading. We have studied the distribution of endogenously expressed thymosin ß10 in cultured human breast cancer cell lines. Both unperturbed monolayer cultures and wound-healing models were examined using double-staining for thymosin ß10 and polymerized (F-) actin. Our findings...... show that thymosin ß10 is expressed in all three-cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-231) studied. No or little staining was detected in confluent cells, whereas strong staining occurred in semiconfluent cells and in cells populating monolayer wounds. Importantly, the distribution of staining...... for thymosin ß10 was inverse of staining for F-actin. These data support a physiological role for thymosin ß10 in sequestration of G-actin as well as in cancer cell motility....

  1. Sex and estrous cycle-dependent rapid protein kinase signaling actions of estrogen in distal colonic cells.

    LENUS (Irish Health Repository)

    O'Mahony, Fiona

    2008-10-01

    Previous studies from our laboratory demonstrated that 17beta-estradiol (E2) rapidly inhibits Cl(-) secretion in rat and human distal colonic epithelium. The inhibition has been shown to occur via targeting of a basolateral K(+) channel identified as the KCNQ1 (KvLQT1) channel. E2 indirectly modulates the channel activity via a cascade of second messengers which are rapidly phosphorylated in response to E2. The anti-secretory mechanism may be the manner by which E2 induces fluid retention in the intestine during periods of high circulating plasma E2. Here we review the sex-dependent and estrous cycle regulation of this novel rapid response to E2. The inhibition of KCNQ1 channel activity and Cl(-) secretion will be of interest in the future in the investigation of the retentive effects of estrogen in female tissue and also in the study of secretory disorders and drugable targets of the intestine.

  2. A new epigenetic marker: The replication-coupled, cell cycle-dependent, dual modification of the histone H4 tail

    Czech Academy of Sciences Publication Activity Database

    Fidlerová, Helena; Kalinová, Jana; Blechová, Miroslava; Velek, Jiří; Raška, Ivan

    2009-01-01

    Roč. 167, č. 1 (2009), s. 76-82 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA304/06/1691 Grant - others:GA MŠk(CZ) LC535 Program:LC Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z40550506 Keywords : epigenetics * H4K16 * H4K20 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.673, year: 2009

  3. local

    Directory of Open Access Journals (Sweden)

    Abílio Amiguinho

    2005-01-01

    Full Text Available The process of socio-educational territorialisation in rural contexts is the topic of this text. The theme corresponds to a challenge to address it having as main axis of discussion either the problem of social exclusion or that of local development. The reasons to locate the discussion in this last field of analysis are discussed in the first part of the text. Theoretical and political reasons are there articulated because the question is about projects whose intentions and practices call for the political both in the theoretical debate and in the choices that anticipate intervention. From research conducted for several years, I use contributions that aim at discuss and enlighten how school can be a potential locus of local development. Its identification and recognition as local institution (either because of those that work and live in it or because of those that act in the surrounding context are crucial steps to progressively constitute school as a partner for development. The promotion of the local values and roots, the reconstruction of socio-personal and local identities, the production of sociabilities and the equation and solution of shared problems were the dimensions of a socio-educative intervention, markedly globalising. This scenario, as it is argued, was also, intentionally, one of transformation and of deliberate change of school and of the administration of the educative territoires.

  4. Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

    Science.gov (United States)

    Lee, Ok Ran; Cho, Hyung-Taeg

    2012-12-01

    Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

  5. LOCAL IMMUNITY BY TISSUE-RESIDENT CD8+ MEMORY T CELLS

    Directory of Open Access Journals (Sweden)

    Thomas eGebhardt

    2012-11-01

    Full Text Available Microbial infection primes a CD8+ cytotoxic T cell response that gives rise to a long-lived population of circulating memory cells able to provide protection against systemic reinfection. Despite this, effective CD8+ T cell surveillance of barrier tissues such as skin and mucosa typically wanes with time, resulting in limited T cell-mediated protection in these peripheral tissues. However, recent evidence suggests that a specialized subset of CD103+ memory T cells can permanently lodge and persist in peripheral tissues, and that these cells can compensate for the loss of peripheral immune surveillance by circulating memory T cells. Here, we review evolving concepts regarding the generation and long-term persistence of these tissue-resident memory T cells (TRM in epithelial and neuronal tissues. We further discuss the role of TRM cells in local infection control and their contribution to localized immune phenomena, in both mice and humans.

  6. Immunohistochemical localization of glucagon and pancreatic polypeptide on rat endocrine pancreas: coexistence in rat islet cells

    Directory of Open Access Journals (Sweden)

    YH Huang

    2009-08-01

    Full Text Available We used immunofluorescence double staining method to investigate the cellular localization of glucagon and pancreatic polypeptide (PP in rat pancreatic islets. The results showed that both A-cells (glucagon-secreting cells and PP-cells (PPsecreting cells were located in the periphery of the islets. However, A-cells and PP-cells had a different regional distribution. Most of A-cells were located in the splenic lobe but a few of them were in the duodenal lobe of the pancreas. In contrast, the majority of PP-cells were found in the duodenal lobe and a few of them were in the splenic lobe of the pancreas. Furthermore, we found that 67.74% A-cells had PP immunoreactivity, 70.92% PP-cells contained glucagon immunoreactivity with immunofluorescence double staining. Our data support the concept of a common precursor stem cell for pancreatic hormone-producing cells.

  7. Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

    International Nuclear Information System (INIS)

    Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

    2008-01-01

    Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism

  8. ANF and exocrine pancreas: ultrastructural autoradiographic localization in acinar cells

    International Nuclear Information System (INIS)

    Chabot, J.G.; Morel, G.; Belles-Isles, M.; Jeandel, L.; Heisler, S.

    1988-01-01

    Atrial natriuretic factor (ANF) binding sites have been recently demonstrated to be present in exocrine pancreas by an in vitro autoradiographic approach. An autoradiographic study was carried out to identify the exocrine cells containing ANF binding sites and to monitor the fate of 125 I-labeled ANF in acinar cells after removal of pancreas at specific time intervals (1-30 min) after intravenous administration. At the light microscopic level, silver grains were found over acinar and centroacinar cells. Concomitant injection of an excess of unlabeled ANF inhibited the binding of labeled peptide by approximately 60%. At the electron microscopic level, the time-course study in acinar cells has revealed that of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria, and zymogen granules, the nucleus had distinct labeling patterns. Plasma membrane was maximally labeled 1 and 2 min after injection with 125 I-ANF. Golgi apparatus was significantly labeled from 2 to 30 min after injection, mitochondria from 1 to 30 min after injection, zymogen granules at 1 and 15 min, and the nucleus only at 30 min. The lysosomal compartment was not labeled during the 30-min observation period. These results suggest that after binding to the plasma membrane, ANF is rapidly internalized and distributed to the intracellular organelles as a function of time. Labeling of the zymogen granules suggests that they may bind ANF and that the atrial peptide may be secreted by acinar cells. The significance of association of radioactivity with mitochondria and nuclei remains to be elucidated but may represent intracellular sites of action of ANF complementary to those on plasma membranes

  9. Localized giant cell tumors in the spinal column radiologic presentation

    International Nuclear Information System (INIS)

    Fernandez Echeverria, M.A.; Parra Blanco, J.A.; Pagola Serrano, M.A.; Mellado Santos, J.M.; Bueno Lopez, J.; Gonzalez Tutor, A.

    1994-01-01

    Given the uncommonness of the location of giant cell tumors (GCT) in the spinal column and the limited number of studies published, we present a case of GCT located in the spinal column, which involved both vertebral bodies and partially destroyed the adjacent rib. (Author)

  10. Local stem cell depletion model for radiation myelitis

    International Nuclear Information System (INIS)

    Yaes, R.J.; Kalend, A.

    1988-01-01

    We propose a model for normal tissue damage based on the assumption that adult mammalian stem cells have limited mobility and, consequently, for each organ, there is a maximum volume (the critical volume, Vc), that can be repopulated and repaired by a single surviving stem cell. This concept is applied to a simple, 1-dimensional model of the spinal cord, where the critical volume is a slice of thickness, t, assumed to be small compared to lengths of spinal cord usually irradiated clinically. The probability of myelitis is explicitly obtained as a function of the dose, dose per fraction, length of cord irradiated, slice thickness, number of stem cells per slice and parameters alpha and beta of the stem cell survival curve. The complication probability is expressed as a triple negative exponential function of dose analogous to the double negative exponential function for tumor control, resulting in a steep dose-response curve with short tails in both the high dose and low dose regions. We show that the model predictions are compatible with the experimental data for radiation myelitis in the rat. We discuss how this concept can be applied to other organs such as skin and to organs composed of structurally and functionally distinct subunits, such as the kidney

  11. Localization and characterization of immunocompetent cells in the human retina

    NARCIS (Netherlands)

    Yang, P.; Das, P. K.; Kijlstra, A.

    2000-01-01

    Recent studies have shown that experimental uveitis can be induced by the appropriate administration of various retinal antigens. Little is known about the in-situ interactions between immune cells in the retina as a prerequisite for understanding the mechanisms involving the presentation of

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

    Directory of Open Access Journals (Sweden)

    Thomas DY

    2006-04-01

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

  13. Funnel for localizing biological cell placement and arrangement

    Energy Technology Data Exchange (ETDEWEB)

    Soscia, David; Benett, William J.; Mukerjee, Erik V.

    2018-03-06

    The present disclosure relates to a funnel apparatus for channeling cells onto a plurality of distinct, closely spaced regions of a seeding surface. The funnel apparatus has a body portion having an upper surface and a lower surface. The body portion forms a plurality of flow paths, at least one of which is shaped to have a decreasing cross-sectional area from the upper surface to the lower surface. The flow paths are formed at the lower surface to enable cells deposited into the flow paths at the upper surface of the funnel apparatus to be channeled into a plurality of distinct, closely spaced regions on the seeding surface positioned adjacent the lower surface.

  14. α-Crystallin localizes to the leading edges of migrating lens epithelial cells

    International Nuclear Information System (INIS)

    Maddala, Rupalatha; Vasantha Rao, P.

    2005-01-01

    α-crystallin (αA and αB) is a major lens protein, which belongs to the small heat-shock family of proteins and binds to various cytoskeletal proteins including actin, vimentin and desmin. In this study, we investigated the cellular localization of αA and αB-crystallins in migrating epithelial cells isolated from porcine lens. Immunofluorescence localization and confocal imaging of αB-crystallin in confluent and in migrating subconfluent cell cultures revealed a distinct pattern of subcellular distribution. While αB-crystallin localization was predominantly cytoplasmic in confluent cultures, it was strongly localized to the leading edges of cell membrane or the lamellipodia in migrating cells. In accordance with this pattern, we found abundant levels of αB-crystallin in membrane fractions compared to cytosolic and nuclear fractions in migrating lens epithelial cells. αA-crystallin, which has 60% sequence identity to αB-crystallin, also exhibited a distribution profile localizing to the leading edge of the cell membrane in migrating lens epithelial cells. Localization of αB-crystallin to the lamellipodia appears to be dependent on phosphorylation of residue serine-59. An inhibitor of p38 MAP kinase (SB202190), but not the ERK kinase inhibitor PD98059, was found to diminish localization of αB-crystallin to the lamellipodia, and this effect was found to be associated with reduced levels of Serine-59 phosphorylated αB-crystallin in SB202190-treated migrating lens epithelial cells. αB-crystallin localization to the lamellipodia was also altered by the treatment with RGD (Arg-Ala-Asp) peptide, dominant negative N17 Rac1 GTPase, cytochalasin D and Src kinase inhibitor (PP2), but not by the Rho kinase inhibitor Y-27632 or the myosin II inhibitor, blebbistatin. Additionally, in migrating lens epithelial cells, αB-crystallin exhibited a clear co-localization with the actin meshwork, β-catenin, WAVE-1, a promoter of actin nucleation, Abi-2, a component of WAVE

  15. Regulation of CD8+ T cell responses to retinal antigen by local FoxP3+ regulatory T cells

    Directory of Open Access Journals (Sweden)

    Scott W McPherson

    2012-06-01

    Full Text Available While pathogenic CD4 T cells are well known mediators of autoimmune uveoretinitis, CD8 T cells can also be uveitogenic. Since preliminary studies indicated that C57BL/6 mice were minimally susceptible to autoimmune uveoretinitis induction by CD8 T cells, the basis of the retinal disease resistance was sought. Mice that express β-galactosidase (βgal on a retina-specific promoter (arrβgal mice were backcrossed to mice expressing green fluorescent protein and diphtheria toxin receptor under control of the Foxp3 promoter (Foxp3-DTR/GFP mice, and to T cell receptor transgenic mice that produce βgal specific CD8 T cells (BG1 mice. These mice were used to explore the role of regulatory T cells in the resistance to retinal autoimmune disease. Experiments with T cells from double transgenic BG1 x Foxp3-DTR/GFP mice transferred into Foxp3-DTR/GFP x arrβgal mice confirmed that the retina was well protected from attempts to induce disease by adoptive transfer of activated BG1 T cells. The successful induction of retinal disease following unilateral intraocular administration of diphtheria toxin to deplete regulatory T cells showed that the protective activity was dependent on local, toxin-sensitive regulatory T cells; the opposite, untreated eye remained disease-free. Although there were very few Foxp3+ regulatory T cells in the parenchyma of quiescent retina, and they did not accumulate in retina, their depletion by local toxin administration led to disease susceptibility. We propose that these regulatory T cells modulate the pathogenic activity of βgal-specific CD8 T cells in the retinas of arrβgal mice on a local basis, allowing immunoregulation to be responsive to local conditions.

  16. Radioresistance of intermediate TCR cells and their localization in the body of mice revealed by irradiation

    International Nuclear Information System (INIS)

    Kimura, Motohiko; Watanabe, Hisami; Ohtsuka, Kazuo; Iiai, Tsuneo; Tsuchida, Masanori; Sato, Shotaro; Abo, Toru

    1993-01-01

    Extrathymic generation of T cells in the liver and in the intestine was recently demonstrated. We investigated herein whether such T cells, especially those in the liver, are present in other organs of mice. This investigation is possible employing our recently introduced method with which even a minor proportion of extrathymic, intermediate T-cell receptor (TCR) cells in organs other than the liver can be identified. Intermediate TCR cells expressed higher levels of IL-2Rβ and lymphocyte function-associated antigen-1 (LFA-1) than bright TCR cells (i.e., T cells of thymic origin) as revealed by two-color staining. Although intermediate TCR cells were present at a small proportion in the spleen and thymus, they predominated in these organs after irradiation (9 Gy) and bone marrow reconstitution, or after low dose irradiation (6 Gy). This was due to that intermediate TCR cells were relatively radioresistant, whereas bright TCR cells were radiosensitive. Microscopic observation and immunochemical staining showed that intermediate TCR cells in the spleen localized in the red pulp and those in the thymus localized in the medulla. These intermediate TCR cells displayed a large light scatter, similar to such cells in the liver. The present results suggest that intermediate TCR cells may proliferate at multiple sites in the body. (author)

  17. Combined local current distribution measurements and high resolution neutron radiography of operating direct methanol fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, Alexander; Wippermann, Klaus [Forschungszentrum Juelich GmbH (Germany). Inst. of Energy Research, IEF-3: Fuel Cells; Sanders, Tilman [RWTH Aachen (DE). Inst. for Power Electronics and Electrical Drives (ISEA); Arlt, Tobias [Helmholtz Centre Berlin (Germany). Inst. for Applied Materials

    2010-07-01

    Neutron radiography allows the investigation of the local fluid distribution in direct methanol fuel cells (DMFCs) under operating conditions. Spatial resolutions in the order of some tens of micrometers at the full test cell area are achieved. This offers the possibility to study practice-oriented, large stack cells with an active area of several hundred cm{sup 2} as well as specially designed, small test cells with an area of some cm{sup 2}. Combined studies of high resolution neutron radiography and segmented cell measurements are especially valuable, because they enable a correlation of local fluid distribution and local performance [1, 2]. The knowledge of this interdependency is essential to optimise the water management and performance respecting a homogeneous fluid, current and temperature distribution and to achieve high performance and durability of DMFCs. (orig.)

  18. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    Science.gov (United States)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

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

    Directory of Open Access Journals (Sweden)

    Nestor Gomez

    2016-09-01

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

  20. The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer

    International Nuclear Information System (INIS)

    Akkiprik, Mustafa; Hu, Limei; Sahin, Aysegul; Hao, Xishan; Zhang, Wei

    2009-01-01

    Insulin-like growth factor binding protein 5 (IGFBP5) has been shown to be associated with breast cancer metastasis in clinical marker studies. However, a major difficulty in understanding how IGFBP5 functions in this capacity is the paradoxical observation that ectopic overexpression of IGFBP5 in breast cancer cell lines results in suppressed cellular proliferation. In cancer tissues, IGFBP5 resides mainly in the cytoplasm; however, in transfected cells, IGFBP5 is mainly located in the nucleus. We hypothesized that subcellular localization of IGFBP5 affects its functions in host cells. To test this hypothesis, we generated wild-type and mutant IGFBP5 expression constructs. The mutation occurs within the nuclear localization sequence (NLS) of the protein and is generated by site-directed mutagenesis using the wild-type IGFBP5 expression construct as a template. Next, we transfected each expression construct into MDA-MB-435 breast cancer cells to establish stable clones overexpressing either wild-type or mutant IGFBP5. Functional analysis revealed that cells overexpressing wild-type IGFBP5 had significantly lower cell growth rate and motility than the vector-transfected cells, whereas cells overexpressing mutant IGFBP5 demonstrated a significantly higher ability to proliferate and migrate. To illustrate the subcellular localization of the proteins, we generated wild-type and mutant IGFBP5-pDsRed fluorescence fusion constructs. Fluorescence microscopy imaging revealed that mutation of the NLS in IGFBP5 switched the accumulation of IGFBP5 from the nucleus to the cytoplasm of the protein. Together, these findings imply that the mutant form of IGFBP5 increases proliferation and motility of breast cancer cells and that mutation of the NLS in IGFBP5 results in localization of IGFBP5 in the cytoplasm, suggesting that subcellular localization of IGFBP5 affects its cell growth and migration functions in the breast cancer cells

  1. Locally Advanced Basal Cell Carcinoma with Intraocular Invasion

    Directory of Open Access Journals (Sweden)

    Georgi Tchernev

    2018-01-01

    Full Text Available We present a 103 - year - old patient, with duration of complaints of about ten years. The initial complaint had been presented as a small nodule, located on the eyebrow, which subsequently ulcerated and encompassed larger regions of the upper and lower eyelids. For the past three years, the patient also had complaints of a worsening of his vision, without seeking for medical help. Within the dermatological examination, an intraocular and periocular localised tumour was established, characterised by a raised peripheral edge and central ulceration. More careful examination revealed that the bulb was fully consumed. The patient refused further diagnosis and treatment. Advanced basal cell carcinomas with intraocular invasion are rare in general. If the patient refuses surgery, radiotherapy and systemic therapy with modern medications such as Vismodegib or Sonidegib are available as treatment options.

  2. Unusual intraconal localization of orbital giant cell angiofibroma.

    Science.gov (United States)

    Ekin, Meryem Altin; Ugurlu, Seyda Karadeniz; Cakalagaoglu, Fulya

    2018-01-01

    Giant cell angiofibroma (GCA) is a recently reported rare soft-tissue tumor that can develop in various sites including orbit. Orbital GCAs were mainly located in the eyelid or extraconal regions such as lacrimal gland and conjunctiva. We report an atypical case of a GCA arising in the intraconal area of the orbit in a 65-year-old male patient. The tumor was excised in total by lateral orbitotomy. Histological and immunohistochemical features were consistent with the diagnosis of GCA. No recurrence was observed during the follow-up of over 2 years. GCA is a rare tumor that should be considered in the differential diagnosis of intraconal orbital tumors. Complete surgical removal is the current optimal treatment option.

  3. Unusual intraconal localization of orbital giant cell angiofibroma

    Directory of Open Access Journals (Sweden)

    Meryem Altin Ekin

    2018-01-01

    Full Text Available Giant cell angiofibroma (GCA is a recently reported rare soft-tissue tumor that can develop in various sites including orbit. Orbital GCAs were mainly located in the eyelid or extraconal regions such as lacrimal gland and conjunctiva. We report an atypical case of a GCA arising in the intraconal area of the orbit in a 65-year-old male patient. The tumor was excised in total by lateral orbitotomy. Histological and immunohistochemical features were consistent with the diagnosis of GCA. No recurrence was observed during the follow-up of over 2 years. GCA is a rare tumor that should be considered in the differential diagnosis of intraconal orbital tumors. Complete surgical removal is the current optimal treatment option.

  4. Local and Long-Range Circuit Connections to Hilar Mossy Cells in the Dentate Gyrus

    Science.gov (United States)

    Sun, Yanjun; Grieco, Steven F.; Holmes, Todd C.

    2017-01-01

    Abstract Hilar mossy cells are the prominent glutamatergic cell type in the dentate hilus of the dentate gyrus (DG); they have been proposed to have critical roles in the DG network. To better understand how mossy cells contribute to DG function, we have applied new viral genetic and functional circuit mapping approaches to quantitatively map and compare local and long-range circuit connections of mossy cells and dentate granule cells in the mouse. The great majority of inputs to mossy cells consist of two parallel inputs from within the DG: an excitatory input pathway from dentate granule cells and an inhibitory input pathway from local DG inhibitory neurons. Mossy cells also receive a moderate degree of excitatory and inhibitory CA3 input from proximal CA3 subfields. Long range inputs to mossy cells are numerically sparse, and they are only identified readily from the medial septum and the septofimbrial nucleus. In comparison, dentate granule cells receive most of their inputs from the entorhinal cortex. The granule cells receive significant synaptic inputs from the hilus and the medial septum, and they also receive direct inputs from both distal and proximal CA3 subfields, which has been underdescribed in the existing literature. Our slice-based physiological mapping studies further supported the identified circuit connections of mossy cells and granule cells. Together, our data suggest that hilar mossy cells are major local circuit integrators and they exert modulation of the activity of dentate granule cells as well as the CA3 region through “back-projection” pathways. PMID:28451637

  5. Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Arai, Rumi; En, Atsuki; Ukekawa, Ryo [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Miki, Kensuke [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ichiban Life Corporation, 1-1-7 Horai-cho, Naka-ku, Yokohama 231-0048 (Japan); Fujii, Michihiko, E-mail: mifuji@yokohama-cu.ac.jp [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ayusawa, Dai [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ichiban Life Corporation, 1-1-7 Horai-cho, Naka-ku, Yokohama 231-0048 (Japan)

    2016-05-13

    5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

  6. Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

    Science.gov (United States)

    Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

    2013-03-01

    Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

  7. Mitochondrial localization of the low level p53 protein in proliferative cells

    Energy Technology Data Exchange (ETDEWEB)

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Oliver, Lisa [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Rincheval, Vincent; Renaud, Flore [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vallette, Francois M. [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Mignotte, Bernard [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vayssiere, Jean-Luc, E-mail: jean-luc.vayssiere@uvsq.fr [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France)

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  8. Mitochondrial localization of the low level p53 protein in proliferative cells

    International Nuclear Information System (INIS)

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie; Oliver, Lisa; Rincheval, Vincent; Renaud, Flore; Vallette, Francois M.; Mignotte, Bernard; Vayssiere, Jean-Luc

    2009-01-01

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  9. Control of cell division and the spatial localization of assembled gene products in Caulobacter crescentus

    International Nuclear Information System (INIS)

    Nathan, P.D.

    1988-01-01

    Experiments are described that examine the role of penicillin-binding proteins (PBPs) in the regulation of cell division in Caulobacter crescentus; and the spatial localization of methyl-accepting chemotaxis proteins (MCPs) in C. crescentus swarmer and predivisional cells. In the analysis of PBP function, in vivo and in vitro assays are used to directly label C. crescentus PBPs with [ 3 H] penicillin G in wild type strain CB15, in a series of conditional cell division mutants and in new temperature sensitive cephalosporin C resistant mutants PC8002 and PC8003. 14 PBPs are characterized and a high molecular weight PBP (PBP 1B) that is required for cell division is identified. PBP 1B competes for β-lactams that induce filament formation and may be a high affinity binding protein. A second high molecular weight PBP (PBP 1C) is also associated with defective cell division. The examination of PBP patterns in synchronous swarmer cells reveals that the in vivo activity of PBP 1B and PBP 1C increases at the time that the cell division pathway is initiated. None of the PBPs, however, appear to be differentially localized in the C. crescentus cell. In the analysis of MCP localization, in vivo and in vitro assays are used to directly label C. crescentus MCPs with methyl- 3 H. MCPs are examined in flagellated and non-flagellated vesicles prepared from cells by immunoaffinity chromatography

  10. Determinates of tumor response to radiation: Tumor cells, tumor stroma and permanent local control

    International Nuclear Information System (INIS)

    Li, Wende; Huang, Peigen; Chen, David J.; Gerweck, Leo E.

    2014-01-01

    Background and purpose: The causes of tumor response variation to radiation remain obscure, thus hampering the development of predictive assays and strategies to decrease resistance. The present study evaluates the impact of host tumor stromal elements and the in vivo environment on tumor cell kill, and relationship between tumor cell radiosensitivity and the tumor control dose. Material and methods: Five endpoints were evaluated and compared in a radiosensitive DNA double-strand break repair-defective (DNA-PKcs −/− ) tumor line, and its DNA-PKcs repair competent transfected counterpart. In vitro colony formation assays were performed on in vitro cultured cells, on cells obtained directly from tumors, and on cells irradiated in situ. Permanent local control was assessed by the TCD 50 assay. Vascular effects were evaluated by functional vascular density assays. Results: The fraction of repair competent and repair deficient tumor cells surviving radiation did not substantially differ whether irradiated in vitro, i.e., in the absence of host stromal elements and factors, from the fraction of cells killed following in vivo irradiation. Additionally, the altered tumor cell sensitivity resulted in a proportional change in the dose required to achieve permanent local control. The estimated number of tumor cells per tumor, their cloning efficiency and radiosensitivity, all assessed by in vitro assays, were used to predict successfully, the measured tumor control doses. Conclusion: The number of clonogens per tumor and their radiosensitivity govern the permanent local control dose

  11. Ultrastructural Localization and Molecular Associations of HCV Capsid Protein in Jurkat T Cells

    Directory of Open Access Journals (Sweden)

    Cecilia Fernández-Ponce

    2018-01-01

    Full Text Available Hepatitis C virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. When expressed in CD4+ T lymphocytes, it can induce modifications in several essential cellular and biological networks. To shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed HCV-core subcellular localization and its associations with host proteins in Jurkat T cells. In order to investigate the intracellular localization of Hepatitis C virus core protein, we have used a lentiviral system to transduce Jurkat T cells and subsequently localize the protein using immunoelectron microscopy techniques. We found that in Jurkat T cells, Hepatitis C virus core protein mostly localizes in the nucleus and specifically in the nucleolus. In addition, we performed pull-down assays combined with Mass Spectrometry Analysis, to identify proteins that associate with Hepatitis C virus core in Jurkat T cells. We found proteins such as NOLC1, PP1γ, ILF3, and C1QBP implicated in localization and/or traffic to the nucleolus. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4+ T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. Thus, in the current work, we show the ultrastructural localization of Hepatitis C virus core and the first profile of HCV core associated proteins in T cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of Hepatitis C virus core protein. Thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying HCV core mediated alterations that had been described in relevant biological processes in CD4+ T cells.

  12. Nuclear localization of the mitochondrial ncRNAs in normal and cancer cells.

    Science.gov (United States)

    Landerer, Eduardo; Villegas, Jaime; Burzio, Veronica A; Oliveira, Luciana; Villota, Claudio; Lopez, Constanza; Restovic, Franko; Martinez, Ronny; Castillo, Octavio; Burzio, Luis O

    2011-08-01

    We have previously shown a differential expression of a family of mitochondrial ncRNAs in normal and cancer cells. Normal proliferating cells and cancer cells express the sense mitochondrial ncRNA (SncmtRNA). In addition, while normal proliferating cells express two antisense mitochondrial ncRNAs (ASncmtRNAs-1 and -2), these transcripts seem to be universally down-regulated in cancer cells. In situ hybridization (ISH) of some normal and cancer tissues reveals nuclear localization of these transcripts suggesting that they are exported from mitochondria. FISH and confocal microscopy, in situ digestion with RNase previous to ISH and electron microscopy ISH was employed to confirm the extra-mitochondrial localization of the SncmtRNA and the ASncmtRNAs in normal proliferating and cancer cells of human and mouse. In normal human kidney and mouse testis the SncmtRNA and the ASncmtRNAs were found outside the organelle and especially localized in the nucleus associated to heterochromatin. In cancer cells, only the SncmtRNA was expressed and was found associated to heterochromatin and nucleoli. The ubiquitous localization of these mitochondrial transcripts in the nucleus suggests that they are new players in the mitochondrial-nuclear communication pathway or retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step on neoplastic transformation and cancer progression.

  13. Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

    Science.gov (United States)

    Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

    2013-10-01

    The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule

  14. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    International Nuclear Information System (INIS)

    Kutty, R. Krishnan; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-01-01

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 27 KKRKAP 276 ) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting

  15. CellMap visualizes protein-protein interactions and subcellular localization

    Science.gov (United States)

    Dallago, Christian; Goldberg, Tatyana; Andrade-Navarro, Miguel Angel; Alanis-Lobato, Gregorio; Rost, Burkhard

    2018-01-01

    Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers. PMID:29497493

  16. Alfalfa Mob1-like proteins are involved in cell proliferation and are localized in the cell division plane during cytokinesis

    International Nuclear Information System (INIS)

    Citterio, Sandra; Piatti, Simonetta; Albertini, Emidio; Aina, Roberta; Varotto, Serena; Barcaccia, Gianni

    2006-01-01

    Mps-one-binder (Mob) proteins play a crucial role in yeast cytokinesis. After cloning two Mob1-like genes, MsMob1-A and MsMob1-B from alfalfa (Medicago sativa L.) we show that, although they are constitutively expressed in roots, stems, leaves, flowers and pods, their transcripts and proteins are mostly produced in actively proliferating tissues. A polyclonal antibody specifically raised against MsMob1 proteins was used for immunolocalization studies in synchronized root tip cells. The subcellular localization of MsMob1-like proteins is demonstrated to be cell cycle-regulated. Cytoplasmic localization is faint and diffused during G 1 and S. It becomes concentrated in punctuate and fibrillar structures in G 2 as well as M phase. At the stage of cytokinesis, the protein is found at the emerging cell plate marking the progressive formation of the septum. Mob1 proteins partially co-localize with microtubules structures functionally related to the spindles and important for cytokinesis in eukaryotic cells. The MsMob1 expression cannot rescue the lethality of the yeast mob1 mutant, suggesting that interaction of Mob1 proteins with their effectors may be species-specific. Localization of Mob1 proteins in the inner layer of the root cap indicates an additional function for this class of proteins in plants, which is likely related to the onset of programmed cell death

  17. Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes.

    Science.gov (United States)

    Johnson, Mary Kathrine; Cooksey, Amanda M; Wise, Dwayne A

    2008-11-01

    CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented. (c) 2008 Wiley-Liss, Inc.

  18. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.

    2008-01-01

    . YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

  19. Multidisciplinary management of the locally advanced unresectable non-small cell lung cancer

    International Nuclear Information System (INIS)

    Cho, Kwan Ho

    2004-01-01

    Locally advanced (Stage III) non-small cell lung cancer (NSCLC) accounts for approximately one third of all cases of NSCLC. Few patients with locally advanced NSCLC present with disease amenable to curative surgical resection. Historically, these patients were treated with primary thoracic radiation therapy (RT) and had poor long term survival rates, due to both progression of local disease and development of distant metastases. Over the last two decades, the use of multidisciplinary approach has improved the outcome for patients with locally advanced NSCLC. Combined chemoradiotherapy is the most favored approach for treatment of locally advanced unresectable NSCLC. There are two basic treatment protocols for administering combined chemotherapy and radiation, sequential versus concurrent. The rationale for using chemotherapy is to eliminate subclinical metastatic disease while improving local control. Sequential use of chemotherapy followed by radiotherapy has improved median and long term survival compared to radiation therapy alone. This approach appears to decrease the risk of distant metastases, but local failure rates remain the same as radiation alone. Concurrent chemoradiotherapy has been studied extensively. The potential advantages of this approach may include sensitization of tumor cells to radiation by the administration of chemotherapy, and reduced overall treatment time compared to sequential therapy; which is known to be important for improving local control in radiation biology. This approach improves survival primarily as a result of improved local control. However, it doesn't seem to decrease the risk of distant metastases probably because concurrent chemoradiation requires dose reductions in chemotherapy due to increased risks of acute morbidity such as acute esophageal toxicity. Although multidisciplinary therapy has led to improved survival rates compared to radiation therapy alone and has become the new standard of care, the optimal therapy of

  20. Cervical cancer stem cells and correlation with radiation response in locally advanced cervical cancer

    International Nuclear Information System (INIS)

    Chopra, Supriya; Goda, Jayant Sastri; Deodhar, Kedar

    2016-01-01

    While tumour-initiating cells (TIC) have been reported across solid tumours, there is dearth of data regarding TICs and radiation response in cervical cancer. From October, 2013- July, 2015 patients with locally advanced cervical cancer were included. Pretreatment biopsy was obtained. IHC was performed for SOX-2,OCT-4, Nanog (ESC), CD44 and Podoplanin (TIC). Semiquantitative scoring was used for IHC. All patients received uniform concurrent chemoradiation and brachytherapy. On follow up, local control and distant relapse was recorded

  1. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  2. Extracellular localization of catalase is associated with the transformed state of malignant cells.

    Science.gov (United States)

    Böhm, Britta; Heinzelmann, Sonja; Motz, Manfred; Bauer, Georg

    2015-12-01

    Oncogenic transformation is dependent on activated membrane-associated NADPH oxidase (NOX). However, the resultant extracellular superoxide anions are also driving the NO/peroxynitrite and the HOCl pathway, which eliminates NOX-expressing transformed cells through selective apoptosis induction. Tumor progression is dependent on dominant interference with intercellular apoptosis-inducing ROS signaling through membrane-associated catalase, which decomposes H2O2 and peroxynitrite and oxidizes NO. Particularly, the decomposition of extracellular peroxynitrite strictly requires membrane-associated catalase. We utilized small interfering RNA (siRNA)-mediated knockdown of catalase and neutralizing antibodies directed against the enzyme in combination with challenging H2O2 or peroxynitrite to determine activity and localization of catalase in cells from three distinct steps of multistage oncogenesis. Nontransformed cells did not generate extracellular superoxide anions and only showed intracellular catalase activity. Transformed cells showed superoxide anion-dependent intercellular apoptosis-inducing ROS signaling in the presence of suboptimal catalase activity in their membrane. Tumor cells exhibited tight control of intercellular apoptosis-inducing ROS signaling through a high local concentration of membrane-associated catalase. These data demonstrate that translocation of catalase to the outside of the cell membrane is already associated with the transformation step. A strong local increase in the concentration of membrane-associated catalase is achieved during tumor progression and is controlled by tumor cell-derived H2O2 and by transglutaminase.

  3. Study of the influence of slab perturbation in the cell on the fuel local burnup

    International Nuclear Information System (INIS)

    Takac, S.; Kocic, A.; Dimitrijevic, Z.; Markovic, H.; Dimitrijevic, V.

    1975-01-01

    The influence of construction material or voids in the fuel element on the fuel burnup was the objective of this study. Experiments were done by cell perturbation method. Theoretical method was developed for calculating the effect of reactor cell perturbation. Obtained results both experimental and theoretical clearly indicate that the minimum quantity of construction material or void cause local increase of neutron flux in the mentioned regions. This increase of flux which amounts to nearly ten percent, and can reach the value of a few tens percent leads to the local increase of fuel burnup [sr

  4. Local myogenic pulp-derived cell injection enhances craniofacial muscle regeneration in vivo.

    Science.gov (United States)

    Jung, J E; Song, M J; Shin, S; Choi, Y J; Kim, K H; Chung, C J

    2017-02-01

    To enhance myogenic differentiation in pulp cells isolated from extracted premolars by epigenetic modification using a DNA demethylation agent, 5-aza-2'-deoxycytidine (5-Aza), and to evaluate the potent stimulatory effect of 5-Aza-treated pulp cell injection for craniofacial muscle regeneration in vivo. Pulp cells were isolated from premolars extracted for orthodontic purposes from four adults (age range, 18-22.1 years). Levels of myogenic differentiation and functional contraction response in vitro were compared between pulp cells with or without pre-treatment of 5-Aza. Changes in muscle regeneration in response to green fluorescent protein (GFP)-labelled myogenic pulp cell injection in vivo were evaluated using a cardiotoxin (CTX)-induced muscle injury model of the gastrocnemius as well as the masseter muscle in mice. Pre-treatment of 5-Aza in pulp cells stimulated myotube formation, myogenic differentiation in terms of desmin and myogenin expression, and the level of collagen gel contraction. The local injection of 5-Aza pre-treated myogenic pulp cells was engrafted into the host tissue and indicated signs of enhanced muscle regeneration in both the gastrocnemius and the masseter muscles. The epigenetic modification of pulp cells from extracted premolars and the local injection of myogenic pulp cells may stimulate craniofacial muscles regeneration in vivo. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Localized extramedullary relapse after autologous hematopoietic stem cell transplantation in multiple myeloma

    International Nuclear Information System (INIS)

    Erkus, Muhan; Meteoglu, Ibrahim; Bolaman, Zahit; Kadikoylu, Gurhan

    2005-01-01

    Extramedullary plasmacytomas are rare manifestation of plasma cell malignancies. After hematopoietic stem cell transplantation HSCT, presentation of localized plasmacytoma with extramedullary growth is very unusual. We report a case of a 56-year-old woman with Dune-Salmon stage IIIA immunoglobulin A-kappa multiple myeloma, which presented 120 days after autologous HSCT with extramedullary plasmacytoma arising from a lymph node in supraclavicular region. The patient had no pretransplant-history related with extramedullary disease. There was no increase of plasma cells in bone marrow or monoclonal protein in urine or serum. Aspiration smears of lymph node revealed a population of plasmacytoid cells at various stages of maturation. The patient was successfully treated with local radiotherapy and has remained progression-free for more than 20 months. (author)

  6. Electrochemical Quantification of Extracellular Local H2O2 Kinetics Originating from Single Cells.

    Science.gov (United States)

    Bozem, Monika; Knapp, Phillip; Mirčeski, Valentin; Slowik, Ewa J; Bogeski, Ivan; Kappl, Reinhard; Heinemann, Christian; Hoth, Markus

    2017-05-15

    H 2 O 2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local H 2 O 2 concentrations ([H 2 O 2 ]) originating from single cells is required. Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H 2 O 2 ] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H 2 O 2 ] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H 2 O 2 ] 5-8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H 2 O 2 to an unstimulated MC, the local [H 2 O 2 ] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H 2 O 2 is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex ® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H 2 O 2 separately. Local extracellular [H 2 O 2 ] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 00, 000-000.

  7. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  8. Local device parameter extraction of a concentrator photovoltaic cell under solar spot illumination

    Energy Technology Data Exchange (ETDEWEB)

    Munji, M.K.; Okullo, W.; van Dyk, E.E.; Vorster, F.J. [Physics Department, Nelson Mandela Metropolitan University, P O Box 77000, Port Elizabeth 6031 (South Africa)

    2010-12-15

    Focused sunlight can act as a localized source of excess minority carriers in a solar cell. Current signal generated by these carriers gives considerable information about the electrical properties of the cell's material. Point by point current-voltage data were measured for a back point-contact concentrator photovoltaic cell when illuminated by focused sunlight. Two numerical curve fitting procedures: a non-linear two-point interval division and particle swarm optimization algorithm were then applied to extract local parameters (i.e. as function of position) from the current-voltage data at each measurement point. Extracted parameters plotted yields relative spatial information about the electrical properties of a solar cell in a two or three dimensional mapping. The curve fitting routines applied to current-voltage data reveal that performance parameters: short circuit current, open circuit voltage, maximum power and fill factor show distinct variations in the vicinity of the observed current reducing feature. The relative values of the diode ideality factors, series resistance, shunt resistance and reverse saturation currents from both methods showed no significant measurable features that could be distinguished. This shows that the observed reduction in photo-induced current was due to severe recombination in the bulk or around the highly diffused point contacts and not the quality of the multiple p-n junctions of the cell. These approaches allow one to obtain a set of parameters at each local point on the cell which are reasonable and representative of the physical system. (author)

  9. Selective individual primary cell capture using locally bio-functionalized micropores.

    Directory of Open Access Journals (Sweden)

    Jie Liu

    Full Text Available BACKGROUND: Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. METHODOLOGY/PRINCIPAL FINDINGS: We locally deposited oligodeoxynucleotide (ODN and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. CONCLUSIONS/SIGNIFICANCE: The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures.

  10. Selective Individual Primary Cell Capture Using Locally Bio-Functionalized Micropores

    Science.gov (United States)

    Liu, Jie; Bombera, Radoslaw; Leroy, Loïc; Roupioz, Yoann; Baganizi, Dieudonné R.; Marche, Patrice N.; Haguet, Vincent; Mailley, Pascal; Livache, Thierry

    2013-01-01

    Background Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. Methodology/Principal Findings We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. Conclusions/Significance The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures. PMID:23469221

  11. Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines

    Directory of Open Access Journals (Sweden)

    Ding-Kun Kang

    Full Text Available Abstract Background: Local anesthetics (LAs are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001, concentration- (p < 0.001, and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

  12. Non-linear elasticity of extracellular matrices enables contractile cells to communicate local position and orientation.

    Directory of Open Access Journals (Sweden)

    Jessamine P Winer

    2009-07-01

    Full Text Available Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning.

  13. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    International Nuclear Information System (INIS)

    Estrela-Lopis, I; Donath, E; Romero, G; Rojas, E; Moya, S E

    2011-01-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al 2 O 3 NPs were found in the cytoplasm. CeO 2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  14. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Science.gov (United States)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  15. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Qiong Deng

    2017-01-01

    Full Text Available The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  16. Improved local and regional control with radiotherapy for Merkel cell carcinoma of the head and neck.

    Science.gov (United States)

    Strom, Tobin; Naghavi, Arash O; Messina, Jane L; Kim, Sungjune; Torres-Roca, Javier F; Russell, Jeffery; Sondak, Vernon K; Padhya, Tapan A; Trotti, Andy M; Caudell, Jimmy J; Harrison, Louis B

    2017-01-01

    We hypothesized that radiotherapy (RT) would improve both local and regional control with Merkel cell carcinoma of the head and neck. A single-institution institutional review board-approved study was performed including 113 patients with nonmetastatic Merkel cell carcinoma of the head and neck. Postoperative RT was delivered to the primary tumor bed (71.7% cases) ± draining lymphatics (33.3% RT cases). Postoperative local RT was associated with improved local control (3-year actuarial local control 89.4% vs 68.1%; p = .005; Cox hazard ratio [HR] 0.18; 95% confidence interval [CI] = 0.06-0.55; p = .002). Similarly, regional RT was associated with improved regional control (3-year actuarial regional control 95.0% vs 66.7%; p = .008; Cox HR = 0.09; 95% CI = 0.01-0.69; p = .02). Regional RT played an important role for both clinical node-negative patients (3-year regional control 100% vs 44.7%; p = .03) and clinical/pathological node-positive patients (3-year regional control 90.9% vs 55.6%; p = .047). Local RT was beneficial for all patients with Merkel cell carcinoma of the head and neck, whereas regional RT was beneficial for clinical node-negative and clinical/pathological node-positive patients. © 2016 Wiley Periodicals, Inc. Head Neck 39: 48-55, 2017. © 2016 Wiley Periodicals, Inc.

  17. Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion.

    Directory of Open Access Journals (Sweden)

    Julie Behr

    Full Text Available This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC and substrate adherent polymorphonuclear neutrophils (PMN is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.

  18. Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion.

    Science.gov (United States)

    Behr, Julie; Gaskin, Byron; Fu, Changliang; Dong, Cheng; Kunz, Robert

    2015-01-01

    This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.

  19. Progression of Intravesical Condyloma Acuminata to Locally Advanced Poorly Differentiated Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    A. Khambati

    2016-07-01

    Full Text Available Condyloma acuminata (CA is a common sexually transmitted disease caused by Human Papilloma Virus (HPV infection. CA of the bladder, however, is an exceedingly rare lesion. We present a rare case of poorly differentiated locally invasive squamous cell carcinoma (SCC arising from recurrent CA of the bladder in an immunocompetent patient and discuss pathophysiology and management of this unusual condition.

  20. Immunocytochemical localization of the elongation factor Tu in E. coli cells

    NARCIS (Netherlands)

    Slot, J.W.; Schilstra, M.J.; Meide, P.H. van der; Posthuma, G.; Cremers, A.F.M.; Bosch, L.

    1984-01-01

    The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labellin technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been

  1. Localization Study of Co-Phthalocyanines in Cells by Raman Micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S.Y.; Arzhantsev, S.Y.; Chikishev, A.Y.; Chikishev, A.Y.; Koroteev, N.I.; Greve, Jan; Otto, Cornelis; Sijtsema, N.M.

    1999-01-01

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  2. Localization study of Co-phthalocyanines in cells by Raman micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S Y; Chikishev, A Y; Koroteev, N I; Greve, J; Otto, C; Sijtsema, N M

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  3. The effect of local breast radiotherapy on circulating CD34+ cells

    International Nuclear Information System (INIS)

    Alajez, Nehad M.; Wang Wei; Biswas, Debashis; Teh, Amy; Sutherland, Robert; Pintilie, Melania; Minden, Mark; Messner, Hans; Fyles, Anthony; Gospodarowicz, Mary; Keating, Armand; Liu, Fei-Fei

    2011-01-01

    The number of circulating CD34 + (hematopoietic stem) cells (HSCs) was observed to decline by 15% in breast cancer patients after starting adjuvant radiation therapy, regardless of age or preceding chemotherapy. These data demonstrate that local radiation therapy can profoundly affect HSC homeostasis, which might have a myriad of important implications.

  4. Button sequestrum in a case of localized Langerhans' cell histiocytosis of the ilium: case report

    International Nuclear Information System (INIS)

    Tordeur, M.; Wybier, M.; Laporte, J.L.; Grenier, P.; Laredo, J.D.

    2000-01-01

    Langerhans' cell histiocytosis (LCH) is characterized by a proliferation of cells exhibiting the same immunohistochemical and ultra-structural characteristics as Langerhans' cells of the epidermis. Eosinophilic granuloma, chronic polyostotic disease (Hand-Schuller-Christian disease) and multisystemic disease (Letterer-Siwe disease) all belong to the spectrum LCH. Osseous lesions are the most common findings. the radiological appearance of skeletal LCH depends on the site and on disease activity. Button sequestrum has been described as uncommon in LCH of the skull and exceedingly rare in LCH at other sites. We report a case of localized LCH of the ilium with a button sequestrum. (author)

  5. Stem cells in Nanomia bijuga (Siphonophora), a colonial animal with localized growth zones.

    Science.gov (United States)

    Siebert, Stefan; Goetz, Freya E; Church, Samuel H; Bhattacharyya, Pathikrit; Zapata, Felipe; Haddock, Steven H D; Dunn, Casey W

    2015-01-01

    Siphonophores (Hydrozoa) have unparalleled colony-level complexity, precision of colony organization, and functional specialization between zooids (i.e., the units that make up colonies). Previous work has shown that, unlike other colonial animals, most growth in siphonophores is restricted to one or two well-defined growth zones that are the sites of both elongation and zooid budding. It remained unknown, however, how this unique colony growth and development is realized at the cellular level. To understand the colony-level growth and development of siphonophores at the cellular level, we characterize the distribution of proliferating cells and interstitial stem cells (i-cells) in the siphonophore Nanomia bijuga. Within the colony, we find evidence that i-cells are present at the tip of the horn, the structure within the growth zone that gives rise to new zooids. Co-localized gene expression of vasa-1, pl10, piwi, nanos-1, and nanos-2 suggests that i-cells persist in the youngest zooid buds and that i-cells become progressively restricted to specific regions within the zooids until they are mostly absent from the oldest zooids. The examined genes remain expressed in gametogenic regions. No evidence for i-cells is found in the stem between maturing zooids. Domains of high cell proliferation include regions where the examined genes are expressed, but also include some areas in which the examined genes were not expressed such as the stem within the growth zones. Cell proliferation in regions devoid of vasa-1, pl10, piwi, nanos-1, and nanos-2 expression indicates the presence of mitotically active epithelial cell lineages and, potentially, progenitor cell populations. We provide the first evidence for i-cells in a siphonophore. Our findings suggest maintenance of i-cell populations at the sites of growth zones and that these sites are the main source of i-cells. This restriction of stem cells to particular regions in the colony, in combination with localized budding

  6. Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-11

    Science.gov (United States)

    Hyun, Young-Min; Chung, Hung-Li; McGrath, James L.; Waugh, Richard E.; Kim, Minsoo

    2009-01-01

    Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. Very Late Antigen-4 (VLA-4, α4β1) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer (FRET) analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence (TIRF) microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells, and is critical for T cell migration on VCAM-1. PMID:19542447

  7. CDKL5 localizes at the centrosome and midbody and is required for faithful cell division.

    Science.gov (United States)

    Barbiero, Isabella; Valente, Davide; Chandola, Chetan; Magi, Fiorenza; Bergo, Anna; Monteonofrio, Laura; Tramarin, Marco; Fazzari, Maria; Soddu, Silvia; Landsberger, Nicoletta; Rinaldo, Cinzia; Kilstrup-Nielsen, Charlotte

    2017-07-24

    The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.

  8. Localized decrease of β-catenin contributes to the differentiation of human embryonic stem cells

    International Nuclear Information System (INIS)

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-01-01

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies

  9. Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

    Science.gov (United States)

    Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

    2013-01-01

    Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

  10. Dickkopf-3, a tissue-derived modulator of local T cell responses

    Directory of Open Access Journals (Sweden)

    Michael eMeister

    2015-02-01

    Full Text Available The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T cell reactivity. Dkk3 is a secreted, mainly tissue derived protein with highest expression in organs considered as immune privileged such as the eye, embryo, placenta and brain. While T cell development and activation status in naïve Dkk3 deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T cell mediated rejection. Moreover, genetic deletion or antibody mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE. This phenotype was accompanied by a change of T cell polarization displayed by an increase of IFNγ producing T cells within in the CNS. In the wild type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4+ and CD8+ T cell responses.

  11. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

    Directory of Open Access Journals (Sweden)

    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

  12. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  13. Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

    Science.gov (United States)

    Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

    1998-01-01

    Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

  14. Local impact of humidification on degradation in polymer electrolyte fuel cells

    Science.gov (United States)

    Sanchez, Daniel G.; Ruiu, Tiziana; Biswas, Indro; Schulze, Mathias; Helmly, Stefan; Friedrich, K. Andreas

    2017-06-01

    The water level in a polymer electrolyte membrane fuel cell (PEMFC) affects the durability as is seen from the degradation processes during operation a PEMFC with fully- and nonhumidified gas streams as analyzed using an in-situ segmented cell for local current density measurements during a 300 h test operating under constant conditions and using ex situ SEM/EDX and XPS post-test analysis of specific regions. The impact of the RH on spatial distribution of the degradation process results from different water distribution giving different chemical environments. Under nonhumidified gas streams, the cathode inlet region exhibits increased degradation, whereas with fully humidified gases the bottom of the cell had the higher performance losses. The degradation and the degree of reversibility produced by Pt dissolution, PTFE defluorination, and contaminants such as silicon (Si) and nickel (Ni) were locally evaluated.

  15. Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data

    Directory of Open Access Journals (Sweden)

    Reema A. Khorshed

    2015-07-01

    Full Text Available Measuring three-dimensional (3D localization of hematopoietic stem cells (HSCs within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components.

  16. Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

    Science.gov (United States)

    Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

    2018-05-08

    We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

  17. Local mechanical properties of bladder cancer cells measured by AFM as a signature of metastatic potential

    Science.gov (United States)

    Abidine, Y.; Laurent, V. M.; Michel, R.; Duperray, A.; Verdier, C.

    2015-10-01

    The rheological properties of bladder cancer cells of different invasivities have been investigated using a microrheological technique well adapted in the range [1-300Hz] of interest to understand local changes in the cytoskeleton microstructure, in particular actin fibres. Drugs disrupting actin and acto-myosin functions were used to study the resistance of such cancer cells. Results on a variety of cell lines were fitted with a model revealing the importance of two parameters, the elastic shear plateau modulus G N 0 as well as the glassy transition frequency f T. These parameters are good markers for invasiveness, with the notable exception of the cell periphery, which is stiffer for less invasive cells, and could be of importance in cancer metastasis.

  18. In-situ Monitoring of Internal Local Temperature and Voltage of Proton Exchange Membrane Fuel Cells

    Directory of Open Access Journals (Sweden)

    Chi-Yuan Lee

    2010-06-01

    Full Text Available The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC that are based on micro-electro-mechanical systems (MEMS. The power density at 0.5 V without a sensor is 450 mW/cm2, and that with a sensor is 426 mW/cm2. Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse.

  19. In-situ monitoring of internal local temperature and voltage of proton exchange membrane fuel cells.

    Science.gov (United States)

    Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

    2010-01-01

    The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm(2), and that with a sensor is 426 mW/cm(2). Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse.

  20. Modelling radiation-induced cell death and tumour re-oxygenation: local versus global and instant versus delayed cell death

    International Nuclear Information System (INIS)

    Gago-Arias, Araceli; Espinoza, Ignacio; Sánchez-Nieto, Beatriz; Aguiar, Pablo; Pardo-Montero, Juan

    2016-01-01

    The resistance of hypoxic cells to radiation, due to the oxygen dependence of radiosensitivity, is well known and must be taken into account to accurately calculate the radiation induced cell death. A proper modelling of the response of tumours to radiation requires deriving the distribution of oxygen at a microscopic scale. This usually involves solving the reaction-diffusion equation in tumour voxels using a vascularization distribution model. Moreover, re-oxygenation arises during the course of radiotherapy, one reason being the increase of available oxygen caused by cell killing, which can turn hypoxic tumours into oxic. In this work we study the effect of cell death kinetics in tumour oxygenation modelling, analysing how it affects the timing of re-oxygenation, surviving fraction and tumour control. Two models of cell death are compared, an instantaneous cell killing, mimicking early apoptosis, and a delayed cell death scenario in which cells can die shortly after being damaged, as well as long after irradiation. For each of these scenarios, the decrease in oxygen consumption due to cell death can be computed globally (macroscopic voxel average) or locally (microscopic). A re-oxygenation model already used in the literature, the so called full re-oxygenation, is also considered. The impact of cell death kinetics and re-oxygenation on tumour responses is illustrated for two radiotherapy fractionation schemes: a conventional schedule, and a hypofractionated treatment. The results show large differences in the doses needed to achieve 50% tumour control for the investigated cell death models. Moreover, the models affect the tumour responses differently depending on the treatment schedule. This corroborates the complex nature of re-oxygenation, showing the need to take into account the kinetics of cell death in radiation response models. (paper)

  1. Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells.

    Science.gov (United States)

    Verbrugghe, Phebe; Kujala, Pekka; Waelput, Wim; Peters, Peter J; Cuvelier, Claude A

    2008-03-01

    The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer's patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer's ring. Immunohistochemistry for clusterin in human Peyer's patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer's patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.

  2. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Science.gov (United States)

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  3. A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

    Science.gov (United States)

    Huberman, Lori B.; Murray, Andrew W.

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  4. Ontogeny and localization of the cells produce IL-2 in healthy animals.

    Science.gov (United States)

    Yamamoto, Mutsumi; Seki, Yoichi; Iwai, Kazuyuki; Ko, Iei; Martin, Alicia; Tsuji, Noriko; Miyagawa, Shuji; Love, Robert B; Iwashima, Makio

    2013-03-01

    IL-2 is a growth factor for activated T cells and is required for maintenance of naturally arising regulatory T cells (nTregs). Mice defective in IL-2/IL-2 receptor signaling pathways have impaired nTregs and suffer from lymphoproliferative disorders, suggesting that IL-2 is present and functional in healthy animals. However, the cellular source of IL-2 is currently unknown. To determine which cells produce IL-2 in healthy animals, we established mice carrying cre gene knock in at the il-2 locus (termed IL-2(cre)). When IL-2(cre) mice were crossed with EGFP reporter mice, EGFP was exclusively expressed by a fraction of CD4 T cells present in both lymphoid and non-lymphoid tissues. Live imaging of IL-2(cre) mice that carry the luciferase reporter showed concentrated localization of luciferase(+) cells in Peyer's patches. These cells were not observed in new born mice but appeared within 3days after birth. Reduction of antigen receptor repertoire by transgene expression reduced their number, indicating that recognition of environmental antigens is necessary for generation of these IL-2 producers in healthy animals. A substantial fraction of EGFP(+) cells also produce IL-10 and IFN-γ, a characteristic profile of type 1 regulatory T cells (Tr1). The data suggest that a group of Tr1 cells have addition roles in immune homeostasis by producing IL-2 along with other cytokines and help maintaining Tregs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

    Science.gov (United States)

    Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

    2014-03-01

    Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

  6. Specific gut commensal flora locally alters T cell tuning to endogenous ligands.

    Science.gov (United States)

    Chappert, Pascal; Bouladoux, Nicolas; Naik, Shruti; Schwartz, Ronald H

    2013-06-27

    Differences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization

    Science.gov (United States)

    Tormin, Ariane; Li, Ou; Brune, Jan Claas; Walsh, Stuart; Schütz, Birgit; Ehinger, Mats; Ditzel, Nicholas; Kassem, Moustapha

    2011-01-01

    Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin−/CD271+/CD45−/CD146+ stem-cell fraction, but also in lin−/CD271+/CD45−/CD146−/low cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34+ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment. PMID:21415267

  8. Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

    International Nuclear Information System (INIS)

    Chubinskiy-Nadezhdin, Vladislav I.; Vasileva, Valeria Y.; Pugovkina, Natalia A.; Vassilieva, Irina O.; Morachevskaya, Elena A.; Nikolsky, Nikolay N.; Negulyaev, Yuri A.

    2017-01-01

    Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca 2+ entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca 2+ -sensitive BK and SK channels was shown. • Local Ca 2+ influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca 2+ ] i . • Functional clustering of SACs and BK channels in stem cell membrane is proposed.

  9. Plasticity in variation of xylem and phloem cell characteristics of Norway spruce under different local conditions

    Directory of Open Access Journals (Sweden)

    Jozica eGricar

    2015-09-01

    Full Text Available There is limited information on intra-annual plasticity of secondary tissues of tree species growing under different environmental conditions. To increase the knowledge about the plasticity of secondary growth, which allows trees to adapt to specific local climatic regimes, we examined climate–radial growth relationships of Norway spruce (Picea abies (L. H. Karst. from three contrasting locations in the temperate climatic zone by analyzing tree-ring widths for the period 1932–2010, and cell characteristics in xylem and phloem increments formed in the years 2009–2011. Variation in the structure of xylem and phloem increments clearly shows that plasticity in seasonal dynamics of cambial cell production and cell differentiation exists on xylem and phloem sides. Anatomical characteristics of xylem and phloem cells are predominantly site-specific characteristics, because they varied among sites but were fairly uniform among years in trees from the same site. Xylem and phloem tissues formed in the first part of the growing season seemed to be more stable in structure, indicating their priority over latewood and late phloem for tree performance. Long-term climate and radial growth analyses revealed that growth was in general less dependent on precipitation than on temperature; however, growth sensitivity to local conditions differed among the sites. Only partial dependence of radial growth of spruce on climatic factors on the selected sites confirms its strategy to adapt the structure of wood and phloem increments to function optimally in local conditions.

  10. Neutron radiography characterization of an operating proton exchange membrane fuel cell with localized current distribution measurements

    International Nuclear Information System (INIS)

    Gagliardo, J.J.; Owejan, J.P.; Trabold, T.A.; Tighe, T.W.

    2009-01-01

    Neutron radiography has proven to be a powerful tool to study and understand the effects of liquid water in an operating fuel cell. In the present work, this experimental method is coupled with locally resolved current and ohmic resistance measurements, giving additional insight into water management and fuel cell performance under a variety of conditions. The effects of varying the inlet humidification level and the current density of the 50 cm 2 cell are studied by simultaneously monitoring electrochemical performance with a 10x10 matrix of current sensors, and liquid water volumes are measured using the National Institute of Standards and Technology (NIST) neutron imaging facility. A counter flow, straight channel proton exchange membrane (PEM) fuel cell is used to demonstrate localized performance loss corresponds to water-filled channels that impede gas transport to the catalyst layer, thereby creating an area that has low current density. Furthermore, certain operating conditions causing excess water accumulation in the channels can result in localized proton resistance increase, a result that can only be accurately observed with combined radiography and distributed electrochemical measurements.

  11. Nonlinear Local Deformations of Red Blood Cell Membranes: Effects of Toxins and Pharmaceuticals (Part 2

    Directory of Open Access Journals (Sweden)

    Alexander M. Chernysh

    2018-01-01

    Full Text Available Modifiers of membranes cause local defects on the cell surface. Measurement of the rigidity at the sites of local defects can provide further information about the structure of defects and mechanical properties of altered membranes.The purpose of the study: a step-by-step study of the process of a nonlinear deformation of red blood cells membranes under the effect of modifiers of different physico-chemical nature.Materials and methods. The membrane deformation of a viscoelastic composite erythrocyte construction inside a cell was studied by the atomic force spectroscopy. Nonlinear deformations formed under the effect of hemin, Zn2+ ions, and verapamil were studied.Results. The process of elastic deformation of the membrane with the indentation of a probe at the sites of local defects caused by modifiers was demonstrated. The probe was inserted during the same step of the piezo scanner z displacement; the probe indentation occured at the different discrete values of h, which are the functions of the membrane structure. At the sites of domains, under the effect of the hemin, tension areas and plasticity areas appeared. A mathematical model of probe indentation at the site of membrane defects is presented.Conclusion. The molecular mechanisms of various types of nonlinear deformations occurring under the effect of toxins are discussed. The results of the study may be of interest both for fundamental researchers of the blood cell properties and for practical reanimatology and rehabilitology. 

  12. Merkel cell tumor of the skin treated with localized radiotherapy: are widely negative margins required?

    Directory of Open Access Journals (Sweden)

    David Parda

    2011-03-01

    Full Text Available Merkel’s cell carcinoma is a rare cutaneous tumor that can affect a wide variety of sites throughout the body. Commonly, it affects the skin alone and the management of limited disease can be confusing since the natural history of the disease involves distant metastasis. Traditional management has required wide local excision with negative margins of resection. We describe a case treated with local therapy alone and review the literature to suggest that complete microscopic excision may not be required if adjuvant radiotherapy is used.

  13. Successful autologous hematopoietic stem cell transplantation for a patient with rapidly progressive localized scleroderma.

    Science.gov (United States)

    Nair, Velu; Sharma, Ajay; Sharma, Sanjeevan; Das, Satyaranjan; Bhakuni, Darshan S; Narayanan, Krishnan; Nair, Vivek; Shankar, Subramanian

    2015-03-01

    Autologous hematopoietic stem cell transplant (HSCT) for rapidly progressive disease has not been reported in localized scleroderma. Our patient, a 16-year-old girl had an aggressive variant of localized scleroderma, mixed subtype (linear-generalized) with Parry Romberg syndrome, with no internal organ involvement, that was unresponsive to immunosuppressive therapy and was causing rapid disfigurement. She was administered autologous HSCT in June 2011 and has maintained drug-free remission with excellent functional status at almost 3.5 years of follow-up. © 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  14. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    Science.gov (United States)

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  15. Cytochemical localization of calcium in cap cells of primary roots of Zea mays L

    Science.gov (United States)

    Moore, R.

    1986-01-01

    The distribution of calcium (Ca) in caps of vertically- and horizontally-oriented roots of Zea mays was monitored to determine its possible role in root graviresponsiveness. A modification of the antimonate precipitation procedure was used to localize Ca in situ. In vertically-oriented roots, the presumed graviperceptive (i.e., columella) cells were characterized by minimal and symmetric staining of the plasmalemma and mitochondria. No precipitate was present in plasmodesmata or cell walls. Within 5 min after horizontal reorientation, staining was associated with the portion of the cell wall adjacent to the distal end of the cell. This asymmetric staining persisted throughout the onset of gravicurvature. No staining of lateral cell walls of columella cells was observed at any stage of gravicurvature, suggesting that a lateral flow of Ca through the columella tissue of horizontally-oriented roots does not occur. The outermost peripheral cells of roots oriented horizontally and vertically secrete Ca through plasmodesmata-like structures in their cell walls. These results are discussed relative to proposed roles of root-cap Ca in root gravicurvature.

  16. Local NSAID infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise

    DEFF Research Database (Denmark)

    Mikkelsen, U R; Langberg, H; Helmark, I C

    2009-01-01

    Despite the widespread consumption of nonsteroidal anti-inflammatory drugs (NSAIDs), the influence of these drugs on muscle satellite cells is not fully understood. The aim of the present study was to investigate the effect of a local NSAID infusion on satellite cells after unaccustomed eccentric...... exercise in vivo in human skeletal muscle. Eight young healthy males performed 200 maximal eccentric contractions with each leg. An NSAID was infused via a microdialysis catheter into the vastus lateralis muscle of one leg (NSAID leg) before, during, and for 4.5 h after exercise, with the other leg working...... cells (CD68(+) or CD16(+) cells) was not significantly increased in either of the legs 8 days after exercise and was unaffected by the NSAID. The main finding in the present study was that the NSAID infusion for 7.5 h during the exercise day suppressed the exercise-induced increase in the number...

  17. Documentation and localization of force-mediated filamin A domain perturbations in moving cells

    Science.gov (United States)

    Nakamura, Fumihiko; Song, Mia; Hartwig, John H.; Stossel, Thomas P.

    2014-08-01

    Endogenously and externally generated mechanical forces influence diverse cellular activities, a phenomenon defined as mechanotransduction. Deformation of protein domains by application of stress, previously documented to alter macromolecular interactions in vitro, could mediate these effects. We engineered a photon-emitting system responsive to unfolding of two repeat domains of the actin filament (F-actin) crosslinker protein filamin A (FLNA) that binds multiple partners involved in cell signalling reactions and validated the system using F-actin networks subjected to myosin-based contraction. Expressed in cultured cells, the sensor-containing FLNA construct reproducibly reported FLNA domain unfolding strikingly localized to dynamic, actively protruding, leading cell edges. The unfolding signal depends upon coherence of F-actin-FLNA networks and is enhanced by stimulating cell contractility. The results establish protein domain distortion as a bona fide mechanism for mechanotransduction in vivo.

  18. Parallel local search for solving Constraint Problems on the Cell Broadband Engine (Preliminary Results

    Directory of Open Access Journals (Sweden)

    Salvator Abreu

    2009-10-01

    Full Text Available We explore the use of the Cell Broadband Engine (Cell/BE for short for combinatorial optimization applications: we present a parallel version of a constraint-based local search algorithm that has been implemented on a multiprocessor BladeCenter machine with twin Cell/BE processors (total of 16 SPUs per blade. This algorithm was chosen because it fits very well the Cell/BE architecture and requires neither shared memory nor communication between processors, while retaining a compact memory footprint. We study the performance on several large optimization benchmarks and show that this achieves mostly linear time speedups, even sometimes super-linear. This is possible because the parallel implementation might explore simultaneously different parts of the search space and therefore converge faster towards the best sub-space and thus towards a solution. Besides getting speedups, the resulting times exhibit a much smaller variance, which benefits applications where a timely reply is critical.

  19. Localized surface plasmon resonance enhanced organic solar cell with gold nanospheres

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Linfang; Wang, Dan; Ye, Yuqian; Qian, Jun; He, Sailing [Centre for Optical and Electromagnetic Research, State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, Hangzhou 310058 (China); Zuo, Lijian; Chen, Hongzheng [Department of Polymer Science and Engineering, State Key Lab of Silicon Materials, Zhejiang University, Hangzhou 310027 (China)

    2011-03-15

    We use gold nanospheres (Au NSs) to improve the performance of polymer organic solar cells. Au NSs with a diameter of about 5 nm or 15 nm were doped into the buffer layer of organic solar cells. We attribute the efficiency improvement to the size-dependent localized surface plasmon resonance (LSPR) effect of Au NSs, which can enhance the light harvest ability of active layer around the Au NSs, and increase the probability of the exciton generation and dissociation. Our results show that solar cells doped with 15 nm-diameter Au NSs exhibit significant improvement of the efficiency (from 1.99% to 2.36%), while solar cells doped with only 5 nm-diameter Au NSs did not give obvious improvement of the performance. (author)

  20. lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1

    DEFF Research Database (Denmark)

    Chakraborty, Debojyoti; Paszkowski-Rogacz, Maciej; Berger, Nicolas

    2017-01-01

    -chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its......-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states....

  1. Actin and Arp2/3 localize at the centrosome of interphase cells

    Energy Technology Data Exchange (ETDEWEB)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  2. Actin and Arp2/3 localize at the centrosome of interphase cells

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. → Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. → Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. → The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  3. Subcellular localization of human neutral ceramidase expressed in HEK293 cells

    International Nuclear Information System (INIS)

    Hwang, Young-ha; Tani, Motohiro; Nakagawa, Tetsuto; Okino, Nozomu; Ito, Makoto

    2005-01-01

    We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence

  4. Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model.

    Science.gov (United States)

    Pauley, Penelope; Matthews, Brya G; Wang, Liping; Dyment, Nathaniel A; Matic, Igor; Rowe, David W; Kalajzic, Ivo

    2014-09-01

    Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

  5. From Cell to Tissue Properties-Modeling Skin Electroporation With Pore and Local Transport Region Formation.

    Science.gov (United States)

    Dermol-Cerne, Janja; Miklavcic, Damijan

    2018-02-01

    Current models of tissue electroporation either describe tissue with its bulk properties or include cell level properties, but model only a few cells of simple shapes in low-volume fractions or are in two dimensions. We constructed a three-dimensional model of realistically shaped cells in realistic volume fractions. By using a 'unit cell' model, the equivalent dielectric properties of whole tissue could be calculated. We calculated the dielectric properties of electroporated skin. We modeled electroporation of single cells by pore formation on keratinocytes and on the papillary dermis which gave dielectric properties of the electroporated epidermis and papillary dermis. During skin electroporation, local transport regions are formed in the stratum corneum. We modeled local transport regions and increase in their radii or density which affected the dielectric properties of the stratum corneum. The final model of skin electroporation accurately describes measured electric current and voltage drop on the skin during electroporation with long low-voltage pulses. The model also accurately describes voltage drop on the skin during electroporation with short high-voltage pulses. However, our results indicate that during application of short high-voltage pulses additional processes may occur which increase the electric current. Our model connects the processes occurring at the level of cell membranes (pore formation), at the level of a skin layer (formation of local transport region in the stratum corneum) with the tissue (skin layers) and even level of organs (skin). Using a similar approach, electroporation of any tissue can be modeled, if the morphology of the tissue is known.

  6. Computational local stiffness analysis of biological cell: High aspect ratio single wall carbon nanotube tip

    Energy Technology Data Exchange (ETDEWEB)

    TermehYousefi, Amin, E-mail: at.tyousefi@gmail.com [Department of Human Intelligence Systems, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology (Kyutech) (Japan); Bagheri, Samira; Shahnazar, Sheida [Nanotechnology & Catalysis Research Centre (NANOCAT), IPS Building, University Malaya, 50603 Kuala Lumpur (Malaysia); Rahman, Md. Habibur [Department of Computer Science and Engineering, University of Asia Pacific, Green Road, Dhaka-1215 (Bangladesh); Kadri, Nahrizul Adib [Department of Biomedical Engineering, Faculty of Engineering, University Malaya, 50603 Kuala Lumpur (Malaysia)

    2016-02-01

    Carbon nanotubes (CNTs) are potentially ideal tips for atomic force microscopy (AFM) due to the robust mechanical properties, nanoscale diameter and also their ability to be functionalized by chemical and biological components at the tip ends. This contribution develops the idea of using CNTs as an AFM tip in computational analysis of the biological cells. The proposed software was ABAQUS 6.13 CAE/CEL provided by Dassault Systems, which is a powerful finite element (FE) tool to perform the numerical analysis and visualize the interactions between proposed tip and membrane of the cell. Finite element analysis employed for each section and displacement of the nodes located in the contact area was monitored by using an output database (ODB). Mooney–Rivlin hyperelastic model of the cell allows the simulation to obtain a new method for estimating the stiffness and spring constant of the cell. Stress and strain curve indicates the yield stress point which defines as a vertical stress and plan stress. Spring constant of the cell and the local stiffness was measured as well as the applied force of CNT-AFM tip on the contact area of the cell. This reliable integration of CNT-AFM tip process provides a new class of high performance nanoprobes for single biological cell analysis. - Graphical abstract: This contribution develops the idea of using CNTs as an AFM tip in computational analysis of the biological cells. The proposed software was ABAQUS 6.13 CAE/CEL provided by Dassault Systems. Finite element analysis employed for each section and displacement of the nodes located in the contact area was monitored by using an output database (ODB). Mooney–Rivlin hyperelastic model of the cell allows the simulation to obtain a new method for estimating the stiffness and spring constant of the cell. Stress and strain curve indicates the yield stress point which defines as a vertical stress and plan stress. Spring constant of the cell and the local stiffness was measured as well

  7. Attempts of local irradiation of cells by microbeam. From ultraviolet to heavy particles

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Yasuhiko [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2002-03-01

    This review describes the history of attempts of local irradiation of cells by microbeam and present status of the study. Local irradiation of cells was attempted as early as in 1912 with use of short {alpha}-particle range and of focused UV beams. After the war, laser microbeams were then developed for microsurgery in embryology. In addition, microbeams of electron generated from the gun and of X-ray collimated were developed. In 1950s, the electron microbeam was generated from Van de Graaff accelerator in Chicago University and proton, deuteron and He-ion microbeams from the cyclotron, in BNL. In 1980s, Gesellschaft fuer Schwerionenforshung (Germany) used heavy ion microbeams from C to U generated from the linear accelerator and PNL, proton to {sup 4}He-ion microbeams from the tandem-electrostatic accelerator. At present in 2002, the equipments for microbeam for cell irradiation are the Van de Graaff accelerators in Gray Cancer Institute (England) and in Columbia University, and the cyclotron in TIARA in Japan. The purpose of the study in TIARA is to develop a system to generate heavy particle microbeams for cell irradiation for analysis of the biological effect of ultra-low fluence, high LET heavy particles like the galactic cosmic ray. Recently, the CHO-KI cell nucleus is irradiated by {sup 40}Ar and {sup 20}Ne ions. (K.H.)

  8. Attempts of local irradiation of cells by microbeam. From ultraviolet to heavy particles

    International Nuclear Information System (INIS)

    Kobayashi, Yasuhiko

    2002-01-01

    This review describes the history of attempts of local irradiation of cells by microbeam and present status of the study. Local irradiation of cells was attempted as early as in 1912 with use of short α-particle range and of focused UV beams. After the war, laser microbeams were then developed for microsurgery in embryology. In addition, microbeams of electron generated from the gun and of X-ray collimated were developed. In 1950s, the electron microbeam was generated from Van de Graaff accelerator in Chicago University and proton, deuteron and He-ion microbeams from the cyclotron, in BNL. In 1980s, Gesellschaft fuer Schwerionenforshung (Germany) used heavy ion microbeams from C to U generated from the linear accelerator and PNL, proton to 4 He-ion microbeams from the tandem-electrostatic accelerator. At present in 2002, the equipments for microbeam for cell irradiation are the Van de Graaff accelerators in Gray Cancer Institute (England) and in Columbia University, and the cyclotron in TIARA in Japan. The purpose of the study in TIARA is to develop a system to generate heavy particle microbeams for cell irradiation for analysis of the biological effect of ultra-low fluence, high LET heavy particles like the galactic cosmic ray. Recently, the CHO-KI cell nucleus is irradiated by 40 Ar and 20 Ne ions. (K.H.)

  9. Changes in Ect2 Localization Couple Actomyosin-Dependent Cell Shape Changes to Mitotic Progression

    OpenAIRE

    Matthews, Helen K.; Delabre, Ulysse; Rohn, Jennifer L.; Guck, Jochen; Kunda, Patricia; Baum, Buzz

    2012-01-01

    Summary As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first...

  10. An Unusual Case of Locally Advanced Glycogen-Rich Clear Cell Carcinoma of the Breast

    Directory of Open Access Journals (Sweden)

    Beatriz Martín-Martín

    2011-09-01

    Full Text Available Glycogen-rich clear cell (GRCC is a rare subtype of breast carcinoma characterized by carcinoma cells containing an optically clear cytoplasm and intracytoplasmic glycogen. We present the case of a 55-year-old woman with a palpable mass in the right breast and clinical signs of locally advanced breast cancer (LABC. The diagnosis of GRCC carcinoma was based on certain histopathological characteristics of the tumor and immunohistochemical analysis. To our knowledge, this is the first case of GRCC LABC with intratumoral calcifications. There is no evidence of recurrence or metastatic disease after 14 months’ follow-up.

  11. Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation.

    Science.gov (United States)

    Coceano, G; Yousafzai, M S; Ma, W; Ndoye, F; Venturelli, L; Hussain, I; Bonin, S; Niemela, J; Scoles, G; Cojoc, D; Ferrari, E

    2016-02-12

    Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young's modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines' elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

  12. Spatial planning via extremal optimization enhanced by cell-based local search

    International Nuclear Information System (INIS)

    Sidiropoulos, Epaminondas

    2014-01-01

    A new treatment is presented for land use planning problems by means of extremal optimization in conjunction to cell-based neighborhood local search. Extremal optimization, inspired by self-organized critical models of evolution has been applied mainly to the solution of classical combinatorial optimization problems. Cell-based local search has been employed by the author elsewhere in problems of spatial resource allocation in combination with genetic algorithms and simulated annealing. In this paper it complements extremal optimization in order to enhance its capacity for a spatial optimization problem. The hybrid method thus formed is compared to methods of the literature on a specific characteristic problem. It yields better results both in terms of objective function values and in terms of compactness. The latter is an important quantity for spatial planning. The present treatment yields significant compactness values as emergent results

  13. Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

    Science.gov (United States)

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

  14. The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

    Directory of Open Access Journals (Sweden)

    Miguel J Xavier

    Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

  15. Near-infrared quantum dots for HER2 localization and imaging of cancer cells.

    Science.gov (United States)

    Rizvi, Sarwat B; Rouhi, Sepideh; Taniguchi, Shohei; Yang, Shi Yu; Green, Mark; Keshtgar, Mo; Seifalian, Alexander M

    2014-01-01

    Quantum dots are fluorescent nanoparticles with unique photophysical properties that allow them to be used as diagnostic, therapeutic, and theranostic agents, particularly in medical and surgical oncology. Near-infrared-emitting quantum dots can be visualized in deep tissues because the biological window is transparent to these wavelengths. Their small sizes and free surface reactive groups that can be conjugated to biomolecules make them ideal probes for in vivo cancer localization, targeted chemotherapy, and image-guided cancer surgery. The human epidermal growth factor receptor 2 gene (HER2/neu) is overexpressed in 25%-30% of breast cancers. The current methods of detection for HER2 status, including immunohistochemistry and fluorescence in situ hybridization, are used ex vivo and cannot be used in vivo. In this paper, we demonstrate the application of near-infrared-emitting quantum dots for HER2 localization in fixed and live cancer cells as a first step prior to their in vivo application. Near-infrared-emitting quantum dots were characterized and their in vitro toxicity was established using three cancer cell lines, ie, HepG2, SK-BR-3 (HER2-overexpressing), and MCF7 (HER2-underexpressing). Mouse antihuman anti-HER2 monoclonal antibody was conjugated to the near-infrared-emitting quantum dots. In vitro toxicity studies showed biocompatibility of SK-BR-3 and MCF7 cell lines with near-infrared-emitting quantum dots at a concentration of 60 μg/mL after one hour and 24 hours of exposure. Near-infrared-emitting quantum dot antiHER2-antibody bioconjugates successfully localized HER2 receptors on SK-BR-3 cells. Near-infrared-emitting quantum dot bioconjugates can be used for rapid localization of HER2 receptors and can potentially be used for targeted therapy as well as image-guided surgery.

  16. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

  17. An iridium oxide microelectrode for monitoring acute local pH changes of endothelial cells.

    Science.gov (United States)

    Ng, Shu Rui; O'Hare, Danny

    2015-06-21

    pH sensors were fabricated by anodically electrodepositing iridium oxide films (AEIROFs) onto microelectrodes on chips and coated with poly(ethyleneimine) (PEI) for mechanical stability. These demonstrate super-Nernstian response to pH from pH 4.0 to 7.7 in chloride-free phosphate buffer. The surface of the chip was coated with fibronectin for the attachment of porcine aortic endothelial cells (PAECs). The working capability of the pH sensor for monitoring acute local pH changes was investigated by stimulating the PAECs with thrombin. Our results show that thrombin induced acute extracellular acidification of PAECs and dissolution of fibronectin, causing the local pH to decrease. The use of PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, reduced extracellular acidification and an increase in local pH was observed. This study shows that our pH sensors can facilitate the investigation of acute cellular responses to stimulation by monitoring the real-time, local pH changes of cells attached to the sensors.

  18. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Science.gov (United States)

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  19. SEU-hardened silicon bipolar and GaAs MESFET SRAM cells using local redundancy techniques

    International Nuclear Information System (INIS)

    Hauser, J.R.

    1992-01-01

    Silicon bipolar and GaAs FET SRAM's have proven to be more difficult to harden with respect to single-event upset mechanisms than have silicon CMOS SRAM's. This is a fundamental property of bipolar and JFET or MESFET device technologies which do not have a high-impedance, nonactive isolation between the control electrode and the current or voltage being controlled. All SEU circuit level hardening techniques applied at the local level must use some type of information storage redundancy so that information loss on one node due to an SEU event can be recovered from information stored elsewhere in the cell. In CMOS technologies, this can be achieved by the use of simple cross-coupling resistors, whereas in bipolar and FET technologies, no such simple approach is possible. Several approaches to the use of local redundancy in bipolar and FET technologies are discussed in this paper. At the expense of increased cell complexity and increased power consumption and write time, several approaches are capable of providing complete SEU hardness at the local cell level

  20. Changes in Ect2 Localization Couple Actomyosin-Dependent Cell Shape Changes to Mitotic Progression

    Science.gov (United States)

    Matthews, Helen K.; Delabre, Ulysse; Rohn, Jennifer L.; Guck, Jochen; Kunda, Patricia; Baum, Buzz

    2012-01-01

    Summary As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division. PMID:22898780

  1. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA in the pig ovary.

    Directory of Open Access Journals (Sweden)

    Milan TomĂĄnek

    2007-01-01

    Full Text Available The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.

  2. Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

    2012-11-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

  3. ADAM28 localizes to HLA-G+ trophoblasts and promotes column cell outgrowth.

    Science.gov (United States)

    De Luca, L C; Le, H T; Mara, D L; Beristain, A G

    2017-07-01

    Trophoblast progenitor cell differentiation towards the extravillous trophoblast (EVT) lineage initiates within proximal regions of anchoring columns of first trimester placental villi. While molecular processes controlling the initial stages of progenitor cell differentiation along the EVT pathway have been described, much remains unknown about factors important in distal column cell differentiation into invasive EVTs. ADAMs are proteases that regulate growth factor signaling, cell-matrix adhesion, and matrix proteolysis, and thus impact many processes relevant in placentation. Global gene expression studies identified the ADAM subtype, ADAM28, to be highly expressed in EVT-like trophoblasts, suggesting that it may play a role in EVT function. This study aims to test the functional importance of ADAM28 in column cell outgrowth and maintenance. ADAM28 mRNA levels and protein localization were determined by qPCR and immunofluorescence microscopy analyses in purified placental villi cell populations and tissues. ADAM28 function in trophoblast column outgrowth was examined using ADAM28-targetting siRNAs in Matrigel-imbedded placental explant cultures. Within placental villi, ADAM28 mRNA levels were highest in HLA-G + column trophoblasts, and consistent with this, ADAM28 was preferentially localized to HLA-G + trophoblasts within distal anchoring columns and decidual tissue. siRNA-directed loss of ADAM28 impaired trophoblast column outgrowth and resulted in increased apoptosis in matrix-invading trophoblasts. Our findings suggest that ADAM28 promotes column outgrowth by providing survival cues within anchoring column cells. This study also provides insight into a possible role for ADAM28 in driving differentiation of column trophoblasts into invasive HLA-G + EVT subsets. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Location of tumor affects local and distant immune cell type and number.

    Science.gov (United States)

    Hensel, Jonathan A; Khattar, Vinayak; Ashton, Reading; Lee, Carnellia; Siegal, Gene P; Ponnazhagan, Selvarangan

    2017-03-01

    Tumors comprise heterogeneous populations of cells, including immune infiltrates that polarize during growth and metastasis. Our preclinical studies on breast cancer (BCa) identified functional differences in myeloid-derived suppressor cells based on tumor microenvironment (TME), prompting variations in host immune response to tumor growth, and dissemination based on tissue type. In order to understand if such variations existed among other immune cells, and if such alteration occurs in response to tumor growth at the primary site or due to bone dissemination, we characterized immune cells, examining localized growth and in the tibia. In addition, immune cells from the spleen were examined from animals of both tumor locations by flow cytometry. The study demonstrates that location of tumor, and not simply the tumor itself, has a definitive role in regulating immune effectors. Among all immune cells characterized, macrophages were decreased and myeloid dendritic cell were increased in both tumor locations. This difference was more evident in subcutaneous tumors. Additionally, spleens from mice with subcutaneous tumors contained greater increases in both macrophages and myeloid dendritic cells than in mice with bone tumors. Furthermore, in subcutaneous tumors there was an increase in CD4 + and CD8 + T-cell numbers, which was also observed in their spleens. These data indicate that alterations in tumor-reactive immune cells are more pronounced at the primary site, and exert a similar change at the major secondary lymphoid organ than in the bone TME. These findings could provide translational insight into designing therapeutic strategies that account for location of metastatic foci.

  5. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    Energy Technology Data Exchange (ETDEWEB)

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: corinne.chadeneau@univ-poitiers.fr [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)

    2014-11-28

    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  6. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    Science.gov (United States)

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPLW515L, MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but was not phosphorylated. We generated disulfide-bonded MPLW515L homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPLW515L with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPLW515L/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling. PMID:19261614

  7. Fatal Metastatic Cutaneous Squamous Cell Carcinoma Evolving from a Localized Verrucous Epidermal Nevus

    Directory of Open Access Journals (Sweden)

    Hassan Riad

    2013-10-01

    Full Text Available A malignant transformation is known to occur in many nevi such as a sebaceous nevus or a basal cell nevus, but a verrucous epidermal nevus has only rarely been associated with neoplastic changes. Keratoacanthoma, multifocal papillary apocrine adenoma, multiple malignant eccrine poroma, basal cell carcinoma and cutaneous squamous cell carcinoma (CSCC have all been reported to develop from a verrucous epidermal nevus. CSCC has also been reported to arise from other nevoid lesions like a nevus comedonicus, porokeratosis, a sebaceous nevus, an oral sponge nevus and an ichthyosiform nevus with CHILD syndrome. Here we report a case of progressive poorly differentiated CSCC arising from a localized verrucous epidermal nevus, which caused both spinal cord and brain metastasis.

  8. Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform.

    Science.gov (United States)

    Hui, Jingjing; Bao, Lei; Li, Siqiao; Zhang, Yi; Feng, Yimei; Ding, Lin; Ju, Huangxian

    2017-07-03

    Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. CMV-specific CD8 T Cell Differentiation and Localization: Implications for Adoptive Therapies

    Directory of Open Access Journals (Sweden)

    Corinne J Smith

    2016-09-01

    Full Text Available Human cytomegalovirus (HCMV is a ubiquitous virus that causes chronic infection, and thus is one of the most common infectious complications of immune suppression. Adoptive transfer of HCMV-specific T cells has emerged as an effective method to reduce the risk for HCMV infection and/or reactivation by restoring immunity in transplant recipients. However, the CMV-specific CD8+ T cell response is comprised of a heterogenous mixture of subsets with distinct functions and localization and it is not clear if current adoptive immunotherapy protocols can reconstitute the full spectrum of CD8+ T cell immunity. The aim of this review is to briefly summarize the role of these T cell subsets in CMV immunity and to describe how current adoptive immunotherapy practices might affect their reconstitution in patients. The bulk of the CMV-specific CD8+ T cell population is made up of terminally differentiated effector T cells with immediate effector function and a short life span. Self-renewing memory T cells within the CMV-specific population retain the capacity to expand and differentiate upon challenge and are important for the long-term persistence of the CD8+ T cell response. Finally mucosal organs, which are frequent sites of CMV reactivation, are primarily inhabited by tissue resident memory T cells, which do not recirculate. Future work on adoptive transfer strategies may need to focus on striking a balance between the formation of these subsets to ensure the development of long lasting and protective immune responses that can access the organs affected by CMV disease.

  10. Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

    Science.gov (United States)

    Stubbs, M; Aiston, S; Agius, L

    2000-12-01

    We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

  11. Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones.

    Science.gov (United States)

    Moran, Yehu; Genikhovich, Grigory; Gordon, Dalia; Wienkoop, Stefanie; Zenkert, Claudia; Ozbek, Suat; Technau, Ulrich; Gurevitz, Michael

    2012-04-07

    Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.

  12. Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

    Science.gov (United States)

    Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

    2003-03-28

    Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

  13. B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

    Directory of Open Access Journals (Sweden)

    MGS Vieira

    2002-10-01

    Full Text Available Biopsies from human localized cutaneous lesions (LCL n = 7 or disseminated lesions (DL n = 8 cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.

  14. Local control rate and prognosis after sequential chemoradiation for small cell carcinoma of the bladder

    International Nuclear Information System (INIS)

    Meijer, Richard P.; Meinhardt, Wim; Poel, Henk G. van der; Rhijn, Bas W. van; Kerst, J. Martijn; Pos, Floris J.; Horenblas, Simon; Bex, Axel

    2013-01-01

    The objectives of this study were to assess the long-term outcome and the risk for local recurrence of patients with small cell carcinoma of the bladder (SCCB) treated with neoadjuvant chemotherapy followed by external beam radiotherapy (sequential chemoradiation). All consecutive patients with primary small cell carcinoma of the bladder (n=66), treated in our institution between 1993 and 2011 were retrospectively evaluated from an institutional database. Only patients with limited disease (Tx-4N0-1M0) small cell carcinoma of the bladder treated with sequential chemoradiation (n=27) were included in this study. Recurrence rates, overall survival and cancer-specific survival were analyzed using the Kaplan-Meier method. Median time to recurrence was 20 months, median overall survival 26 months, 5-year overall survival 22.2%, median cancer-specific survival 47 months and 5-year cancer-specific survival 39.6%. For complete responders after neoadjuvant chemotherapy (n=19), median cancer-specific survival was 52 months with a 5-year cancer-specific survival 45.9% versus a median cancer-specific survival of 22 months and 5-year cancer-specific survival 0.0% for incomplete responders (n=8; P=0.034). Eight patients (29.6%) underwent transurethral resections (TUR-BT) for local recurrences in the bladder. At the end of follow up, four patients had undergone cystectomy for recurrence of disease resulting in a bladder-preservation rate of 85.2%. Median time to local recurrence was 29 months and median time to distant recurrence was 10 months. Sequential chemoradiation for limited disease small cell carcinoma of the bladder results in a reasonable outcome with a high bladder preservation rate. Response to neoadjuvant chemotherapy represents a significant prognostic factor in this patient population. (author)

  15. Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells

    DEFF Research Database (Denmark)

    Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna

    2010-01-01

    -activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia....

  16. TRX-1 Regulates SKN-1 Nuclear Localization Cell Non-autonomously in Caenorhabditis elegans.

    Science.gov (United States)

    McCallum, Katie C; Liu, Bin; Fierro-González, Juan Carlos; Swoboda, Peter; Arur, Swathi; Miranda-Vizuete, Antonio; Garsin, Danielle A

    2016-05-01

    The Caenorhabditis elegans oxidative stress response transcription factor, SKN-1, is essential for the maintenance of redox homeostasis and is a functional ortholog of the Nrf family of transcription factors. The numerous levels of regulation that govern these transcription factors underscore their importance. Here, we add a thioredoxin, encoded by trx-1, to the expansive list of SKN-1 regulators. We report that loss of trx-1 promotes nuclear localization of intestinal SKN-1 in a redox-independent, cell non-autonomous fashion from the ASJ neurons. Furthermore, this regulation is not general to the thioredoxin family, as two other C. elegans thioredoxins, TRX-2 and TRX-3, do not play a role in this process. Moreover, TRX-1-dependent regulation requires signaling from the p38 MAPK-signaling pathway. However, while TRX-1 regulates SKN-1 nuclear localization, classical SKN-1 transcriptional activity associated with stress response remains largely unaffected. Interestingly, RNA-Seq analysis revealed that loss of trx-1 elicits a general, organism-wide down-regulation of several classes of genes; those encoding for collagens and lipid transport being most prevalent. Together, these results uncover a novel role for a thioredoxin in regulating intestinal SKN-1 nuclear localization in a cell non-autonomous manner, thereby contributing to the understanding of the processes involved in maintaining redox homeostasis throughout an organism. Copyright © 2016 by the Genetics Society of America.

  17. Patterns of failure after involved field radiotherapy for locally advanced esophageal squamous cell carcinoma.

    Science.gov (United States)

    Li, Duo-Jie; Li, Hong-Wei; He, Bin; Wang, Geng-Ming; Cai, Han-Fei; Duan, Shi-Miao; Liu, Jing-Jing; Zhang, Ya-Jun; Cui, Zhen; Jiang, Hao

    2016-01-01

    To retrospectively analyze the patterns of failure and the treatment effects of involved-field irradiation (IFI) on patients treated with locally advanced esophageal squamous cell carcinoma (ESCC) and to determine whether IFI is practicable in these patients. A total of 79 patients with locally advanced ESCC underwent three dimensional conformal (3D)CRT) or intensity modulated radiotherapy (IMRT) using IFI or elective nodal irradiation (ENI) according to the target volume. The patterns of failure were defined as local/regional, in-field, out)of)field regional lymph node (LN) and distant failure. With a median follow)up of 32.0 months, failures were observed in 66 (83.6%) patients. The cumulative incidence of local/regional failure (55.8 vs 52.8%) and in)field regional lymph node failure (25.6 vs 19.4%) showed no statistically significant difference between the IFI and the ENI group (p=0.526 and 0.215, respectively). Out)of)field nodal relapse rate of only 7.0% was seen in the IFI group. Three)year survival rates for the ENI and IFI group were 22.2 and 18.6%, respectively (p=0.240), and 3)year distant metastasis rates were 27.8 and 32.6%, respectively (p=0.180). The lung V10, V20, V30 and mean lung dose of the ENI group were greater than those of the IFI group, while the mean lung dose and V10 had statistically significant difference. The patterns of failure and survival rates in the IFI group were similar as in the ENI group; the regional recurrence and distant metastasis are the main cause of treatment failure. IFI is feasible for locally advanced ESCC. Further investigation is needed to increase local control and decrease distant metastasis in these patients.

  18. Combined modality therapy for locally advanced non-small cell lung carcinoma

    International Nuclear Information System (INIS)

    Recine, D.; Rowland, K.; Reddy, S.; Lee, M.S.; Bonomi, P.; Taylor, S.; Faber, L.P.; Warren, W.; Kittle, C.F.; Hendrickson, F.R.

    1990-01-01

    Multi-modality treatment consisting of cisplatin, VP-16, and 5-fluorouracil chemotherapy given concomitantly with external beam radiation was used to treat 64 patients with locally advanced Stage III non-small cell lung carcinoma. This regimen was used in a preoperative fashion for four cycles in patients considered surgically resectable and with curative intent for six cycles in the remainder of patients. The clinical response rate for the entire group was 84% and the overall local control rate was 74%. The median survival was 13 months with a median follow-up for live patients of 19 months. The actuarial 3-year survival and disease-free survival rates were 30% and 23%, respectively. Histologic complete response was 39% and appeared to predict for survival. The 3-year actuarial survival and disease-free survival rates for 23 resected patients were 69% and 45%, respectively, with the complete histologic responders having a disease-free survival of 78%. The pattern of first recurrence did not appear to differ by histology or presence of lymph nodes in this subset of patients. The actuarial 3-year survival and disease-free survival rates for inoperable patients receiving six cycles of treatment were 18% and 23%, respectively. The local control was 67% with the majority of these patients having Stage IIIB disease. The Mountain International staging system appeared to predict for operability, local recurrence, and survival. This concomitant treatment regimen is feasible, with the major toxicities being leukopenia, nausea, and vomiting

  19. Functioning islet cell tumor of the pancreas. Localization with dynamic spiral CT

    International Nuclear Information System (INIS)

    Chung, M.J.; Choi, B.I.; Han, J.K.; Chung, J.W.; Han, M.C.; Bae, S.H.

    1997-01-01

    Purpose: The purpose of this study was to evaluate the usefulness of dynamic spiral CT, including multidimensional reformation, in the detection and localization of islet cell tumors of the pancreas. Material and Methods: Seven patients with histopathologically proven functioning islet cell tumors of the pancreas were studied with 2-phase contrast-enhanced spiral CT. Scanning of the arterial phase and late phase was started 30 s and 180 s, respectively, after injection of 100 ml of contrast medium at a rate of 3 ml/s. Results: Axial images in the arterial phase depicted the lesions in 5 patients, but in the late phase in only one patient. Multiplanar reformatted images of the arterial phase depicted the lesions in all 7 patients. Maximal intensity projection images demonstrated all lesions with information of their relationship to the vascular structure. Conclusion: Dynamic spiral CT with scanning during the arterial phase and retrospective multidimensional reformation is useful for preoperative detection and localization of small islet cell tumors of the pancreas. (orig.)

  20. Chemotherapy related toxicity in locally advanced non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Bahl Amit

    2006-01-01

    Full Text Available Background: For inoperable non-small cell lung cancer combined chemotherapy and radiotherapy plays an important role as a therapeutic modality. The aim of the present study was to analyze neoadjuvant chemotherapy related acute toxicity in locally advanced lung cancer (stage IIIA and IIIB in Indian patients using Cisplatin and Etoposide combination chemotherapy. Material and methods: Forty patients of locally advanced Non small cell lung cancer received three cycles neoadjuvant chemotherapy using Injection Cisplatin and Etoposide. The patients were taken for Radical radiotherapy to a dose of 60 Gray over 30 fractions in conventional fractionation after completing chemotherapy. Chemotherapy associated toxicity was assessed using common toxicity criteria (CTC v2.0 Results: Forty patients were available for final evaluation. Median age of presentation of patients was fifty-six years. Thirteen patients had Non small cell lung cancer stage IIIA while twenty-seven patients had Stage IIIB disease. Anemia was the most common hematological toxicity observed (seen in 81% of patients. Nausea and vomiting were the most common non -hematological toxicity seen. Sensory neuropathy was seen in 38%of patients. 88% patients developed alopecia. Seven patients developed febrile neutropenias. Conclusion: Neo-adjuvant chemotherapy using Cisplatin and Etoposide continues to be a basic regimen in the Indian set up despite availability of higher molecules, since it is cost effective, well tolerated and therapeutically effective. Blood transfusions, growth factors and supportive care can be used effectively to over come toxicity associated with this regimen.

  1. Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

    Science.gov (United States)

    Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

    2013-06-15

    Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

    Science.gov (United States)

    Eryilmaz, Marion; Schmitt, Eberhard; Krufczik, Matthias; Theda, Franziska; Lee, Jin-Ho; Cremer, Christoph; Bestvater, Felix; Schaufler, Wladimir; Hausmann, Michael; Hildenbrand, Georg

    2018-01-22

    In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

  3. Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

    Science.gov (United States)

    Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2013-01-01

    Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328

  4. Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex Trunculus digestive cells

    Science.gov (United States)

    2011-01-01

    Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. Conclusion The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids. PMID:21631952

  5. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    International Nuclear Information System (INIS)

    Zettergren, Eric; Swamy, Tushar; Niedre, Mark; Runnels, Judith; Lin, Charles P

    2012-01-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument. (paper)

  6. Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging.

    Science.gov (United States)

    Managò, Stefano; Migliaccio, Nunzia; Terracciano, Monica; Napolitano, Michela; Martucci, Nicola M; De Stefano, Luca; Rendina, Ivo; De Luca, Anna Chiara; Lamberti, Annalisa; Rea, Ilaria

    2018-04-01

    Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

    Science.gov (United States)

    Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

    2013-07-02

    Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Stereotactic Body Radiotherapy for Localized Ureter Transitional Cell Carcinoma: Three Case Reports

    Directory of Open Access Journals (Sweden)

    Yoshiyasu Maehata

    2015-01-01

    Full Text Available The gold standard management for ureter transitional cell carcinoma (UTCC is radical nephroureterectomy with excision of the bladder cuff. However, some patients cannot undergo this procedure for several reasons. In the case reports described herein, we performed stereotactic body radiotherapy (SBRT on three patients with inoperable or surgery-rejected localized UTCC. Two out of the three patients did not develop local recurrence or distant metastasis during the observation period. However, recurrence was detected in the bladder of one patient 22 months after the treatment. No acute or late adverse events occurred in any of the three patients. SBRT may become one of the treatment options for inoperable or surgery-rejected UTCC patients.

  9. Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

  10. Targeted therapy for localized non-small-cell lung cancer: a review

    Directory of Open Access Journals (Sweden)

    Paleiron N

    2016-07-01

    Full Text Available Nicolas Paleiron,1 Olivier Bylicki,2 Michel André,1 Emilie Rivière,1 Frederic Grassin,1 Gilles Robinet,3 Christos Chouaïd4 On behalf of the GFPC Group 1Chest Department, HIA Clermont Tonnerre, Brest, 2Chest Department, HIA Percy, Clamart, 3Chest Department, CHU de Brest, Brest, 4GRC OncoEst, Université Paris XII, Paris, France Abstract: Targeted therapies have markedly improved the management of patients with advanced non-small-cell lung cancer (NSCLC, but their efficacy in localized NSCLC is less well established. The aim of this review is to analyze trials of targeted therapies in localized NSCLC. In patients with wild-type EGFR, tyrosine kinase inhibitors have shown no efficacy in Phase III trials. Few data are available for EGFR-mutated localized NSCLC, as routine biological profiling is not recommended. Available studies are small, often retrospectives, and/or conducted in a single-center making it difficult to draw firm conclusions. Ongoing prospective Phase III trials are comparing adjuvant tyrosine kinase inhibitor administration versus adjuvant chemotherapy. By analogy with the indication of bevacizumab in advanced NSCLC, use of antiangiogenic agents in the perioperative setting is currently restricted to nonsquamous NSCLC. Several trials of adjuvant or neoadjuvant bevacizumab are planned or ongoing, but for the moment there is no evidence of efficacy. Data on perioperative use of biomarkers in early-stage NSCLC come mainly from small, retrospective, uncontrolled studies. Assessment of customized adjuvant or neoadjuvant therapy in localized NSCLC (with or without oncogenic driver mutations is a major challenge. Keywords: targeted therapy, non-small-cell lung cancer, adjuvant, neo-adjuvant, surgery 

  11. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    International Nuclear Information System (INIS)

    Zhao, Zhuo; Wang, Hao; Lin, Marina; Groban, Leanne

    2015-01-01

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

  12. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

    2015-03-27

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

  13. LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β.

    Science.gov (United States)

    Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

    2012-11-14

    Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer's disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA.

  14. Experimentally and numerically investigating cell performance and localized characteristics for a high-temperature proton exchange membrane fuel cell

    International Nuclear Information System (INIS)

    Su Ay; Ferng, Yuh Ming; Shih, Jah Ching

    2009-01-01

    This paper is to experimentally and numerically investigate the cell performance and the localized characteristics associated with a high-temperature proton exchange membrane fuel cell (PEMFC). Three experiments are carried out in order to study the performance of the PEMFC with different operating conditions and to validate the numerical simulation model. The model proposed herein is a three-dimensional (3-D) computational fluid dynamics (CFD) non-isothermal model that essentially consists of thermal-hydraulic equations and electrochemical model. The performance curves of the PEMFC predicted by the present model agree with the experimental measured data. In addition, both the experiments and the predictions precisely demonstrate the enhanced effects of inlet gas temperature and system pressure on the PEMFC performance. Based on the simulation results, the localized characteristics within a PEMFC can be reasonably captured. These parameters include the fuel gas distribution, liquid water saturation distribution, membrane conductivity distribution, temperature variation, and current density distribution etc. As the PEMFC is operated at the higher current density, the fuel gas would be insufficiently supplied to the catalyst layer, consequently causing the decline in the generation of power density. This phenomenon is so called mass transfer limitation, which can be precisely simulated by the present CFD model.

  15. Management of Locally Advanced Renal Cell Carcinoma with Invasion of the Duodenum

    Directory of Open Access Journals (Sweden)

    Andrew T. Schlussel

    2013-01-01

    Full Text Available Renal cell carcinoma (RCC is rare but aggressive, with greater than 20% of patients presenting with stage III or IV, disease. Surgical resection of the primary tumor regardless of stage is the treatment of choice, and en bloc resection of involved organs provides the only potential chance for cure. This case report describes a patient with metastatic right-sided RCC with invasion of the inferior vena cava and duodenum managed by en block resection and pancreaticoduodenectomy. This report will review the workup and treatment of locally advanced RCC, as well as the role of cytoreductive nephrectomy in the setting of metastatic disease.

  16. The impact of locally multiply damaged sites (LMDS) induced by ionizing radiation in mammalian cells

    International Nuclear Information System (INIS)

    Averbeck, D.; Boucher, D.

    2006-01-01

    Monte Carlo calculations have shown that ionising radiations produce a specific type of clustered cell damage called locally multiply damaged sites or LMDS. These lesions consist of closely positioned single-strand breaks, (oxidative) base damage and DNA double-strand breaks (DSB) in between one helical turn of DNA. As specific markers of radiation-induced damage these lesions are likely to condition biological responses and are thus of great interest for radiation protection. Calculations indicate that there should be more LMDS induced by high than by low LET radiation, and they should be absent in un-irradiated cells. Processes like K-shell activation and local Auger electron emission can be expected to add complex DSB or LMDS, producing significant chromosomal damage. In the discussion of the specificity of ionising radiation in comparison to other genotoxic agents, many arguments have been put forward that these lesions should be particularly deleterious for living cells. Complex lesions of that type should represent big obstacles for DNA repair and give rise to high lethality. Moreover, cellular attempts to repair them could accentuate harm, leading to mutations, genetic instability and cancer. In vitro experiments with oligonucleotides containing an artificially introduced set of base damage and SSB in different combinations have shown that depending on the close positioning of the damage on DNA, repair enzymes, and even whole cell extracts, are unable to repair properly and may stimulate mis-repair. Pulsed field gel electrophoresis (PFGE) in conjunction with enzymatic treatments has been used to detect LMDS in mammalian cells after high and low LET radiation. In order to further define the importance of LMDS for radiation induced cellular responses, we studied the induction of LMDS as a function of radiation dose and dose rate in mammalian cells (CHO and MRC5) using 137 Cs gamma-radiation. Using PFGE and specific glycosylases to convert oxidative damage into

  17. The impact of locally multiply damaged sites (LMDS) induced by ionizing radiation in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Averbeck, D.; Boucher, D. [Institut Curie-Section de Recherche, UMR2027 CNRS, LCR-V28 du CEA, Centre Universitaire, 91405 Orsay Cedex (France)

    2006-07-01

    Monte Carlo calculations have shown that ionising radiations produce a specific type of clustered cell damage called locally multiply damaged sites or LMDS. These lesions consist of closely positioned single-strand breaks, (oxidative) base damage and DNA double-strand breaks (DSB) in between one helical turn of DNA. As specific markers of radiation-induced damage these lesions are likely to condition biological responses and are thus of great interest for radiation protection. Calculations indicate that there should be more LMDS induced by high than by low LET radiation, and they should be absent in un-irradiated cells. Processes like K-shell activation and local Auger electron emission can be expected to add complex DSB or LMDS, producing significant chromosomal damage. In the discussion of the specificity of ionising radiation in comparison to other genotoxic agents, many arguments have been put forward that these lesions should be particularly deleterious for living cells. Complex lesions of that type should represent big obstacles for DNA repair and give rise to high lethality. Moreover, cellular attempts to repair them could accentuate harm, leading to mutations, genetic instability and cancer. In vitro experiments with oligonucleotides containing an artificially introduced set of base damage and SSB in different combinations have shown that depending on the close positioning of the damage on DNA, repair enzymes, and even whole cell extracts, are unable to repair properly and may stimulate mis-repair. Pulsed field gel electrophoresis (PFGE) in conjunction with enzymatic treatments has been used to detect LMDS in mammalian cells after high and low LET radiation. In order to further define the importance of LMDS for radiation induced cellular responses, we studied the induction of LMDS as a function of radiation dose and dose rate in mammalian cells (CHO and MRC5) using {sup 137}Cs gamma-radiation. Using PFGE and specific glycosylases to convert oxidative damage

  18. Localization of cells containing sedimented amyloplasts in the shoots of normal and lazy rice seedlings.

    Science.gov (United States)

    Abe, K; Takahashi, H; Suge, H

    1994-12-01

    We have examined the localization of the cells containing sedimented amyloplasts (putative statocytes) and its relation to the graviresponding sites in the shoots of normal and lazy rice seedlings. All graviresponsive organs of the shoots of normal rice seedlings, the mesocotyl, the coleoptile and the leaf-sheath base, were found to possess the statocytes. This is the first indication that mesocotyl senses gravity by its own cells in inducing gravitropic bending in rice seedlings. In lazy-Kamenoo, although the shoots lost their gravitropic response with the advance of age, sedimentation of amyloplasts itself might not be attributable to the agravitropic growth of the shoots, because, including those of the leaf-sheath bases that had lost their response to gravity, sedimented amyloplasts appeared to be identical to those of normal Kamenoo and of younger seedlings of lazy-Kamenoo whose gravitropism is still apparent.

  19. Locally advanced and metastatic basal cell carcinoma: molecular pathways, treatment options and new targeted therapies.

    Science.gov (United States)

    Ruiz Salas, Veronica; Alegre, Marta; Garcés, Joan Ramón; Puig, Lluis

    2014-06-01

    The hedgehog (Hh) signaling pathway has been identified as important to normal embryonic development in living organisms and it is implicated in processes including cell proliferation, differentiation and tissue patterning. Aberrant Hh pathway has been involved in the pathogenesis and chemotherapy resistance of different solid and hematologic malignancies. Basal cell carcinoma (BCC) and medulloblastoma are two well-recognized cancers with mutations in components of the Hh pathway. Vismodegib has recently approved as the first inhibitor of one of the components of the Hh pathway (smoothened). This review attempts to provide current data on the molecular pathways involved in the development of BCC and the therapeutic options available for the treatment of locally advanced and metastatic BCC, and the new targeted therapies in development.

  20. Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

    Science.gov (United States)

    Yokobori, Kosuke; Kobayashi, Kaoru; Azuma, Ikuko; Akita, Hidetaka; Chiba, Kan

    2017-10-01

    Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  1. Asymmetric Localization of Cdx2 mRNA during the First Cell-Fate Decision in Early Mouse Development

    Directory of Open Access Journals (Sweden)

    Maria Skamagki

    2013-02-01

    Full Text Available A longstanding question in mammalian development is whether the divisions that segregate pluripotent progenitor cells for the future embryo from cells that differentiate into extraembryonic structures are asymmetric in cell-fate instructions. The transcription factor Cdx2 plays a key role in the first cell-fate decision. Here, using live-embryo imaging, we show that localization of Cdx2 transcripts becomes asymmetric during development, preceding cell lineage segregation. Cdx2 transcripts preferentially localize apically at the late eight-cell stage and become inherited asymmetrically during divisions that set apart pluripotent and differentiating cells. Asymmetric localization depends on a cis element within the coding region of Cdx2 and requires cell polarization as well as intact microtubule and actin cytoskeletons. Failure to enrich Cdx2 transcripts apically results in a significant decrease in the number of pluripotent cells. We discuss how the asymmetric localization and segregation of Cdx2 transcripts could contribute to multiple mechanisms that establish different cell fates in the mouse embryo.

  2. Recent Developments of the Local Effect Model (LEM) - Implications of clustered damage on cell transformation

    Science.gov (United States)

    Elsässer, Thilo

    Exposure to radiation of high-energy and highly charged ions (HZE) causes a major risk to human beings, since in long term space explorations about 10 protons per month and about one HZE particle per month hit each cell nucleus (1). Despite the larger number of light ions, the high ionisation power of HZE particles and its corresponding more complex damage represents a major hazard for astronauts. Therefore, in order to get a reasonable risk estimate, it is necessary to take into account the entire mixed radiation field. Frequently, neoplastic cell transformation serves as an indicator for the oncogenic potential of radiation exposure. It can be measured for a small number of ion and energy combinations. However, due to the complexity of the radiation field it is necessary to know the contribution to the radiation damage of each ion species for the entire range of energies. Therefore, a model is required which transfers the few experimental data to other particles with different LETs. We use the Local Effect Model (LEM) (2) with its cluster extension (3) to calculate the relative biological effectiveness (RBE) of neoplastic transformation. It was originally developed in the framework of hadrontherapy and is applicable for a large range of ions and energies. The input parameters for the model include the linear-quadratic parameters for the induction of lethal events as well as for the induction of transformation events per surviving cell. Both processes of cell inactivation and neoplastic transformation per viable cell are combined to eventually yield the RBE for cell transformation. We show that the Local Effect Model is capable of predicting the RBE of neoplastic cell transformation for a broad range of ions and energies. The comparison of experimental data (4) with model calculations shows a reasonable agreement. We find that the cluster extension results in a better representation of the measured RBE values. With this model it should be possible to better

  3. High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells. A relation between localization and function?

    NARCIS (Netherlands)

    Kooij, A.; Bosch, K. S.; Frederiks, W. M.; van Noorden, C. J.

    1992-01-01

    The localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells

  4. Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines

    Directory of Open Access Journals (Sweden)

    Marion Eryilmaz

    2018-01-01

    Full Text Available In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex, a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR and alternative non-homologous end joining (a-NHEJ. We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

  5. Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells

    Science.gov (United States)

    Jiang, L; Dong, P; Zhang, Z; Li, C; Li, Y; Liao, Y; Li, X; Wu, Z; Guo, S; Mai, S; Xie, D; Liu, Z; Zhou, F

    2015-01-01

    Bladder cancer (BC) is very common and associated with significant morbidity and mortality, though the molecular underpinnings of its origination and progression remain poorly understood. In this study, we demonstrate that Prohibitin 1 (PHB) was overexpressed in human BC tissues and that PHB upregulation was associated with poor prognosis. We also found that PHB was necessary and sufficient for BC cell proliferation. Interestingly, the overexpressed PHB was primarily found within mitochondria, and we provide the first direct evidence that phosphorylation by Akt at Thr258 of PHB induces this mitochondrial localization. Inhibiton of Akt reverses these effects and inhibited the proliferation of BC cells. Finally, the phosphorylation of PHB was required for BC cell proliferation, further implicating the importance of the Akt in BC. Taken together, these findings identify the Akt/PHB signaling cascade as a novel mechanism of cancer cell proliferation and provide the scientific basis for the establishment of PHB as a new prognostic marker and treatment target for BC. PMID:25719244

  6. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    International Nuclear Information System (INIS)

    Stalker, A.; Hermo, L.; Antakly, T.

    1989-01-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor

  7. Brain local and regional neuroglial alterations in Alzheimer's Disease: cell types, responses and implications.

    Science.gov (United States)

    Toledano, Adolfo; Álvarez, María-Isabel; Toledano-Díaz, Adolfo; Merino, José-Joaquín; Rodríguez, José Julio

    2016-01-01

    From birth to death, neurons are dynamically accompanied by neuroglial cells in a very close morphological and functional relationship. Three families have been classically considered within the CNS: astroglia, oligodendroglia and microglia. Many types/subtypes (including NGR2+ cells), with a wide variety of physiological and pathological effects on neurons, have been described using morphological and immunocytochemical criteria. Glio-glial, glio-neuronal and neuro-glial cell signaling and gliotransmission are phenomena that are essential to support brain functions. Morphofunctional changes resulting from the plasticity of all the glial cell types parallel the plastic neuronal changes that optimize the functionality of neuronal circuits. Moreover, neuroglia possesses the ability to adopt a reactive status (gliosis) in which, generally, new functions arise to improve and restore if needed the neural functionality. All these features make neuroglial cells elements of paramount importance when attempting to explain any physiological or pathological processes in the CNS, because they are involved in both, neuroprotection/neurorepair and neurodegeneration. There exist diverse and profound, regional and local, neuroglial changes in all involutive processes (physiological and pathological aging; neurodegenerative disorders, including Alzheimer ´s disease -AD-), but today, the exact meaning of such modifications (the modifications of the different neuroglial types, in time and place), is not well understood. In this review we consider the different neuroglial cells and their responses in order to understand the possible role they fulfill in pathogenesis, diagnosis and treatment (preventive or palliative) of AD. The existence of differentiated and/or concurrent pathogenic and neuro-protective/neuro-restorative astroglial and microglial responses is highlighted.

  8. Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

    OpenAIRE

    G Nicholas Llewellyn; Ian B Hogue; Jonathan R Grover; Akira Ono

    2010-01-01

    T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. ...

  9. Isolated (localized) idiopathic granulomatous (giant cell) vasculitis in an intramuscular lipoma.

    Science.gov (United States)

    Fernando Val-Bernal, J; Val, Daniel; Calvo, Ignacio; Francisca Garijo, M

    2006-01-01

    Isolated (localized) idiopathic granulomatous vasculitis (IGV) is an uncommon, heterogeneous, and poorly defined group of disorders characterized by infiltration of the arterial wall caused by compactly grouped mononuclear phagocytes, with or without giant cells, in segmental distribution. We report on a 55-year-old woman with IGV limited to an intramuscular lipoma of the left thigh. The vasculitis was identified incidentally upon microscopic examination of the removed tumor. The IGV was centered on two medium-sized arteries, accompanied by narrowing of the lumens, and not associated with secondary changes such as infart or postinfart fibrosis. The inflammatory infiltrate was rich in T-lymphocytes and macrophages, with the presence of giant cells. The patient was asymptomatic and well in a follow-up period of 2 months, during which she was not treated. To our knowledge, this is the first report of lipoma involvement in localized IGV. It is important to distinguish cases of isolated intratumorous IGV from systemic disease, because the latter implies a poor prognosis and requires an aggressive treatment.

  10. Anatomical localization of commensal bacteria in immune cell homeostasis and disease.

    Science.gov (United States)

    Fung, Thomas C; Artis, David; Sonnenberg, Gregory F

    2014-07-01

    The mammalian gastrointestinal (GI) tract is colonized by trillions of beneficial commensal bacteria that are essential for promoting normal intestinal physiology. While the majority of commensal bacteria are found in the intestinal lumen, many species have also adapted to colonize different anatomical locations in the intestine, including the surface of intestinal epithelial cells (IECs) and the interior of gut-associated lymphoid tissues. These distinct tissue localization patterns permit unique interactions with the mammalian immune system and collectively influence intestinal immune cell homeostasis. Conversely, dysregulated localization of commensal bacteria can lead to inappropriate activation of the immune system and is associated with numerous chronic infectious, inflammatory, and metabolic diseases. Therefore, regulatory mechanisms that control proper anatomical containment of commensal bacteria are essential to maintain tissue homeostasis and limit pathology. In this review, we propose that commensal bacteria associated with the mammalian GI tract can be anatomically defined as (i) luminal, (ii) epithelial-associated, or (iii) lymphoid tissue-resident, and we discuss the role and regulation of these microbial populations in health and disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jose F Abisambra

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

  12. Phase precession through acceleration of local theta rhythm: a biophysical model for the interaction between place cells and local inhibitory neurons.

    Science.gov (United States)

    Castro, Luísa; Aguiar, Paulo

    2012-08-01

    Phase precession is one of the most well known examples within the temporal coding hypothesis. Here we present a biophysical spiking model for phase precession in hippocampal CA1 which focuses on the interaction between place cells and local inhibitory interneurons. The model's functional block is composed of a place cell (PC) connected with a local inhibitory cell (IC) which is modulated by the population theta rhythm. Both cells receive excitatory inputs from the entorhinal cortex (EC). These inputs are both theta modulated and space modulated. The dynamics of the two neuron types are described by integrate-and-fire models with conductance synapses, and the EC inputs are described using non-homogeneous Poisson processes. Phase precession in our model is caused by increased drive to specific PC/IC pairs when the animal is in their place field. The excitation increases the IC's firing rate, and this modulates the PC's firing rate such that both cells precess relative to theta. Our model implies that phase coding in place cells may not be independent from rate coding. The absence of restrictive connectivity constraints in this model predicts the generation of phase precession in any network with similar architecture and subject to a clocking rhythm, independently of the involvement in spatial tasks.

  13. Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    HU JIAN

    2009-01-01

    Full Text Available Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1 in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC. The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS and nuclear export signal (NES are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30 patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

  14. Delayed esophageal perforation from stereotactic body radiation therapy for locally recurrent central nonsmall cell lung cancer

    Directory of Open Access Journals (Sweden)

    Sandeep Sainathan

    2014-01-01

    Full Text Available Stereotactic body radiation therapy (SBRT is a novel form of external beam radiation therapy. It is used to treat early and locally recurrent nonsmall cell lung cancer (NSLC in medically inoperable patients. It uses high dose, hypofractionated radiotherapy, with targeting of the tumor by precise spatial localization, thus minimizing injury to surrounding tissues. It can be safely used to ablate NSLC in both central and peripheral locations. We present two cases of delayed esophageal perforation after SBRT for locally recurrent central NSLC. The perforations occurred several months after the therapy. They were treated with covered esophageal stents, with mortality, due to the perforation in one of the patients. SBRT should be judiciously used to ablate centrally located NSLC and patients who develop episodes of esophagitis during or after SBRT, need to be closely followed with endoscopy to look for esophageal ulcerations. These ulcers should be closely followed for healing as these may degenerate into full thickness perforations several months after SBRT.

  15. Business Cycle Dependent Unemployment Insurance

    DEFF Research Database (Denmark)

    Andersen, Torben M.; Svarer, Michael

    The consequences of business cycle contingencies in unemployment insurance systems are considered in a search-matching model allowing for shifts between "good" and "bad" states of nature. We show that not only is there an insurance argument for such contingencies, but there may also be an incentive...

  16. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells

    International Nuclear Information System (INIS)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J.

    1988-01-01

    Dopamine, generated locally from L-dopa, inhibits Na + -K + -ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na + -K + -ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (Qo 2 ) and 86 Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive Qo 2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive Qo 2 in a concentration-dependent manner, with half-maximal inhibition (K 0.5 ) of 5 x 10 -7 M and a maximal inhibition of 14.1 ± 1.5% at 10 -4 M. L-Dopa also blunted the nystatin-stimulated Qo 2 in a concentration-dependent manner, indicating the L-dopa directly inhibits Na + -K + -ATPase activity and not sodium entry. Ouabain-sensitive 86 Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive Qo 2 and 86 Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive Qo 2 at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na + -K + -ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner

  17. Radiotherapy is associated with significant improvement in local and regional control in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    Kang, Susan H; Haydu, Lauren E; Goh, Robin Yeong Hong; Fogarty, Gerald B

    2012-01-01

    Merkel cell carcinoma (MCC) is a rare tumour of skin. This study is a retrospective audit of patients with MCC from St Vincent’s and Mater Hospital, Sydney, Australia. The aim of this study was to investigate the influence of radiotherapy (RT) on the local and regional control of MCC lesions and survival of patients with MCC. The data bases in anatomical pathology, RT and surgery. We searched for patients having a diagnosis of MCC between 1996 and 2007. Patient, tumour and treatment characteristics were collected and analysed. Univariate survival analysis of categorical variables was conducted with the Kaplan-Meier method together with the Log-Rank test for statistical significance. Continuous variables were assessed using the Cox regression method. Multivariate analysis was performed for significant univariate results. Sixty seven patients were found. Sixty two who were stage I-III and were treated with radical intent were analysed. 68% were male. The median age was 74 years. Forty-two cases (68%) were stage I or II, and 20 cases (32%) were stage III. For the subset of 42 stage I and II patients, those that had RT to their primary site had a 2-year local recurrence free survival of 89% compared with 36% for patients not receiving RT (p<0.001). The cumulative 2-year regional recurrence free survival for patients having adjuvant regional RT was 84% compared with 43% for patients not receiving this treatment (p<0.001). Immune status at initial surgery was a significant predictor for OS and MCCSS. In a multivariate analysis combining macroscopic size (mm) and immune status at initial surgery, only immune status remained a significant predictor of overall survival (HR=2.096, 95% CI: 1.002-4.385, p=0.049). RT is associated with significant improvement in local and regional control in Merkel cell carcinoma. Immunosuppression is an important factor in overall survival

  18. Predictors and Patterns of Local, Regional, and Distant Failure in Squamous Cell Carcinoma of the Vulva.

    Science.gov (United States)

    Bogani, Giorgio; Cromi, Antonella; Serati, Maurizio; Uccella, Stefano; Donato, Violante Di; Casarin, Jvan; Naro, Edoardo Di; Ghezzi, Fabio

    2017-06-01

    To identify factors predicting for recurrence in vulvar cancer patients undergoing surgical treatment. We retrospectively evaluated data of consecutive patients with squamous cell vulvar cancer treated between January 1, 1990 and December 31, 2013. Basic descriptive statistics and multivariable analysis were used to design predicting models influencing outcomes. Five-year disease-free survival (DFS) and overall survival (OS) were analyzed using the Cox model. The study included 101 patients affected by vulvar cancer: 64 (63%) stage I, 12 (12%) stage II, 20 (20%) stage III, and 5 (5%) stage IV. After a mean (SD) follow-up of 37.6 (22.1) months, 21 (21%) recurrences occurred. Local, regional, and distant failures were recorded in 14 (14%), 6 (6%), and 3 (3%) patients, respectively. Five-year DFS and OS were 77% and 82%, respectively. At multivariate analysis only stromal invasion >2 mm (hazard ratio: 4.9 [95% confidence interval, 1.17-21.1]; P=0.04) and extracapsular lymph node involvement (hazard ratio: 9.0 (95% confidence interval, 1.17-69.5); P=0.03) correlated with worse DFS, although no factor independently correlated with OS. Looking at factors influencing local and regional failure, we observed that stromal invasion >2 mm was the only factor predicting for local recurrence, whereas lymph node extracapsular involvement predicted for regional recurrence. Stromal invasion >2 mm and lymph node extracapsular spread are the most important factors predicting for local and regional failure, respectively. Studies evaluating the effectiveness of adjuvant treatment in high-risk patients are warranted.

  19. Does endoscopic ultrasound improve detection of locally recurrent anal squamous-cell cancer?

    Science.gov (United States)

    Peterson, Carrie Y; Weiser, Martin R; Paty, Philip B; Guillem, Jose G; Nash, Garrett M; Garcia-Aguilar, Julio; Patil, Sujata; Temple, Larissa K

    2015-02-01

    Evaluating patients for recurrent anal cancer after primary treatment can be difficult owing to distorted anatomy and scarring. Many institutions incorporate endoscopic ultrasound to improve detection, but the effectiveness is unknown. The aim of this study is to compare the effectiveness of digital rectal examination and endoscopic ultrasound in detecting locally recurrent disease during routine follow-up of patients with anal cancer. This study is a retrospective, single-institution review. This study was conducted at an oncologic tertiary referral center. Included were 175 patients with nonmetastatic anal squamous-cell cancer, without persistent disease after primary chemoradiotherapy, who had at least 1 posttreatment ultrasound and examination by a colorectal surgeon. The primary outcomes measured were the first modality to detect local recurrence, concordance, crude cancer detection rate, sensitivity, specificity, and predictive value. Eight hundred fifty-five endoscopic ultrasounds and 873 digital rectal examinations were performed during 35 months median follow-up. Overall, ultrasound detected 7 (0.8%) mesorectal and 32 (3.7%) anal canal abnormalities; digital examination detected 69 (7.9%) anal canal abnormalities. Locally recurrent disease was found on biopsy in 8 patients, all detected first or only with digital examination. Four patients did not have an ultrasound at the time of diagnosis of recurrence. The concordance of ultrasound and digital examination in detecting recurrent disease was fair at 0.37 (SE, 0.08; 95% CI, 0.21-0.54), and there was no difference in crude cancer detection rate, sensitivity, specificity, and negative or positive predictive values. The heterogeneity of follow-up timing and examinations is not standardized in this study but is reflective of general practice. Endoscopic ultrasound did not provide any advantage over digital rectal examination in identifying locally recurrent anal cancer, and should not be recommended for

  20. [Planned neck dissection in the treatment of locally advanced head and neck squamous cell carcinoma].

    Science.gov (United States)

    Jiang, L; Lou, J L; Wang, K J; Fang, M Y; Fu, Z F

    2018-02-07

    Objective: To investigate the value of planned neck dissection combined with induction chemotherapy and concurrent chemoradiotherapy in regional control and the outcome of locally advanced head and neck squamous cell carcinoma. Methods: A prospective randomized controlled study totally enrolled sixty-four patients of head and neck squamous cell carcinomas(include oropharynx, hypopharynx, and larynx) in stages Ⅳa-Ⅳb with lymph node metastase was were N2-N3. All patients firstly received 2-3 cycles of induction chemotherapy(ICT), then divided into two groups randomly, according to the efficacy of ICT. Group A(the study group) received planned neck dissection(PND) and concurrent chemoradiotherapy(CCRT). Group B(the control group) received concurrent chemoradiotherapy(CCRT). The differences in clinicopathologic features, local recurrence(LR), regional recurrence(RR), disease-free survival(DFS), and overall survival(OS) between the two groups were estimated. SPSS 19.0 software was used to analyze the data. Results: Group A enrolled twenty-one patients, and group B enrolled forty-three patients.The follow-up of all patients were 4-55 months, median follow-up time was 22 months. In study group, two-year OS and DFS were 80.9% and 68.3%, respectively. In control group, two-year OS and DFS were 90.7% and 67.1%, respectively. There was no significant difference in gender( P =0.215), age( P =0.828), primary tumor site( P =0.927), LR( P =0.126), DFS( P =0.710), and OS( P =0.402) between the two groups, while the RR(χ(2)=5.640, P squamous cell carcinoma.

  1. Biologic Evaluation of Diabetes and Local Recurrence in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Yang, Xuebin; Liu, Yongjun; Mani, Haresh; Olson, Jeffrey; Clawson, Gary; Caruso, Carla; Bruggeman, Richard; Varlotto, John M; Zander, Dani S; Rassaei, Negar

    2017-01-01

    A recent multicenter study led by our institution demonstrated that local recurrence of non-small cell lung cancer (NSCLC) was significantly more frequent in patients with diabetes, raising the possibility of different tumor biology in diabetics. Epithelial-to-mesenchymal transition (EMT) plays a key role in local tumor recurrence and metastasis. In the present study, we investigated differences of tumor microenvironment between patients with and without diabetes by examining expression of EMT markers. Seventy-nine NSCLC patients were selected from the cohort of our early multicenter study. These patients were classified into 4 groups: 39 with adenocarcinoma with (n = 19) and without (n = 20) diabetes, and 40 with squamous cell carcinoma with (n = 20) and without (n = 20) diabetes. Immunohistochemical expression of eight EMT markers was analyzed, including transforming growth factor-beta (TGF-β), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), vimentin, E-cadherin, N-cadherin, HtrA1, and beta-catenin. Five markers (E-cadherin, HtrA1, TGF-β, IGF-1R and vimentin) demonstrated significantly higher expression in diabetics than in non-diabetics in both histology types. N-cadherin had higher expression in diabetics, though the difference did not reach statistical significance. EGFR showed a higher expression in diabetics in squamous cell carcinoma only. Beta-catenin was the only marker with no difference in expression between diabetics versus non-diabetics. Our findings suggest that diabetes is associated with enhanced EMT in NSCLC, which may contribute to growth and invasiveness of NSCLC.

  2. Accelerated superfractionated radiotherapy with concomitant boost for locally advanced head-and-neck squamous cell carcinomas

    International Nuclear Information System (INIS)

    Morris, Monica M.; Schmidt-Ullrich, Rupert K.; DiNardo, L.; Manning, Matthew A.; Silverman, L.; Clay, L.; Johnson, Christopher R.; Amir, Cyrus

    2002-01-01

    Purpose: A growing body of evidence supports the efficacy of accelerated superfractionated radiotherapy with concomitant boost for advanced head-and-neck carcinomas. This study represents a single-institution experience, performed to identify the factors influencing tumor control, survival, and toxicity. Methods and Materials: Between 1988 and 1999, 133 patients with primary squamous cell head-and-neck carcinoma underwent accelerated superfractionated radiotherapy using a concomitant boost. The concomitant boost in this regimen was delivered using reduced fields delivered 3 times weekly in a twice-daily schedule during the final phase. The total radiation dose ranged from 64.8 Gy to 76.5 Gy (mean 71.1). Patients were evaluated in follow-up for local control and late toxicity. Multivariate analysis of treatment and patient parameters was performed to evaluate their influence on toxicity, local control, and overall survival. Results: With a mean follow-up of 37 months, the actuarial overall survival rate for the entire group at 5 years was 24% and the local control rate was 57%. The tumor volume was the most significant predictor of local control, such that each 1-cm 3 increase in volume was associated with a 1% decrease in local control. For patients with tumor volumes ≤30 cm 3 vs. >30 cm 3 , the 5-year disease-specific survival rate was 52% and 27% (p = 0.004) and locoregional control rate was 76% and 26% (p<0.001), respectively. Seventy-six patients with a minimum of 12 months and median of 39 months toxicity follow-up were studied for late effects. None of these patients experienced Grade 4 or 5 toxicity. The actuarial rate of significant toxicity (Grade III or greater) was 32% at 5 years. Of the toxicities observed, xerostomia (19%) was the most common. Multivariate analysis revealed N stage and dose as independent predictors of Grade 3 effects. Conclusion: The locoregional control and survival for patients in this institutional experience compare favorably to

  3. Combination of systemic chemotherapy with local stem cell delivered S-TRAIL in resected brain tumors.

    Science.gov (United States)

    Redjal, Navid; Zhu, Yanni; Shah, Khalid

    2015-01-01

    Despite advances in standard therapies, the survival of glioblastoma multiforme (GBM) patients has not improved. Limitations to successful translation of new therapies include poor delivery of systemic therapies and use of simplified preclinical models which fail to reflect the clinical complexity of GBMs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and we have tested its efficacy by on-site delivery via engineered stem cells (SC) in mouse models of GBM that mimic the clinical scenario of tumor aggressiveness and resection. However, about half of tumor lines are resistant to TRAIL and overcoming TRAIL-resistance in GBM by combining therapeutic agents that are currently in clinical trials with SC-TRAIL and understanding the molecular dynamics of these combination therapies are critical to the broad use of TRAIL as a therapeutic agent in clinics. In this study, we screened clinically relevant chemotherapeutic agents for their ability to sensitize resistant GBM cell lines to TRAIL induced apoptosis. We show that low dose cisplatin increases surface receptor expression of death receptor 4/5 post G2 cycle arrest and sensitizes GBM cells to TRAIL induced apoptosis. In vivo, using an intracranial resection model of resistant primary human-derived GBM and real-time optical imaging, we show that a low dose of cisplatin in combination with synthetic extracellular matrix encapsulated SC-TRAIL significantly decreases tumor regrowth and increases survival in mice bearing GBM. This study has the potential to help expedite effective translation of local stem cell-based delivery of TRAIL into the clinical setting to target a broad spectrum of GBMs. © 2014 AlphaMed Press.

  4. Local regulation of haemopoietic stem cell proliferation in mice following irradiation

    International Nuclear Information System (INIS)

    Ali, A.M.; Riches, A.C.; Wright, E.G.

    1989-01-01

    Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment. (author)

  5. Local ATP generation by brain-type creatine kinase (CK-B facilitates cell motility.

    Directory of Open Access Journals (Sweden)

    Jan W P Kuiper

    Full Text Available BACKGROUND: Creatine Kinases (CK catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. CONCLUSION/SIGNIFICANCE: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

  6. NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

    DEFF Research Database (Denmark)

    Iversen, Marie Beck; Jensen, Simon Kok; Hansen, Anne Louise

    2015-01-01

    . The marine sponge derived lipid αGalCer has recently been demonstrated as a specific agonist for activation of human and murine NKT cells. In the present study we investigated the applicability of αGalCer pre-treatment for immune protection against intra-vaginal HSV-2 infection. We found that C57BL/6 WT mice...... that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells...... in vaginal tissue and elevated levels of IFN-γ in the vaginal tissue and in vaginal fluids 24h after αGalCer pre-treatment. Collectively our data demonstrate a protective effect of αGalCer induced activation of NKT cells in the innate immune protection against viral infection....

  7. A Tale of Three Cell Types: Alkaloid Biosynthesis Is Localized to Sieve Elements in Opium Poppy

    Science.gov (United States)

    Bird, David A.; Franceschi, Vincent R.; Facchini, Peter J.

    2003-01-01

    Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3′-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants. PMID:14508000

  8. Localization of glucagon and insulin cells and its variation with respect to physiological events in Eutropis carinata

    Directory of Open Access Journals (Sweden)

    Vidya. R. Chandavar

    2011-06-01

    Full Text Available The aim of the present investigation was to localize glucagon and insulin immunoreactive (IR cells of pancreas during annual seasonal cycle of reproduction and to find out whether they had any effect on the regulation of plasma glucose level in the skink Eutropis carinata. Immunolocalized pancreatic cells revealed significantly different mean numbers in different periods of reproduction. The numbers of glucagon-IR and insulin-IR cells were highest in recrudescent period which was corresponded with low plasma glucose level. Unlike other lizards the arrangement of insulin cells in the central core and glucagon cells at the periphery was absent instead glucagon-IR and insulin-IR cells were paracrine in arrangement. Among the two immunoreactive cells glucagon-IR cells were predominant. Morphological differences between two cell types were observed by electron microscopy after staining with uranyl acetate and lead citrate. Plasma glucose showed cyclic change being highest during reproductive period.

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

    Science.gov (United States)

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

    2017-10-15

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

  10. Influence of the local absorber layer thickness on the performance of ZnO nanorod solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Belaidi, Abdelhak; Dittrich, Thomas; Kieven, David; Tornow, Julian; Schwarzburg, Klaus; Lux-Steiner, Martha [Helmholtz-Zentrum Berlin fuer Materialien und Energie, Berlin (Germany)

    2008-08-15

    The local absorber layer thickness (d{sub local}) of solar cells with extremely thin absorber was changed between 10 nm and 70 nm. As a model system, ZnO nanorod arrays (electron conductor) with fixed internal surface area coated with In{sub 2}S{sub 3} (absorber) and impregnated with CuSCN (transparent hole conductor) were applied. The performance of the small area solar cells depended critically on d{sub local}. The highest short circuit current density was reached for the lowest d{sub local}. In contrast, the highest open circuit voltage was obtained for the highest d{sub local}. A maximum energy conversion efficiency of 3.4% at AM1.5 was achieved. Limiting factors are discussed.(copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  11. Enabling cell-cell communication via nanopore formation: structure, function and localization of the unique cell wall amidase AmiC2 of Nostoc punctiforme.

    Science.gov (United States)

    Büttner, Felix M; Faulhaber, Katharina; Forchhammer, Karl; Maldener, Iris; Stehle, Thilo

    2016-04-01

    To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N-acetylmuramoyl-l-alanine amidase AmiC2 (Npun_F1846; EC 3.5.1.28) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high-resolution crystal structure (1.12 Å) of its catalytic domain (AmiC2-cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2-cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2-cat. An inhibitory α-helix, as found in the Escherichia coli AmiC(E. coli) structure, is absent. Taken together, our data provide insight into the cell-biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell-cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases. The AmiC2-cat structure has been deposited in the Protein Data Bank under accession number 5EMI. © 2016 Federation of European Biochemical Societies.

  12. Activity of ABCG2 Is Regulated by Its Expression and Localization in DHT and Cyclopamine-Treated Breast Cancer Cells.

    Science.gov (United States)

    Chua, Vivian Y L; Larma, Irma; Harvey, Jennet; Thomas, Marc A; Bentel, Jacqueline M

    2016-10-01

    Elevated expression of the efflux transporter, ATP-binding cassette subfamily G isoform 2 (ABCG2) on the plasma membrane of cancer cells contributes to the development of drug resistance and is a key characteristic of cancer stem cells. In this study, gene expression analysis identified that treatment of the MCF-7 and T-47D breast cancer cell lines with the androgen, 5α-dihydrotestosterone (DHT), and the Hedgehog signaling inhibitor, cyclopamine downregulated ABCG2 mRNA levels. In MCF-7 cells, and in Hoechst 33342(lo) /CD44(hi) /CD24(lo) breast cancer stem-like cells isolated from MCF-7 cultures, ABCG2 was accumulated in cell-to-cell junction complexes and in large cytoplasmic aggresome-like vesicles. DHT treatments, which decreased cellular ABCG2 protein levels, led to diminished ABCG2 localization in both cell-to-cell junction complexes and in cytoplasmic vesicles. In contrast, cyclopamine, which did not alter ABCG2 protein levels, induced accumulation of ABCG2 in cytoplasmic vesicles, reducing its localization in cell-to-cell junction complexes. The reduced localization of ABCG2 at the plasma membrane of MCF-7 cells was associated with decreased efflux of the ABCG2 substrate, mitoxantrone, and increased sensitivity of cyclopamine-treated cultures to the cytotoxic effects of mitoxantrone. Together, these findings indicate that DHT and cyclopamine reduce ABCG2 activity in breast cancer cells by distinct mechanisms, providing evidence to advocate the adjunct use of analogous pharmaceutics to increase or prolong the efficacy of breast cancer treatments. J. Cell. Biochem. 117: 2249-2259, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Streptococcus pneumoniae PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance

    NARCIS (Netherlands)

    Peters, Katharina; Schweizer, Inga; Beilharz, Katrin; Stahlmann, Christoph; Veening, Jan-Willem; Hakenbeck, Regine; Denapaite, Dalia

    The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of

  14. Improved radiotherapy for locally advanced Non-Small Cell Lung Carcinoma (NSCLC) patients

    DEFF Research Database (Denmark)

    Ottosson, Wiviann

    to comply with the DIBH technique. For DIBH, the patients are guided to hold their breath almost at their maximum inspiration level during imaging and treatment. This leads to reduction of the breathing motion which decreases the movement of the tumor and OARs. It also expands the lung tissue which...... be reduced by the DIBH method for the lung cancer patients. The overall aim of the clinical part of this thesis was to clarify the potential benefit of offering DIBH gating, compared to free-breathing (FB), for lung cancer patients. Particularly, the benefits for locally advanced non-small cell lung cancer...... (NSCLC) patients were explored. For the dosimetric part of the thesis, the dosimetric aspects of correct dose calculations in heterogeneous patient-like geometries were studied. The clinical aspects of DIBH were evaluated in three different studies, where planning and setup verification images acquired...

  15. Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells

    International Nuclear Information System (INIS)

    Berry, Andrea M.; Flock, Kelly E.; Loh, Horace H.; Ko, Jane L.

    2006-01-01

    Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis

  16. Rapid and coordinated processing of global motion images by local clusters of retinal ganglion cells.

    Science.gov (United States)

    Matsumoto, Akihiro; Tachibana, Masao

    2017-01-01

    Even when the body is stationary, the whole retinal image is always in motion by fixational eye movements and saccades that move the eye between fixation points. Accumulating evidence indicates that the brain is equipped with specific mechanisms for compensating for the global motion induced by these eye movements. However, it is not yet fully understood how the retina processes global motion images during eye movements. Here we show that global motion images evoke novel coordinated firing in retinal ganglion cells (GCs). We simultaneously recorded the firing of GCs in the goldfish isolated retina using a multi-electrode array, and classified each GC based on the temporal profile of its receptive field (RF). A moving target that accompanied the global motion (simulating a saccade following a period of fixational eye movements) modulated the RF properties and evoked synchronized and correlated firing among local clusters of the specific GCs. Our findings provide a novel concept for retinal information processing during eye movements.

  17. Inhibition of DNA chain elongation in Chinese hamster cells by damage localized behind the replication fork

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Hur, E [Israel Atomic Energy Commission, Beersheba. Nuclear Research Center-Negev; Hagan, M P [Armed Forces Radiobiology Research Inst., Bethesda, MD (USA)

    1984-05-01

    Chinese hamster fibroblasts were pulse labelled with 5-bromodeoxyuridine and exposed at time intervals (Tsub(i)) to near-ultraviolet (U.V.A.) light in the presence of a bisbenzimidazole derivative (Hoechst 33342). The sensitivity of the cells in terms of colony forming ability fluctuated depending on Tsub(i). Inhibition of DNA synthesis also depended on Tsub(i) and was maximal when Tsub(i)=O. Using the alkaline elution technique it was shown that the effect of a large dose of light was to inhibit both initiation and elongation of DNA chains. These effects were most pronounced for Tsub(i)=O. It is concluded that DNA damage in an active replicon can inhibit initiation of new replicons and that damage localized behind the replication fork can retard elongation of nascent DNA chains. This effect on chain elongation decreases with increased distance of the damage from the replication fork.

  18. Estudo quantitativo das células de Langerhans em carcinomas basocelulares com maior e menor potencial de agressividade local Quantitative study of Langerhans cells in basal cell carcinoma with higher or lower potential of local aggressiveness

    Directory of Open Access Journals (Sweden)

    Itamar Santos

    2010-04-01

    Full Text Available FUNDAMENTOS - O carcinoma basocelular localiza-se principalmente em áreas expostas ao sol, apresentando formas clínicas e histológicas diferentes, algumas com grande e outras com pequena agressividade local. Células de Langerhans participam ativamente do sistema imune da pele. OBJETIVO - Avaliar quantitativamente as células de Langerhans sobrepostas aos carcinomas basocelulares de maior e menor potencial de agressividade local, assim como nas respectivas epidermes sãs adjacentes. MÉTODOS - Dois grupos com 14 preparações histológicas cada. No primeiro, carcinoma basocelular de menor potencial de agressividade local e, no segundo, carcinoma basocelular de maior potencial. Empregou-se a imunoistoquímica com proteína S100 para identificação das células de Langerhans. Utilizando microscópio óptico em aumento de 400 vezes e a grade morfométrica de Weibel, foram contadas as células de Langerhans presentes em sete campos, obtendo-se a média em cada lâmina. Foi utilizado teste estatístico de Wilcoxon para análise estatística. RESULTADOS - No grupo de menor potencial de agressividade local, na epiderme sã adjacente houve aumento significativo no número de células de Langerhans comparado ao da epiderme sobreposta ao carcinoma basocelular (p d 0,05. No grupo de maior potencial de agressividade local, não houve diferença com significado estatístico (p > 0,05. CONCLUSÃO - O maior número de células de Langerhans na epiderme sã vizinha à lesão tumoral de menor potencial de agressividade local poderia representar uma maior resistência imunológica da epiderme, limitando a agressividade da neoplasia.BACKGROUNDS - Basal cell carcinoma affects areas of the body that have been exposed to the sun, and this disorder has different clinical and histopathologic presentations. Some of these forms have a higher potential of local aggressiveness, while others have a lower potential. Langerhans cells actively participate in the skin

  19. CEP55 overexpression predicts poor prognosis in patients with locally advanced esophageal squamous cell carcinoma.

    Science.gov (United States)

    Jiang, Wenpeng; Wang, Zhou; Jia, Yang

    2017-01-01

    Development of esophageal squamous cell carcinoma (ESCC) involves alterations in multiple genes with corresponding proteins. Recent studies have demonstrated that centrosomal protein 55 (CEP55) shares certain features with oncogenes, and CEP55 overexpression is associated with the development and progression of malignant tumors. The present study aimed to analyze, for the first time, whether CEP55 expression is related to clinicopothalogic features in the esophageal squamous cell carcinoma (ESCC), as well as patient survival. A total of 110 patients with mid-thoracic ESCC who suffered from Ivor-Lewis were enrolled. The CEP55 expression profile of these patients in tumour tissues and corresponding healthy esophageal mucosa (CHEM) was detected by immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction analyses. Correlations between CEP55 expression and clinicopathological factors were analyzed using χ 2 test. The log-rank test was employed to calculate survival rate. A Cox regression multivariate analysis was performed to determine independent prognostic factors. The results demonstrated that CEP55 expression in ESCC was significantly higher than that of CHEM (POverexpression of CEP55 was significantly associated with differentiation degree (P=0.022), T stage (P=0.019), lymph node metastasis (P=0.033), clinicopathological staging (P=0.002) and tumor recurrence (P=0.021) in locally advanced ESCC patients. In addition, CEP55 overexpression was significantly associated with reduced overall survival of patients after surgery (P=0.012). The 5-year survival rate of patients without CEP55 overexpression was significantly higher than that of patients with CEP55 overexpression (P=0.012). Therefore, these findings suggest that CEP55 overexpression correlates with poor prognosis in locally advanced ESCC patients.

  20. Laparoscopic resection of tumor recurrence after radical nephrectomy for localized renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Lessandro Curcio

    2014-06-01

    Full Text Available Introduction Local recurrence of Renal Cell Carcinoma (RCC after radical nephrectomy is a rare event. Some known risk factors are: clinical/pathological stage, locorregional disease and lyimph node positivity. Since up to 30-40% of patients can achieve a disease-free status, we show a case (video in which we performed a laparoscopic excision of a local RCC, taking advantage of all the well-known benefits of laparoscopy.Case report A 56 years old female with a history of open radical nephrectomy two years before was diagnosed with a mass at the time of surveillance CT imaging during follow-up. The suspected local recurrence was 12cm, and vascularized predominantly by tributaries originating from the iliac vessels. There was no other site of disease (i.e. brain, lung, liver, bones and laboratory tests were normal. Laparoscopic approach was approached, by inserting 4 trocars (2 of 10 and 2 of 5mm with the patient in the lateral position.Result The procedure lasted 130 minutes, with 220mL of estimated bleeding; the larger vessels were ligated with polymer clips (Hem-o-lok and the smaller handled by ultrasonic clamp. The specimen was removed by a small incision below the umbilicus in an appropriate bag. The patient was feed in the first postoperative day and discharged on the third day. Histopathology revealed sarcoma, with a high degree of mitosis, and negative surgical margins. She was referred to medical oncology for adjuvant therapy consideration.Conclusion The laparoscopic resection of recurrent tumor should be encouraged in highly selected cases. The minimally invasive method, with its known advantages, especially for more debilitated patients, can be advantageous when applied to suitable cases.

  1. Laparoscopic resection of tumor recurrence after radical nephrectomy for localized renal cell carcinoma.

    Science.gov (United States)

    Curcio, Lessandro; Cunha, Antonio Claudio; Renteria, Juan; Presto, Daniel

    2014-01-01

    Local recurrence of Renal Cell Carcinoma (RCC) after radical nephrectomy is a rare event. Some known risk factors are: clinical/pathological stage, locorregional disease and lyimph node positivity. Since up to 30-40% of patients can achieve a disease-free status, we show a case (video) in which we performed a laparoscopic excision of a local RCC, taking advantage of all the well-known benefits of laparoscopy. A 56 years old female with a history of open radical nephrectomy two years before was diagnosed with a mass at the time of surveillance CT imaging during follow-up. The suspected local recurrence was 12 cm, and vascularized predominantly by tributaries originating from the iliac vessels. There was no other site of disease (i.e. brain, lung, liver, bones) and laboratory tests were normal. Laparoscopic approach was approached, by inserting 4 trocars (2 of 10 and 2 of 5mm) with the patient in the lateral position. The procedure lasted 130 minutes, with 220 mL of estimated bleeding; the larger vessels were ligated with polymer clips (Hem-o-lok) and the smaller handled by ultrasonic clamp. The specimen was removed by a small incision below the umbilicus in an appropriate bag. The patient was feed in the first postoperative day and discharged on the third day. Histopathology revealed sarcoma, with a high degree of mitosis, and negative surgical margins. She was referred to medical oncology for adjuvant therapy consideration. The laparoscopic resection of recurrent tumor should be encouraged in highly selected cases. The minimally invasive method, with its known advantages, especially for more debilitated patients, can be advantageous when applied to suitable cases.

  2. Local photodynamic therapy delays recurrence of equine periocular squamous cell carcinoma compared to cryotherapy.

    Science.gov (United States)

    Giuliano, Elizabeth A; Johnson, Philip J; Delgado, Cherlene; Pearce, Jacqueline W; Moore, Cecil P

    2014-07-01

    (i) To report the successful treatment of 10 cases of equine periocular squamous cell carcinoma (PSCC) with surgical excision and photodynamic therapy (PDT) using verteporfin. (ii) To evaluate time to first tumor recurrence between PDT-treated horses and horses treated with surgical excision and cryotherapy. A total of 24 equine PSCC cases were included: group 1 (n = 14) had excision and cryotherapy (1993–2003), group 2 (n = 10), excision and local PDT (2006–2010). Evaluated data: signalment, treatment method, tumor location, size, and time to first recurrence. Groups were compared via chi-square test for categorical variables and Wilcoxon rank-sum test for numeric variables. Time to tumor recurrence was examined using Kaplan–Meier product-limit survival analysis. Of 24 cases, nine breeds were affected. Mean age at treatment in years: 14 (range 5–24) in group 1; 11 (range 8–18) in group 2. Median tumor size: 163 mm2 (range 20–625 mm2) in group 1; 195 mm2 (range 45–775 mm2) in group 2. Signalment, tumor laterality, and size were not significantly different between groups. Time to recurrence was significantly different between groups (Logrank test, P = 0.0006). In group 1, 11/14 horses had tumor regrowth with median time to recurrence in months: 10 (range 1–44). In group 2 (minimum follow-up of 25 months; range 25–50), no horse demonstrated tumor recurrence after one treatment with excision and PDT. This represents the first report of local PDT using verteporfin for treatment of equine PSCC. Following surgery, the likelihood of tumor recurrence was significantly reduced with local PDT compared with cryotherapy. © 2013 American College of Veterinary Ophthalmologists.

  3. Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

    Science.gov (United States)

    Thayer, S S; Huffaker, R C

    1984-05-01

    The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

  4. COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

    Science.gov (United States)

    Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

    2001-05-01

    To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

  5. Delayed postoperative radiation therapy in local control of squamous cell carcinoma of the tongue and floor of the mouth

    Energy Technology Data Exchange (ETDEWEB)

    Amar, Ali; Chedid, Helma Maria; Curioni, Otavio Alberto; Rapoport, Abrao, E-mail: arapoport@uol.com.br [Universidade de Sao Paulo (USP), Sao Paulo, SP (Brazil); Dedivitis, Rogerio Aparecido; Cernea, Claudio Roberto; Brandao, Lenine Garcia [Hospital Heliopolis, Sao aulo, SP (Brazil)

    2014-10-15

    Objective: to evaluate the effect of time between surgery and postoperative radiation therapy on local recurrence of squamous cell carcinoma of the tongue and floor of the mouth. Methods: a total of 154 patients treated between 1996 and 2007 were selected considering local recurrence rate and time of the adjuvant radiotherapy. Results: local recurrence was diagnosed in 54 (35%) patients. Radiation therapy reduced the rate of local recurrences, although with no statistical significance. The time between surgery and initiation of postoperative radiotherapy did not significantly influence the risk of local recurrence in patients referred to adjuvant treatment (p=0.49). Conclusion: in the presence of risk factors for local recurrence, a short delay in starting the adjuvant radiation therapy does not contraindicate its performance. (author)

  6. Menstrual Cycle Dependent Variability for Serum Tumor Markers CEA, AFP, CA 19-9, CA 125 and CA 15-3 in Healthy Women

    Directory of Open Access Journals (Sweden)

    Ayşe Binnur Erbağci

    1999-01-01

    Full Text Available Information on menstrual cycle dependent variation of tumor markers in healthy women is a subject of diagnostic efficiency and has an impact in elucidating the normal function of these markers. In this study midfollicular and midluteal concentrations of serum CEA, AFP, CA 19-9, CA 125, CA 15-3 and their relations with LH, FSH, prolactin, estradiol and progesterone were evaluated during ovulatory cycles in a group of 23 healthy female individuals. Samples were collected on the 7th and 21st day of the same menstrual cycle. Tumor marker and hormone concentrations were determined with chemiluminescence or electrochemiluminescence EIA methods. A significant phase-dependent difference was observed for CA 15-3, midluteal concentrations (mean ± SEM; 26.33 ± 1.56 U/ml higher than the midfollicular (mean ± SEM; 19.27 ± 1.49 U/ml concentrations (p < 0.001. But an obvious difference for other tumor markers investigated did not exist. Significant correlations of follicular and luteal CA 125 levels with body mass index of the subjects were observed (r:0.52, p < 0.05 and r:0.57, p < 0.005, respectively.

  7. Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

    Science.gov (United States)

    Llewellyn, G Nicholas; Hogue, Ian B; Grover, Jonathan R; Ono, Akira

    2010-10-28

    T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.

  8. Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

    Directory of Open Access Journals (Sweden)

    G Nicholas Llewellyn

    2010-10-01

    Full Text Available T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS, are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.

  9. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. (Harvard Medical School, Boston, MA (USA) Karolinska Institute, Stockholm (Sweden))

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  10. Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

    Science.gov (United States)

    Fuentes, Federico

    2009-01-01

    PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity. PMID:19158394

  11. Ultrastructural autoradiographic localization of exogenous arachidonic acid in cultured endothelial and smooth muscle cells

    International Nuclear Information System (INIS)

    Tasca, S.I.; Galis, Z.

    1988-01-01

    The uptake and intracellular localization of exogenous arachidonic acid (AA) were investigated in cultured endothelial (EC) and smooth muscle cells (SMC) isolated from bovine aorta. The [ 14 C]AA uptake was assessed biochemically and by light and electron microscopic autoradiography. The highest values of silver grain surface density were associated with the mitochondria, lysosomes, and the Golgi apparatus of the EC. The grain linear density was greater on the nuclear envelope than on plasmalemma. On SMC, the grain density was highest on lipid droplets whereas the linear densities of the nuclear envelope and plasmalemma were similar. The share of each subcellular compartment in the AA distribution was estimated as the percentage of the individual silver grain count out of the total cell-associated radioactivity. The results showed that cytoplasm (including endoplasmic reticulum, ribosomes, and small vesicles) made the main contribution followed by the nucleus and at lower values by other organelles. These subcompartments may represent the intracellular sites from which AA could be mobilized for prostanoid synthesis by EC and SMC. (author)

  12. Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

    International Nuclear Information System (INIS)

    Clarke, J.T.; Cook, H.W.; Spence, M.W.

    1985-01-01

    To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-[1- 3 H]galactose or [ 3 H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [ 3 H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid

  13. The influence of local electric fields on photoinduced absorption in dye-sensitized solar cells.

    Science.gov (United States)

    Cappel, Ute B; Feldt, Sandra M; Schöneboom, Jan; Hagfeldt, Anders; Boschloo, Gerrit

    2010-07-07

    The dye-sensitized solar cell (DSC) challenges conventional photovoltaics with its potential for low-cost production and its flexibility in terms of color and design. Transient absorption spectroscopy is widely used to unravel the working mechanism of DSCs. A surprising, unexplained feature observed in these studies is an apparent bleach of the ground-state absorption of the dye, under conditions where the dye is in the ground state. Here, we demonstrate that this feature can be attributed to a change of the local electric field affecting the absorption spectrum of the dye, an effect related to the Stark effect first reported in 1913. We present a method for measuring the effect of an externally applied electric field on the absorption of dye monolayers adsorbed on flat TiO(2) substrates. The measured signal has the shape of the first derivative of the absorption spectra of the dyes and reverses sign along with the reversion of the direction of the change in dipole moment upon excitation relative to the TiO(2) surface. A very similar signal is observed in photoinduced absorption spectra of dye-sensitized TiO(2) electrodes under solar cell conditions, demonstrating that the electric field across the dye molecules changes upon illumination. This result has important implications for the analysis of transient absorption spectra of DSCs and other molecular optoelectronic devices and challenges the interpretation of many previously published results.

  14. Spontaneous, local diastolic subsarcolemmal calcium releases in single, isolated guinea-pig sinoatrial nodal cells.

    Science.gov (United States)

    Sirenko, Syevda G; Yang, Dongmei; Maltseva, Larissa A; Kim, Mary S; Lakatta, Edward G; Maltsev, Victor A

    2017-01-01

    Uptake and release calcium from the sarcoplasmic reticulum (SR) (dubbed "calcium clock"), in the form of spontaneous, rhythmic, local diastolic calcium releases (LCRs), together with voltage-sensitive ion channels (membrane clock) form a coupled system that regulates the action potential (AP) firing rate. LCRs activate Sodium/Calcium exchanger (NCX) that accelerates diastolic depolarization and thus participating in regulation of the time at which the next AP will occur. Previous studies in rabbit SA node cells (SANC) demonstrated that the basal AP cycle length (APCL) is tightly coupled to the basal LCR period (time from the prior AP-induced Ca2+ transient to the diastolic LCR occurrence), and that this coupling is further modulated by autonomic receptor stimulation. Although spontaneous LCRs during diastolic depolarization have been reported in SANC of various species (rabbit, cat, mouse, toad), prior studies have failed to detect LCRs in spontaneously beating SANC of guinea-pig, a species that has been traditionally used in studies of cardiac pacemaker cell function. We performed a detailed investigation of whether guinea-pig SANC generate LCRs and whether they play a similar key role in regulation of the AP firing rate. We used two different approaches, 2D high-speed camera and classical line-scan confocal imaging. Positioning the scan-line beneath sarcolemma, parallel to the long axis of the cell, we found that rhythmically beating guinea-pig SANC do, indeed, generate spontaneous, diastolic LCRs beneath the surface membrane. The average key LCR characteristics measured in confocal images in guinea-pig SANC were comparable to rabbit SANC, both in the basal state and in the presence of β-adrenergic receptor stimulation. Moreover, the relationship between the LCR period and APCL was subtended by the same linear function. Thus, LCRs in guinea-pig SANC contribute to the diastolic depolarization and APCL regulation. Our findings indicate that coupled-clock system

  15. Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements.

    Science.gov (United States)

    Basketter, David; Kolle, Susanne N; Schrage, Arnhild; Honarvar, Naveed; Gamer, Armin O; van Ravenzwaay, Bennard; Landsiedel, Robert

    2012-08-01

    The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach. Copyright © 2011 John Wiley & Sons, Ltd.

  16. Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

    Directory of Open Access Journals (Sweden)

    Cory H.T. Boone

    2017-08-01

    glutathione to combat the oxidative cellular environment, glycolytic to PPP cycling of carbon generating NADPH, obstruction of carbon flow through the TCA cycle, decreased ATP generation, and metacaspase dependent apoptotic cell death. Keywords: Local anesthetic toxicity, Oxidative stress, Metabolomics profiling, Apoptotic cell death pathways, Flow cytometry, Mass spectrometry

  17. A Spatial Control for Correct Timing of Gene Expression during the Escherichia coli Cell Cycle

    Directory of Open Access Journals (Sweden)

    Yuan Yao

    2016-12-01

    Full Text Available Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function of cell cycle. We find that the TorR protein localizes to the old poles of the Escherichia coli cells, forming a functional focus. The TorR focus co-localizes with the nucleoid in a cell-cycle-dependent manner, and consequently regulates transcription of a number of genes. Formation of one TorR focus at the old poles of cells requires interaction with the MreB and DnaK proteins, and ATP, suggesting that TorR delivery requires cytoskeleton organization and ATP. Further, absence of the protein–protein interactions and ATP leads to loss in function of TorR as a transcription factor. We propose a mechanism for timing of cell-cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of the cell cycle by limiting its own spatial distribution.

  18. Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

    Science.gov (United States)

    Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

    2013-01-01

    Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

  19. Marked differences in immunocytological localization of [3H]estradiol-binding protein in rat pancreatic acinar tumor cells compared to normal acinar cells

    International Nuclear Information System (INIS)

    Beaudoin, A.R.; Grondin, G.; St Jean, P.; Pettengill, O.; Longnecker, D.S.; Grossman, A.

    1991-01-01

    [ 3 H]Estradiol can bind to a specific protein in normal rat pancreatic acinar cells. Electron microscopic immunocytochemical analysis has shown this protein to be localized primarily in the rough endoplasmic reticulum and mitochondria. Rat exocrine pancreatic tumor cell lines, whether grown in tissue culture (AR42J) or as a tumor mass after sc injection into rats (DSL-2), lacked detectable amounts of this [ 3 H]estradiol-binding protein (EBP), as determined by the dextran-coated charcoal assay. Furthermore, primary exocrine pancreatic neoplasms induced with the carcinogen azaserine contained little or no detectable [ 3 H]estradiol-binding activity. However, electron immunocytochemical studies of transformed cells indicated the presence of material that cross-reacted with antibodies prepared against the [ 3 H]EBP. The immunopositive reaction in transformed cells was localized almost exclusively in lipid granules. Such lipid organelles in normal acinar cells, although present less frequently than in transformed cells, have never been observed to contain EBP-like immunopositive material. Presumably, the aberrant localization of EBP in these acinar tumor cells results in loss of function of this protein, which in normal pancreatic acinar cells appears to exert a modulating influence on zymogen granule formation and the process of secretion

  20. Two-way regulation between cells and aligned collagen fibrils: local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells.

    Science.gov (United States)

    Li, Wen; Zhu, Bofan; Strakova, Zuzana; Wang, Rong

    2014-08-08

    It has been well established that an aligned matrix provides structural and signaling cues to guide cell polarization and cell fate decision. However, the modulation role of cells in matrix remodeling and the feedforward effect on stem cell differentiation have not been studied extensively. In this study, we report on the concerted changes of human decidua parietalis placental stem cells (hdpPSCs) and the highly ordered collagen fibril matrix in response to cell-matrix interaction. With high-resolution imaging, we found the hdpPSCs interacted with the matrix by deforming the cell shape, harvesting the nearby collagen fibrils, and reorganizing the fibrils around the cell body to transform a 2D matrix to a localized 3D matrix. Such a unique 3D matrix prompted high expression of β-1 integrin around the cell body that mediates and facilitates the stem cell differentiation toward neural cells. The study offers insights into the coordinated, dynamic changes at the cell-matrix interface and elucidates cell modulation of its matrix to establish structural and biochemical cues for effective cell growth and differentiation. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues.

    Science.gov (United States)

    Amoyel, Marc; Hillion, Kenzo-Hugo; Margolis, Shally R; Bach, Erika A

    2016-11-01

    Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on loss of access to these signals. Here, we demonstrate that the differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot differentiate properly. We find that an insulin peptide produced by somatic cells immediately outside of the stem cell niche acts locally to promote somatic differentiation through Insulin-like receptor (InR) activation. These results indicate that there is a local 'differentiation' niche that upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, we demonstrate that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, we show that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that the local production of positive and negative InR signals regulates the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation are actively induced by signaling. © 2016. Published by The Company of Biologists Ltd.

  2. The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death.

    Science.gov (United States)

    Yu, Xiaoli; Tang, Junli; Wang, Qunqing; Ye, Wenwu; Tao, Kai; Duan, Shuyi; Lu, Chenchen; Yang, Xinyu; Dong, Suomeng; Zheng, Xiaobo; Wang, Yuanchao

    2012-10-01

    • The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  3. Computer algorithms for automated detection and analysis of local Ca2+ releases in spontaneously beating cardiac pacemaker cells.

    Directory of Open Access Journals (Sweden)

    Alexander V Maltsev

    Full Text Available Local Ca2+ Releases (LCRs are crucial events involved in cardiac pacemaker cell function. However, specific algorithms for automatic LCR detection and analysis have not been developed in live, spontaneously beating pacemaker cells. In the present study we measured LCRs using a high-speed 2D-camera in spontaneously contracting sinoatrial (SA node cells isolated from rabbit and guinea pig and developed a new algorithm capable of detecting and analyzing the LCRs spatially in two-dimensions, and in time. Our algorithm tracks points along the midline of the contracting cell. It uses these points as a coordinate system for affine transform, producing a transformed image series where the cell does not contract. Action potential-induced Ca2+ transients and LCRs were thereafter isolated from recording noise by applying a series of spatial filters. The LCR birth and death events were detected by a differential (frame-to-frame sensitivity algorithm applied to each pixel (cell location. An LCR was detected when its signal changes sufficiently quickly within a sufficiently large area. The LCR is considered to have died when its amplitude decays substantially, or when it merges into the rising whole cell Ca2+ transient. Ultimately, our algorithm provides major LCR parameters such as period, signal mass, duration, and propagation path area. As the LCRs propagate within live cells, the algorithm identifies splitting and merging behaviors, indicating the importance of locally propagating Ca2+-induced-Ca2+-release for the fate of LCRs and for generating a powerful ensemble Ca2+ signal. Thus, our new computer algorithms eliminate motion artifacts and detect 2D local spatiotemporal events from recording noise and global signals. While the algorithms were developed to detect LCRs in sinoatrial nodal cells, they have the potential to be used in other applications in biophysics and cell physiology, for example, to detect Ca2+ wavelets (abortive waves, sparks and

  4. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    Energy Technology Data Exchange (ETDEWEB)

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-07-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

  5. Factors predicting radiation pneumonitis in locally advanced non-small cell lung cancer

    International Nuclear Information System (INIS)

    Kim, Myung Soo; Lee, Ji Hae; Ha, Bo Ram; Lee, Re Na

    2011-01-01

    Thoracic radiotherapy is a major treatment modality of stage III non-small cell lung cancer. The normal lung tissue is sensitive to radiation and radiation pneumonitis is the most important dose-limiting complication of thoracic radiation therapy. This study was performed to identify the clinical and dosimetric parameters related to the risk of radiation pneumonitis after definitive radiotherapy in stage III non-small cell cancer patients. The medical records were reviewed for 49 patients who completed definitive radiation therapy for locally advanced non-small cell lung cancer from August 2000 to February 2010. Radiation therapy was delivered with the daily dose of 1.8 Gy to 2.0 Gy and the total radiation dose ranged from 50.0 Gy to 70.2 Gy (median, 61.2 Gy). Elective nodal irradiation was delivered at a dose of 45.0 Gy to 50.0 Gy. Seven patients (14.3%) were treated with radiation therapy alone and forty two patients (85.7%) were treated with chemotherapy either sequentially or concurrently. Twenty-five cases (51.0%) out of 49 cases experienced radiation pneumonitis. According to the radiation pneumonitis grade, 10 (20.4%) were grade 1, 9 (18.4%) were grade 2, 4 (8.2%) were grade 3, and 2 (4.1%) were grade 4. In the univariate analyses, no clinical factors including age, sex, performance status, smoking history, underlying lung disease, tumor location, total radiation dose and chemotherapy were associated with grade ≥2 radiation pneumonitis. In the subgroup analysis of the chemotherapy group, concurrent rather than sequential chemotherapy was significantly related to grade ≥2 radiation pneumonitis comparing sequential chemotherapy. In the univariate analysis with dosimetric factors, mean lung dose (MLD), V20, V30, V40, MLDipsi, V20ipsi, V30ipsi, and V40ipsi were associated with grade ≥2 radiation pneumonitis. In addition, multivariate analysis showed that MLD and V30 were independent predicting factors for grade ≥2 radiation pneumonitis. Concurrent

  6. Renal angiographic and computed tomographic evaluation of local extension of renal cell carcinoma

    International Nuclear Information System (INIS)

    Masuda, Fujio; Onishi, Tetsuro; Sasaki, Tadamasa; Arai, Yoshikazu; Shoji, Ryo

    1981-01-01

    In 23 cases of renal cell carcinoma, the degree of local invasion of carcinoma was diagnosed using renal angiography and CT, and compared with the findings obtained by operation or autopsy. Among 5 cases in which the tumor was confined to the renal capsule, accurate diagnosis could be established with renal angiography in 4 cases and with CT in all of 5 cases. Both renal angiography and CT provided correct diagnosis in 7 of 8 cases in which the tumor showed infiltration extending to the perinephric fat. Out of 5 cases with tumor invasion of renal vein or inferior vena cava, diagnosis could be established correctly by renal angiography and CT in 3 cases. Among the remaining 2 cases the diagnosis could be established by renal angiography and CT in one each case. Among 5 cases with metastases to the regional lymph nodes, diagnosis could be established by renal angiography in only 2 cases, while all of 5 cases could be diagnosed by CT. In 3 cases where the tumor invaded an ajacent organ beyond Gerota's fastia, renal angiography could diagnose in none of the 3 cases while with CT all of 3 cases could successfully be diagnosed. The consistency of degree of local invasion as revealed by renal angiography and CT was seen in 15 of 23 cases (65%) for renal agniography and 20 of 23 cases (87%) for CT, indicating superiority of CT in this respect. In particular, CT appears to be more superior to renal angiography for determining whether the tumor confined to Gerota's fastia or it infiltrated over it. Both combined use of renal angiography and CT, the degree of infiltration of tumor could be diagnosed correctly in 22 of 23 cases (96%). (author)

  7. Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

    Science.gov (United States)

    Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

    2001-10-01

    Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

  8. Propagation of localized structures in relativistic magnetized electron-positron plasmas using particle-in-cell simulations

    Energy Technology Data Exchange (ETDEWEB)

    López, Rodrigo A. [Departamento de Física, Facultad de Ciencias Físicas y Matemáticas, Universidad de Concepción, Concepción 4070386 (Chile); Muñoz, Víctor [Departamento de Física, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago (Chile); Viñas, Adolfo F. [Geospace Physics Laboratory, Heliophysics Science Division, NASA Goddard Space Flight Center, Greenbelt, Maryland 20771 (United States); Valdivia, Juan A. [Departamento de Física, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago (Chile); Centro para el Desarrollo de la Nanociencia y la Nanotecnología (CEDENNA), Santiago 9170124 (Chile)

    2015-09-15

    We use a particle-in-cell simulation to study the propagation of localized structures in a magnetized electron-positron plasma with relativistic finite temperature. We use as initial condition for the simulation an envelope soliton solution of the nonlinear Schrödinger equation, derived from the relativistic two fluid equations in the strongly magnetized limit. This envelope soliton turns out not to be a stable solution for the simulation and splits in two localized structures propagating in opposite directions. However, these two localized structures exhibit a soliton-like behavior, as they keep their profile after they collide with each other due to the periodic boundary conditions. We also observe the formation of localized structures in the evolution of a spatially uniform circularly polarized Alfvén wave. In both cases, the localized structures propagate with an amplitude independent velocity.

  9. Propagation of localized structures in relativistic magnetized electron-positron plasmas using particle-in-cell simulations

    International Nuclear Information System (INIS)

    López, Rodrigo A.; Muñoz, Víctor; Viñas, Adolfo F.; Valdivia, Juan A.

    2015-01-01

    We use a particle-in-cell simulation to study the propagation of localized structures in a magnetized electron-positron plasma with relativistic finite temperature. We use as initial condition for the simulation an envelope soliton solution of the nonlinear Schrödinger equation, derived from the relativistic two fluid equations in the strongly magnetized limit. This envelope soliton turns out not to be a stable solution for the simulation and splits in two localized structures propagating in opposite directions. However, these two localized structures exhibit a soliton-like behavior, as they keep their profile after they collide with each other due to the periodic boundary conditions. We also observe the formation of localized structures in the evolution of a spatially uniform circularly polarized Alfvén wave. In both cases, the localized structures propagate with an amplitude independent velocity

  10. Localized squamous-cell cancer of the esophagus: retrospective analysis of three treatment schedules

    International Nuclear Information System (INIS)

    Delcambre, Corinne; Jacob, Jacques Henry; Pottier, Didier; Gignoux, Marc; Ollivier, Jean Michel; Vie, Brigitte; Roussel, Alain; Segol, Philippe

    2001-01-01

    Background and purpose: A retrospective study comparing chemotherapy and radiation, esophagectomy alone versus preoperative radiochemotherapy and surgery in localized squamous-cell esophageal carcinoma. Materials and methods: Between 1989 and 1995, 139 patients (40 stage I, 77 stage IIA and 22 stage IIB according to the UICC 78 TNM classification) were treated in two different institutions. They were divided into three groups according to the treatment proposed: E group (treatment by esophagectomy; n=30), RCT+E group (treatment by preoperative radiochemotherapy and esophagectomy; n=46), RCT group (treatment by radiochemotherapy; n=63). Factors like age, tumor localization and stage were similar in all groups. An intention to treat analysis was made. Results: The E group showed no postoperative mortality, while in the RCT+E group, the surgery mortality was 12.8%. The mortality after RCT was 1.7%. After preoperative radiochemotherapy, a pathological complete response was observed in 25% of cases and the curative resection rate was higher (82% after RCT+E versus 60% after E). The 5-year survival difference between the three groups was not relevant (E group, 12.6%; RCT group, 25.8%; RCT+E group, 38.7%). The median survival was 29, 24 and 28.5 months, respectively. The event-free survival was identical for the E group and the RCT group. For patients treated by radiochemotherapy, local and/or distant relapses were significantly reduced by esophagectomy (relapses occurred in 51% of patients in the RCT+E group versus 75% in the RCT group, P=0.017). Palliative care (dilatations, prosthesis, gastrostomy or jejunostomy) to improve dysphagia was necessary for 38% of patients treated by exclusive radiochemotherapy versus 11% of patients treated by surgery (P=0.001). Conclusions: Treatments by esophagectomy or radiochemotherapy were not significantly different. Preoperative radiochemotherapy and surgery lead to a higher survival rate than exclusive radiochemotherapy, however

  11. Gemcitabine, cisplatin, and hyperfractionated accelerated radiotherapy for locally advanced non-small cell lung cancer.

    Science.gov (United States)

    Zwitter, Matjaz; Kovac, Viljem; Smrdel, Uros; Strojan, Primoz

    2006-09-01

    Due to potent radiosensitization and potential serious or fatal toxicity, concurrent gemcitabine and irradiation should only be applied within clinical trials. We here present experience from a phase I-II clinical trial for patients with locally advanced non-small cell lung cancer (NSCLC) treated with hyperfractionated accelerated radiotherapy and concurrent low-dose gemcitabine. Eligible patients had locally advanced inoperable NSCLC without pleural effusion, Eastern Cooperative Oncology Group performance status 0-1, were chemotherapy naïve and had no previous radiotherapy to the chest, and had adequate hematopoietic, liver, and kidney function. Routine brain computed tomography was not performed, and positron emission tomography/computed tomography was not available. Treatment consisted of three parts: induction chemotherapy with gemcitabine and cisplatin in standard doses, local treatment with concurrent chemotherapy and radiotherapy, and consolidation chemotherapy. Patients were irradiated with opposed AP-PA and oblique fields, using 2.5-D treatment planning. Although corrections for inhomogeneous tissue were made, volume of total lung receiving > or =20 Gy (V20) could not be determined. The trial started as phase I, aimed to determine the dose-limiting toxicity and maximal tolerated dose (MTD) for concurrent hyperfractionated radiotherapy (1.4 Gy twice daily) and gemcitabine 55 mg/m twice weekly as a radiosensitizer. Phase II of the trial then continued at the level of MTD. Twenty-eight patients with NSCLC, nine patients with stage IIIA, 16 patients with IIIB, and three patients with an inoperable recurrence after previous surgery, entered the trial. The first 12 patients entered Phase I of the trial at the initial level of 42 Gy in 30 fractions in 3 weeks. Dose-limiting toxicity was acute esophagitis; 47.6 Gy in 34 fractions in 3.5 weeks was the MTD for this regimen of concurrent chemotherapy and radiotherapy. In phase II of the trial, this dose was applied

  12. Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T Cell Mediated Tumor Control in the Genital Tract

    Science.gov (United States)

    Sun, Yun-Yan; Peng, Shiwen; Han, Liping; Qiu, Jin; Song, Liwen; Tsai, Yachea; Yang, Benjamin; Roden, Richard B.S.; Trimble, Cornelia L.; Hung, Chien-Fu; Wu, T-C

    2015-01-01

    Purpose Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Experimental Design Here we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than IM delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16+ cervical cancer (TC-1 luc). Results We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8+ T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8+ T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8+ T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8+ T cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Conclusions Our results support future clinical translation using cervicovaginal TA-HPV vaccination. PMID:26420854

  13. Chronic low-dose UVA irradiation induces local suppression of contact hypersensitivity, Langerhans cell depletion and suppressor cell activation in C3H/HeJ mice

    International Nuclear Information System (INIS)

    Bestak, Rosa; Halliday, G.M.

    1996-01-01

    It has previously been demonstrated that chronic low-dose solar-simulated UV radiation could induce both local and systemic immunosuppression as well as tolerance to a topically applied hapten. In this study, we have used a chronic low-dose UV-irradiation protocol to investigate the effects of UVA on the skin immune system of C3H/HeJ mice. Irradiation with UVA+B significantly suppressed the local and systemic primary contact hypersensitivity (CHS) response to the hapten 2,4,6-trinitrochlorobenzene. Furthermore, UVA+B reduced Langerhans cell (LC) and dendritic epidermal T cell (DETC) densities in chronically UV-irradiated mice. Ultraviolet A irradiation induced local, but not systemic, immunosuppression and reduced LC (32%) but not DETC from the epidermis compared to the shaved control animals. Treatment of mice with both UVA+B and UVA radiation also induced an impaired secondary CHS response, and this tolerance was transferable with spleen cells. (Author)

  14. Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

    Science.gov (United States)

    Yoshida, Saishu; Kato, Takako; Kanno, Naoko; Nishimura, Naoto; Nishihara, Hiroto; Horiguchi, Kotaro; Kato, Yukio

    2017-10-01

    Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

  15. Adjuvant therapy for locally advanced renal cell cancer: A systematic review with meta-analysis

    Directory of Open Access Journals (Sweden)

    Lima Carmen SP

    2011-03-01

    Full Text Available Abstract Background Many adjuvant trials have been undertaken in an attempt to reduce the risk of recurrence among patients who undergo surgical resection for locally advanced renal cancer. However, no clear benefit has been identified to date. This systematic review was conducted to examine the exact role of adjuvant therapy in renal cancer setting. Methods Randomized controlled trials were searched comparing adjuvant therapy (chemotherapy, vaccine, immunotherapy, biochemotherapy versus no active treatment after surgery among renal cell cancer patients. Outcomes were overall survival (OS, disease-free survival (DFS, and severe toxicities. Risk ratios (RR, hazard ratios (HR and 95% confidence intervals were calculated using a fixed-effects meta-analysis. Heterogeneity was measured by I2. Different strategies of adjuvant treatment were evaluated separately. Results Ten studies (2,609 patients were included. Adjuvant therapy provided no benefits in terms of OS (HR 1.07; 95%CI 0.89 to 1.28; P = 0.48 I2 = 0% or DFS (HR 1.03; 95%CI 0.87 to 1.21; P = 0.77 I2 = 15% when compared to no treatment. No subgroup analysis (immunotherapy, vaccines, biochemotherapy and hormone therapy had relevant results. Toxicity evaluation depicted a significantly higher frequency of serious adverse events in the adjuvant group. Conclusions This analysis provided no support for the hypothesis that the agents studied provide any clinical benefit for renal cancer patients although they increase the risk of toxic effects. Randomized trials are underway to test targeted therapies, which might open a new therapeutic frontier. Until these trials yield results, no adjuvant therapy can be recommended for patients who undergo surgical resection for renal cell cancer.

  16. Comparison of in vitro cell binding characteristics of four monoclonal antibodies and their individual tumor localization properties in mice

    International Nuclear Information System (INIS)

    Andrew, S.M.; Johnstone, R.W.; Russell, S.M.; McKenzie, I.F.; Pietersz, G.A.

    1990-01-01

    Although many antibodies are being used for imaging studies, it is not clear which in vitro properties of antibodies will best reflect their in vivo characteristics. The ability to correlate in vitro binding characteristics of monoclonal antibodies to tumor antigens with their in vivo localization characteristics, particularly with respect to tumor localization properties, is desirable for rapid selection of monoclonal antibodies with potential for clinical use. The in vitro binding characteristics of three monoclonal antibodies to the murine Ly-2.1 antigen and one to the Ly-3.1 antigen have been studied on cultured tumor cells bearing these antigens. The association and dissociation rate constants, apparent affinity, and immunoreactivity of each antibody in vitro were compared with their ability to localize the s.c. tumors from the same cell line growing in Ly-2.1-/Ly-3.1-mice. The antibody with the highest affinity and fastest association rate localized to tumor at the earliest time (16-20 h after injection) and had the highest percentage of the injected dose/g in the tumor (greater than 25%). The antibody with the lowest affinity showed significantly less localization to tumor cells, compared with the other three antibodies. The ranking of the antibodies by affinity agreed with the ranking in terms of their ability to localize to tumors, but the in vitro immunoreactivity of the antibodies, as measured by a cell binding assay, did not correlate with their tumor localization properties. Immunoscintigraphic studies did not precisely correlate with biodistribution data or in vitro binding characteristics, because tumors could be satisfactorily imaged with each antibody, although it was noted that the antibody with the highest affinity gave the best image

  17. Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements.

    Science.gov (United States)

    Kolle, Susanne N; Basketter, David; Schrage, Arnhild; Gamer, Armin O; van Ravenzwaay, Bennard; Landsiedel, Robert

    2012-08-01

    In a previous study, the predictive capacity of a modified local lymph node assay (LLNA) based on cell counts, the LNCC, was demonstrated to be closely similar to that of the original assay. In addition, a range of substances, including some technical/commercial materials and a range of agrochemical formulations (n = 180) have also been assessed in both methods in parallel. The results in the LNCC and LLNA were generally consistent, with 86% yielding an identical classification outcome. Discordant results were associated with borderline data and were evenly distributed between the two methods. Potency information derived from each method also demonstrated good consistency (n = 101), with 93% of predictions being close. Skin irritation was observed only infrequently and was most commonly associated with positive results; it was not associated with the discordant results. Where different vehicles were used with the same test material, the effect on sensitizing activity was modest, consistent with historical data. Analysis of positive control data indicated that the LNCC and LLNA displayed similar levels of biological variation. When taken in combination with the previously published results on LLNA Performance Standard chemicals, it is concluded that the LNCC provides a viable non-radioactive alternative to the LLNA for the assessment of substances, including potency predictions, as well as for the evaluation of preparations. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Palliative radiotherapy in asymptomatic patients with locally advanced, unresectable, non-small cell lung cancer

    International Nuclear Information System (INIS)

    Reinfuss, M.; Skolyszewski, J.; Kowalska, T.; Rzepecki, W.; Kociolek, D.

    1993-01-01

    Between 1983 and 1990, 332 patients with non-small cell lung cancer (NSCLC) were referred to short-time, split-course palliative thoracic radiotherapy. The group consisted of patients with locally advanced (III o ), unresectable cancer, not suitable for curative radiotherapy, asymptomatic or having only minimal symptoms related to intrathoracic tumor. The therapeutic plan involved two series of irradiation. Tumor dose delivered in each series was 20 Gy given in five daily fractions over five treatment days. There were four weeks interval between series. Of 332 patients initially qualified to thoracic radiotherapy only 170 patients received the treatment; the other 162 patients were not irradiated because of treatment refusal or logistic problems concerning therapy. They made the control group of the study, receiving the best possible symptomatic care. Twelve-month survivals in the radiotherapy and control groups were 32.4% and 9.3%, respectively; 24-month survivals 11.2% and 0%, respectively. Improvement of survival after palliative thoracic radiotherapy was observed only in patients with clinical stage IIIA and Karnofsky's performance status (KPS) ≥ 70. (orig.) [de

  19. Dye-sensitized solar cells using natural dyes as sensitizers from Malaysia local fruit `Buah Mertajam'

    Science.gov (United States)

    Hambali, N. A. M. Ahmad; Roshidah, N.; Hashim, M. Norhafiz; Mohamad, I. S.; Saad, N. Hidayah; Norizan, M. N.

    2015-05-01

    We experimentally demonstrate the high conversion efficiency, low cost, green technology and easy to fabricate dye-sensitized solar cells (DSSCs) using natural anthocyanin dyes as sensitizers. The DSSCs was fabricated by using natural anthocyanin dyes which were extracted from different parts of the plants inclusive `Buah Mertajam', `Buah Keriang Dot', `Bunga Geti', Hibiscus, Red Spinach and Henna. The natural anthocyanin dyes that found in flower, leaves and fruits were extracted by the simple procedures. This anthocyanin dye is used to replace the expensive chemical synthetic dyes due to its ability to effectively attach into the surface of Titanium dioxide (TiO2). A natural anthocyanin dyes molecule adsorbs to each particle of the TiO2 and acts as the absorber of the visible light. A natural anthocyanin dye from Buah Mertajam shows the best performance with the conversion efficiency of 5.948% and fill factor of 0.708 followed by natural anthocyanin dyes from `Buah Keriang Dot', `Bunga Geti', Hibiscus, Red Spinach and Henna. Buah Mertajam or scientifically known as eriglossum rubiginosum is a local Malaysia fruit.

  20. Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

    Science.gov (United States)

    Uheda, Eiji; Maejima, Kazuhiro

    2009-10-15

    In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly.

  1. Claudin-4 Undergoes Age-Dependent Change in Cellular Localization on Pig Jejunal Villous Epithelial Cells, Independent of Bacterial Colonization

    Directory of Open Access Journals (Sweden)

    J. Alex Pasternak

    2015-01-01

    Full Text Available Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs. In the present study, FcRn gene (FCGRT was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases.

  2. Blistering in ALD Al2O3 passivation layers as rear contacting for local Al BSF Si solar cells

    NARCIS (Netherlands)

    Vermang, B.; Goverde, J.C.; Uruena, A.; Lorenz, A.; Cornagliotti, E.; Rothschild, A.; John, J.; Poortmans, J.; Mertens, R.

    2012-01-01

    Random Al back surface field (BSF) p-type Si solar cells are presented, where a stack of Al2O3 and SiNx is used as rear surface passivation layer containing blisters. It is shown that no additional contact opening step is needed, since during co-firing local Al BSFs are induced at the location of

  3. Concurrent versus Sequential Chemoradiotherapy with Cisplatin and Vinorelbine in Locally Advanced Non-Small Cell Lung Cancer: A Randomized Study

    Czech Academy of Sciences Publication Activity Database

    Zatloukal, P.; Petruželka, L.; Zemanová, M.; Havel, L.; Janků, F.; Judas, L.; Kubík, A.; Křepela, E.; Fiala, P.; Pecen, Ladislav

    2004-01-01

    Roč. 46, - (2004), s. 87-98 ISSN 0169-5002 Institutional research plan: CEZ:AV0Z1030915 Keywords : concurrent chemoradiotherapy * sequential chemoradiotherapy * locally advanced non-small cell lung cancer * cisplatin * vinorelbine Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 2.914, year: 2004

  4. Modulation of Olfactory Bulb Network Activity by Serotonin: Synchronous Inhibition of Mitral Cells Mediated by Spatially Localized GABAergic Microcircuits

    Science.gov (United States)

    Schmidt, Loren J.; Strowbridge, Ben W.

    2014-01-01

    Although inhibition has often been proposed as a central mechanism for coordinating activity in the olfactory system, relatively little is known about how activation of different inhibitory local circuit pathways can generate coincident inhibition of principal cells. We used serotonin (5-HT) as a pharmacological tool to induce spiking in ensembles…

  5. Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Costello, F.; Huhtanen, M.

    1996-01-01

    Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP...

  6. Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors

    Science.gov (United States)

    Zallocchi, Marisa; Meehan, Daniel T.; Delimont, Duane; Askew, Charles; Garrige, Suneetha; Gratton, Michael Anne; Rothermund-Franklin, Christie A.; Cosgrove, Dominic

    2009-01-01

    The Usher syndrome 3A (CLRN1) gene encodes clarin-1, which is a member of the tetraspanin family of transmembrane proteins. Although identified more than 6 years ago, little is known about its localization or function in the eye and ear. We developed a polyclonal antibody that react with all clarin-1 isoforms and used it to characterize protein expression in cochlea and retina. In the cochlea, we observe clarin-1expression in the stereocilia of P0 mice, and in synaptic terminals present at the base of the auditory hair cells from E18 to P6. In the retina, clarin-1 localizes to the connecting cilia, inner segment of photoreceptors and to the ribbon synapses. RT-PCR from P0 cochlea and P28 retina show mRNAs encoding only isoforms 2 and 3. Western-blots show that only isoform 2 is present in protein extracts from these same tissues. We examined clarin-1 expression in the immortomouse-derived hair cell line UB/OC-1. Only isoform 2 is expressed in UB/OC-1 at both mRNA and protein levels, suggesting this isoform is biologically relevant to hair cell function. The protein co-localizes with microtubules and post-transgolgi vesicles. The sub-cellular localization of clarin-1 in hair cells and photoreceptors suggests it functions at both the basal and apical poles of neurosensoriepithelia. PMID:19539019

  7. Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

    Science.gov (United States)

    Aron Badin, R; Vadori, M; Vanhove, B; Nerriere-Daguin, V; Naveilhan, P; Neveu, I; Jan, C; Lévèque, X; Venturi, E; Mermillod, P; Van Camp, N; Dollé, F; Guillermier, M; Denaro, L; Manara, R; Citton, V; Simioni, P; Zampieri, P; D'avella, D; Rubello, D; Fante, F; Boldrin, M; De Benedictis, G M; Cavicchioli, L; Sgarabotto, D; Plebani, M; Stefani, A L; Brachet, P; Blancho, G; Soulillou, J P; Hantraye, P; Cozzi, E

    2016-07-01

    Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  8. Salvage surgery for local failures after stereotactic ablative radiotherapy for early stage non-small cell lung cancer

    International Nuclear Information System (INIS)

    Verstegen, Naomi E.; Maat, Alexander P. W. M.; Lagerwaard, Frank J.; Paul, Marinus A.; Versteegh, Michel I; Joosten, Joris J.; Lastdrager, Willem; Smit, Egbert F.; Slotman, Ben J.; Nuyttens, Joost J. M. E.; Senan, Suresh

    2016-01-01

    The literature on surgical salvage, i.e. lung resections in patients who develop a local recurrence following stereotactic ablative radiotherapy (SABR), is limited. We describe our experience with salvage surgery in nine patients who developed a local recurrence following SABR for early stage non-small cell lung cancer (NSCLC). Patients who underwent surgical salvage for a local recurrence following SABR for NSCLC were identified from two Dutch institutional databases. Complications were scored using the Dindo-Clavien-classification. Nine patients who underwent surgery for a local recurrence were identified. Median time to local recurrence was 22 months. Recurrences were diagnosed with CT- and/or 18FDG-PET-imaging, with four patients also having a pre-surgical pathological diagnosis. Extensive adhesions were observed during two resections, requiring conversion from a thoracoscopic procedure to thoracotomy during one of these procedures. Three patients experienced complications post-surgery; grade 2 (N = 2) and grade 3a (N = 1), respectively. All resection specimens showed viable tumor cells. Median length of hospital stay was 8 days (range 5–15 days) and 30-day mortality was 0 %. Lymph node dissection revealed mediastinal metastases in 3 patients, all of whom received adjuvant therapy. Our experience with nine surgical procedures for local recurrences post-SABR revealed two grade IIIa complications, and a 30-day mortality of 0 %, suggesting that salvage surgery can be safely performed after SABR

  9. Evaluation of expansile nanoparticle tumor localization and efficacy in a cancer stem cell-derived model of pancreatic peritoneal carcinomatosis

    Science.gov (United States)

    Herrera, Victoria LM; Colby, Aaron H; Tan, Glaiza AL; Moran, Ann M; O’Brien, Michael J; Colson, Yolonda L; Ruiz-Opazo, Nelson; Grinstaff, Mark W

    2016-01-01

    Aim: To evaluate the tumor localization and efficacy pH-responsive expansile nanoparticles (eNPs) as a drug delivery system for pancreatic peritoneal carcinomatosis (PPC) modeled in nude rats. Methods & materials: A Panc-1-cancer stem cell xeno1graft model of PPC was validated in vitro and in vivo. Tumor localization was tracked via in situ imaging of fluorescent eNPs. Survival of animals treated with paclitaxel-loaded eNPs (PTX-eNPs) was evaluated in vivo. Results: The Panc-1-cancer stem cell xenograft model recapitulates significant features of PPC. Rhodamine-labeled eNPs demonstrate tumor-specific, dose- and time-dependent localization to macro- and microscopic tumors following intraperitoneal injection. PTX-eNPs are as effective as free PTX in treating established PPC; but, PTX-eNPs result in fewer side effects. Conclusion: eNPs are a promising tool for the detection and treatment of PPC. PMID:27078118

  10. Noise genetics: inferring protein function by correlating phenotype with protein levels and localization in individual human cells.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    2014-03-01

    Full Text Available To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.

  11. Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

    Directory of Open Access Journals (Sweden)

    Felipe O Bendezú

    Full Text Available The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

  12. Cell-free DNA levels and correlation to stage and outcome following treatment of locally advanced rectal cancer.

    Science.gov (United States)

    Boysen, Anders Kindberg; Wettergren, Yvonne; Sorensen, Boe Sandahl; Taflin, Helena; Gustavson, Bengt; Spindler, Karen-Lise Garm

    2017-11-01

    Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery. The level of cell free DNA was quantified by digital droplet PCR and expressed as copy number of beta 2 microglobulin. We found a median level of cell free DNA in the AJCC stages I-III of 3100, 8300, and 10,700 copies/mL respectively. For patients with 12 sampled lymph nodes or above, the median level of cell free DNA were 2400 copies/mL and 4400 copies/mL (p = 0.04) for node negative and node positive disease respectively. The median follow-up was 39 months and 11 recurrences were detected (15%). The median level for patients with recurrent disease was 13,000 copies/mL compared to 5200 copies/mL for non-recurrent patients (p = 0.08). We have demonstrated a correlation between the level of total cell free DNA and the pathologic stage and nodal involvement. Furthermore, we have found a trend towards a correlation with the risk of recurrence following resection of localized rectal cancer.

  13. Estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract of female rats upon colon-bladder cross-sensitization.

    Directory of Open Access Journals (Sweden)

    Xiao-Qing Pan

    Full Text Available Co-morbidity of bladder, bowel, and non-specific pelvic pain symptoms is highly prevalent in women. Little evidence is present on modulation of pelvic pain syndromes by sex hormones, therefore, the objective of this study was to clarify the effects of hormonal fluctuations within the estrous cycle on regulatory neuropeptides in female rats using a model of neurogenic bladder dysfunction. The estrous cycle in female rats (Sprague-Dawley, 230-250 g was assessed by vaginal smears and weight of uterine horns. Neurogenic bladder dysfunction was induced by a single inflammatory insult to the distal colon. Protein expression of calcitonin gene related peptide (CGRP, substance P (SP, nerve growth factor (NGF, and brain derived neurotrophic factor (BDNF in the pelvic organs, sensory ganglia and lumbosacral spinal cord was compared in rats in proestrus (high estrogen vs diestrus (low estrogen. Under normal physiological conditions, concentration of SP and CGRP was similar in the distal colon and urinary bladder during all phases of the estrous cycle, however, acute colitis induced a significant up-regulation of CGRP content in the colon (by 63% and urinary bladder (by 54%, p≤0.05 to control of rats in proestrus. These changes were accompanied by a significant diminution of CGRP content in L6-S2 DRG after colonic treatment, likely associated with its release in the periphery. In rats with high estrogen at the time of testing (proestrus, experimental colitis caused a significant up-regulation of BDNF colonic content from 26.1±8.5 pg/ml to 83.4±32.5 pg/ml (N = 7, p≤0.05 to control and also induced similar effects on BDNF in the urinary bladder which was also up-regulated by 5-fold in rats in proestrus (p≤0.05 to respective control. Our results demonstrate estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract upon colon-bladder cross-sensitization, which may contribute to pain fluctuations in female patients

  14. Thorax irradiation triggers a local and systemic accumulation of immunosuppressive CD4+ FoxP3+ regulatory T cells

    International Nuclear Information System (INIS)

    Wirsdörfer, Florian; Cappuccini, Federica; Niazman, Muska; Leve, Simone de; Westendorf, Astrid M; Lüdemann, Lutz; Stuschke, Martin; Jendrossek, Verena

    2014-01-01

    Lymphocyte infiltration is a common feature of radiation-induced pneumonitis and fibrosis, but their contribution to the pathogenic processes is still unclear. Here, we addressed the impact of thorax irradiation on the T cell compartment with a focus on immunosuppressive regulatory T cells (Treg). C57BL/6 wild type mice (WT) received anesthesia only (sham controls, 0 Gy) or were exposed to a single dose of whole thorax irradiation (15 Gy). Immune cells from lung tissue, spleen, and cervical lymph nodes were collected 10 to 84 days post-irradiation and phenotypically characterized by flow cytometry. Whole thorax irradiation provoked an increased influx of CD3+ T cells at 42 and 84 days post-irradiation. In contrast, local irradiation caused a sustained reduction in CD3+ T cells in peripheral lymphoid tissues. Interestingly, we observed a significant local and systemic increase in the fraction of CD4+ T cells expressing the transcription factor forkhead box P3 (FoxP3), the phenotypic marker for murine Treg, at day 21 post-irradiation. The accumulation of Treg was associated with increased levels of T cells expressing surface proteins characteristic for recruitment and immunosuppressive activity, e.g. CD103, CTLA-4 and CD73. Importantly, Treg isolated at this time point were able to suppress CD4+ effector T cells to a similar extent as Treg isolated from control mice. The response of the adaptive immune system to whole thorax irradiation is characterized by local immunoactivation and systemic immunosuppression. The transient accumulation of immunosuppressive CD4+ FoxP3+ Treg may be required to protect the lung against excessive inflammation-induced tissue damage. Further investigations shall define the mechanisms underlying the accumulation of Treg and their role for the pathogenesis of radiation-induced lung disease

  15. Studies on the intracellular localization of hHR23B

    International Nuclear Information System (INIS)

    Katiyar, Samiksha; Lennarz, William J.

    2005-01-01

    Yeast Rad23, originally identified as a DNA repair protein, has been proposed to participate in other cellular functions, i.e., the proteasome-degradation pathway, the process of spindle pole body duplication and as a component of the anaphase checkpoint. Two human homologs of yeast Rad23, hHR23A and hHR23B, exhibit high sequence homology with yRad23 and also have been shown to be involved in DNA repair and proteasome-dependent degradation. Previous studies on the intracellular localization of hHR23A and hHR23B revealed their predominant localization in the nucleus during interphase and in the cytoplasm during mitosis. We have analyzed the localization of hHR23B during all the phases of the cell cycle using immunofluorescence. Unlike previous studies, our results suggest localization of hHR23B in the nucleus as well as in the cytoplasm during G1 phase. The nuclear levels of hHR23B decrease during S-phase of the cell cycle. When the cell enters mitosis, hHR23B relocalizes in the cytoplasm without association with chromatin. These results indicate that the intracellular distribution hHR23B is cell cycle dependent

  16. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    Science.gov (United States)

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine…

  17. Cytological Characteristics of Mucose Cell and Vaginal Temperature and pH During Estrous Cycle in Local Sheep

    Directory of Open Access Journals (Sweden)

    Siti Darodjah Rasad

    2017-09-01

    Full Text Available Aim of this study was to examine the characteristics cytology of mucous cell-,temperature- and pH vagina during estrous cycle in local sheep.  31local sheep were synchronized with vaginal sponge consist of 20  mg  progesterone hormone before carried out observations of cytology of cells from the vaginal mucose through vaginal swabs, temperature and pH of the vagina.  Vaginal swabs were collected daily at 7 am for a weeks.Vaginal temperature and pH measurement is carried out twice a day, at 07.00 am and 15.00 pm for a weeks after vaginal swabs. Smears of the swab were then prepared on glass slide and they were stained with Giemsa.  Vaginal epithelial cells; Parabasal, intermediate and superficial cells were counted and their percentages during pro-estrous, estrous and di-estrous were determined. Di-estrous was characterized by the absent of superficial cells in the epithelial vagina. Pro-estrous was characterized by the increasing progressively of intermediate/superficial cells in epithelial vagina, whereas estrous was characterized by the presence of superficial/cornification cells in most epithelial vagina. Based on the dominance of superficial cell, the number of sheep identified as estrous is highest on third day, with 52%.  Observation on vaginal temperature also resulting that the highest temperature values obtained on the third day of 39,08±0.28°C.  It could be effected of the vaginal pH during the observation. Underthe influence ofestrogen, the epithelial vaginalcellssynthesizeand accumulateglycogenin large quantitiesdepositedin the lumen ofvagina. Vaginal bacteriametabolizethe glycogenformlactic acid, which causesvaginal pHis low.The pH conditions prevent from pathogenic microorganisms and fungi. Increased estrogenal so cause cell proliferation through the thickening of the epithelium lining of the vagina so that the cells differentiate.Increasing of glycogenin the superficial cells, and  ceratin cells found in the cytoplasm of

  18. Retrospective analysis of multidisciplinary therapy for locally advanced squamous cell carcinoma of the maxillary sinus

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Hiroshi; Seo, Yuji; Nakajima, Kaori; Miyano, Takashi [Asahikawa Medical Univ., Hokkaido (Japan); Kikuchi, Yuzou [Kanazawa Univ. (Japan). School of Medicine

    2002-06-01

    The purpose of this study was to retrospectively investigate the efficacy of multidisciplinary therapy (concomitant radiotherapy and intra-arterial infusion of 5-fluorouracil (5-FU) followed by maxillectomy) in the treatment of squamous cell carcinoma of the maxillary sinus. We reviewed 71 patient records with locally advanced but respectable carcinoma of the maxillary sinus treated by means of multidisciplinary therapy between 1978 through 1997. The clinical T factor for these patients, according to the UICC definitions (1997), was 12 for T2, 46 for T3, and 13 for T4. Twelve patients were diagnosed as node-positive at initial presentation. Intra-arterial 5-FU was delivered via a superficial temporal artery in accordance with radiotherapy, and the cumulative 5-FU dose ranged from 2,900 mg to 5,250 mg (median 5,000 mg). The total radiotherapy dose ranged from 29 Gy to 48 Gy (median 48 Gy) with conventional fractionation. Patients underwent radical maxillectomy thereafter. The 5-year overall survival rate and disease-specific survival rate of all the patients were 58% and 68%, respectively. There was no significant correlation of clinical T factor or N factor with disease-specific survival on univariate and multivariate analysis. The overall treatment-related mortality rate was 3.7%. Radiation cataract later developed in all evaluable patients whose lenses were within the treatment volume. About a half of the operable T4 patients survived over 5 years by means of the above-mentioned multidisciplinary therapy. This multidisciplinary therapy should be compared to treatment with a combination of surgery and postoperative chemoradiotherapy. (author)

  19. Retrospective analysis of multidisciplinary therapy for locally advanced squamous cell carcinoma of the maxillary sinus

    International Nuclear Information System (INIS)

    Yoshida, Hiroshi; Seo, Yuji; Nakajima, Kaori; Miyano, Takashi; Kikuchi, Yuzou

    2002-01-01

    The purpose of this study was to retrospectively investigate the efficacy of multidisciplinary therapy (concomitant radiotherapy and intra-arterial infusion of 5-fluorouracil (5-FU) followed by maxillectomy) in the treatment of squamous cell carcinoma of the maxillary sinus. We reviewed 71 patient records with locally advanced but respectable carcinoma of the maxillary sinus treated by means of multidisciplinary therapy between 1978 through 1997. The clinical T factor for these patients, according to the UICC definitions (1997), was 12 for T2, 46 for T3, and 13 for T4. Twelve patients were diagnosed as node-positive at initial presentation. Intra-arterial 5-FU was delivered via a superficial temporal artery in accordance with radiotherapy, and the cumulative 5-FU dose ranged from 2,900 mg to 5,250 mg (median 5,000 mg). The total radiotherapy dose ranged from 29 Gy to 48 Gy (median 48 Gy) with conventional fractionation. Patients underwent radical maxillectomy thereafter. The 5-year overall survival rate and disease-specific survival rate of all the patients were 58% and 68%, respectively. There was no significant correlation of clinical T factor or N factor with disease-specific survival on univariate and multivariate analysis. The overall treatment-related mortality rate was 3.7%. Radiation cataract later developed in all evaluable patients whose lenses were within the treatment volume. About a half of the operable T4 patients survived over 5 years by means of the above-mentioned multidisciplinary therapy. This multidisciplinary therapy should be compared to treatment with a combination of surgery and postoperative chemoradiotherapy. (author)

  20. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    Directory of Open Access Journals (Sweden)

    Irene L Ibañez

    Full Text Available The Cyclin-dependent kinase inhibitor 1B (p27Kip1 is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2O(2 in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p at serine 10 (S10 and at threonine 198 (T198 because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2O(2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2O(2 (0.1 µM to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2O(2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization

  1. Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Al-Shammari AM

    2014-05-01

    Full Text Available Ahmed M Al-Shammari,1 Farah E Ismaeel,2 Shahlaa M Salih,2 Nahi Y Yaseen11Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Researches, Mustansiriya University, 2Departments of Biotechnology, College of Science, Al-Nahrain University, Baghdad, IraqAbstract: Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5 was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5 cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05 on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72

  2. Growth-dependent catalase localization in Exiguobacterium oxidotolerans T-2-2T reflected by catalase activity of cells.

    Science.gov (United States)

    Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

    2013-01-01

    A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2(T), exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.

  3. Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

    Science.gov (United States)

    Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

    2013-01-01

    A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state. PMID:24204687

  4. Radiological response and survival in locally advanced non-small-cell lung cancer patients treated with three-drug induction chemotherapy followed by radical local treatment.

    Science.gov (United States)

    Bonanno, Laura; Zago, Giulia; Marulli, Giuseppe; Del Bianco, Paola; Schiavon, Marco; Pasello, Giulia; Polo, Valentina; Canova, Fabio; Tonetto, Fabrizio; Loreggian, Lucio; Rea, Federico; Conte, PierFranco; Favaretto, Adolfo

    2016-01-01

    If concurrent chemoradiotherapy cannot be performed, induction chemotherapy followed by radical-intent surgical treatment is an acceptable option for non primarily resectable non-small-cell lung cancers (NSCLCs). No markers are available to predict which patients may benefit from local treatment after induction. This exploratory study aims to assess the feasibility and the activity of multimodality treatment, including triple-agent chemotherapy followed by radical surgery and/or radiotherapy in locally advanced NSCLCs. We retrospectively collected data from locally advanced NSCLCs treated with induction chemotherapy with carboplatin (area under the curve 6, d [day]1), paclitaxel (200 mg/m(2), d1), and gemcitabine (1,000 mg/m(2) d1, 8) for three to four courses, followed by radical surgery and/or radiotherapy. We analyzed radiological response and toxicity. Estimated progression-free survival (PFS) and overall survival (OS) were correlated to response, surgery, and clinical features. In all, 58 NSCLCs were included in the study: 40 staged as IIIA, 18 as IIIB (according to TNM Classification of Malignant Tumors-7th edition staging system). A total of 36 (62%) patients achieved partial response (PR), and six (10%) progressions were recorded. Grade 3-4 hematological toxicity was observed in 36 (62%) cases. After chemotherapy, 37 (64%) patients underwent surgery followed by adjuvant radiotherapy, and two patients received radical-intent radiotherapy. The median PFS and OS were 11 months and 23 months, respectively. Both PFS and OS were significantly correlated to objective response (P<0.0001) and surgery (P<0.0001 and P=0.002). Patients obtaining PR and receiving local treatment achieved a median PFS and OS of 35 and 48 months, respectively. Median PFS and OS of patients not achieving PR or not receiving local treatment were 5-7 and 11-15 months, respectively. The extension of surgery did not affect the outcome. The multimodality treatment was feasible, and triple

  5. Colitic scid mice fed Lactobacillus spp. show an ameliorated gut histopathology and an altered cytokine profile by local T cells

    DEFF Research Database (Denmark)

    Møller, Peter Lange; Paerregaard, Anders; Gad, Monika

    2005-01-01

    BACKGROUND: Scid mice transplanted with CD4 T blast cells develop colitis. We investigated if the disease was influenced in colitic mice treated with antibiotic and fed Lactobacillus spp. METHODS: Colitic scid mice were treated for 1 week with antibiotics (vancomycin/meropenem) followed or not fo......-gamma production than mice not fed probiotics. CONCLUSIONS: Our data suggest that probiotics added to the drinking water may ameliorate local histopathological changes and influence local cytokine levels in colitic mice but not alter the colitis-associated weight loss....

  6. Prospective identification and skeletal localization of cells capable of multilineage differentiation in vivo.

    Science.gov (United States)

    Taichman, Russell S; Wang, Zhuo; Shiozawa, Yusuke; Jung, Younghun; Song, Junhui; Balduino, Alex; Wang, Jincheng; Patel, Lalit R; Havens, Aaron M; Kucia, Magdalena; Ratajczak, Mariusz Z; Krebsbach, Paul H

    2010-10-01

    A prospective in vivo assay was used to identify cells with potential for multiple lineage differentiation. With this assay, it was first determined that the 5-fluorouracil resistant cells capable of osseous tissue formation in vivo also migrated toward stromal derived factor-1 (SDF-1) in vitro. In parallel, an isolation method based on fluorescence-activated cell sorting was employed to identify a very small cell embryonic-like Lin-/Sca-1+CD45- cell that with as few as 500 cells was capable of forming bone-like structures in vivo. Differential marrow fractionation studies determined that the majority of the Lin-Sca-1+CD45- cells reside in the subendosteal regions of marrow. To determine whether these cells were capable of differentiating into multiple lineages, stromal cells harvested from Col2.3 Delta TK mice were implanted with a gelatin sponge into SCID mice to generate thymidine kinase sensitive ossicles. At 1.5 months, 2,000 green fluorescent protein (GFP)+ Lin-Sca-1+CD45- cells were injected into the ossicles. At harvest, colocalization of GFP-expressing cells with antibodies to the osteoblast-specific marker Runx-2 and the adipocyte marker PPAP gamma were observed. Based on the ability of the noncultured cells to differentiate into multiple mesenchymal lineages in vivo and the ability to generate osseous tissues at low density, we propose that this population fulfills many of the characteristics of mesenchymal stem cells.

  7. Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues.

    Directory of Open Access Journals (Sweden)

    Diana Fleissner

    Full Text Available BACKGROUND: In contrast to intestinal CD4(+ regulatory T cells (T(regs, the generation and function of immunomodulatory intestinal CD8(+ T cells is less well defined. To dissect the immunologic mechanisms of CD8(+ T cell function in the mucosa, reactivity against hemagglutinin (HA expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied. METHODOLOGY AND PRINCIPAL FINDINGS: HA-specific CD8(+ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3(+ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8(+Foxp3(+ T cells. Antigen-experienced CD8(+ T cells in this transgenic mouse model suppressed the proliferation of CD8(+ and CD4(+ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8(+ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4(+ T(reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8(+Foxp3(+ T cells. CONCLUSION AND SIGNIFICANCE: We demonstrate that gut specific antigen presentation is sufficient to induce CD8(+ T(regsin vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.

  8. Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues.

    Science.gov (United States)

    Fleissner, Diana; Hansen, Wiebke; Geffers, Robert; Buer, Jan; Westendorf, Astrid M

    2010-10-20

    In contrast to intestinal CD4(+) regulatory T cells (T(regs)), the generation and function of immunomodulatory intestinal CD8(+) T cells is less well defined. To dissect the immunologic mechanisms of CD8(+) T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied. HA-specific CD8(+) T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3(+) and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8(+)Foxp3(+) T cells. Antigen-experienced CD8(+) T cells in this transgenic mouse model suppressed the proliferation of CD8(+) and CD4(+) T cells in vitro. Gene expression analysis of suppressive HA-specific CD8(+) T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4(+) T(reg) subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8(+)Foxp3(+) T cells. We demonstrate that gut specific antigen presentation is sufficient to induce CD8(+) T(regs)in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.

  9. CD147-mediated chemotaxis of CD4+CD161+ T cells may contribute to local inflammation in rheumatoid arthritis.

    Science.gov (United States)

    Lv, Minghua; Miao, Jinlin; Zhao, Peng; Luo, Xing; Han, Qing; Wu, Zhenbiao; Zhang, Kui; Zhu, Ping

    2018-01-01

    CD161 is used as a surrogate marker for Th17 cells, which are implicated in the pathogenesis of rheumatoid arthritis (RA). In this study, we evaluated the percentage, clinical significance, and CD98 and CD147 expression of CD4 + CD161 + T cells. The potential role of CD147 and CD98 in cyclophilin A-induced chemotaxis of CD4 + CD161 + T cells was analyzed. Thirty-seven RA patients, 15 paired synovial fluid (SF) of RA, and 22 healthy controls were recruited. The cell populations and surface expression of CD98 and CD147 were analyzed by flow cytometry. Spearman's rank correlation coefficient and multiple linear regression were applied to calculate the correlations. Chemotaxis assay was used to investigate CD4 + CD161 + T cell migration. We found that the percentage of CD4 + CD161 + T cells and their expression of CD147 and CD98 in SF were higher than in the peripheral blood of RA patients. Percentage of SF CD4 + CD161 + T cells was positively correlated with 28-Joint Disease Activity Score (DAS28). CD147 monoclonal antibody (HAb18) attenuated the chemotactic ability of CD4 + CD161 + T cells. An increased CD4 + CD161 + T cell percentage and expression of CD147 and CD98 were shown in RA SF. Percentage of SF CD4 + CD161 + T cells can be used as a predictive marker of disease activity in RA. CD147 block significantly decreased the chemotactic index of CD4 + CD161 + cells induced by cyclophilin A (CypA). These results imply that the accumulation of CD4 + CD161 + T cells in SF and their high expression of CD147 may be associated with CypA-mediated chemotaxis and contribute to local inflammation in RA.

  10. Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

    2009-01-01

    Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study...... in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep...... into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP...

  11. Development and experimental basis of local subretinal technique of xenogenic’s injection stem cells labelled by magnetic perticles

    Directory of Open Access Journals (Sweden)

    Yu. A. Belyy

    2014-10-01

    Full Text Available Purpose: is to develop a technique for local subretinal injection of xenogeneic stem cells labeled with magnetic particles and to prove experimentally its effectiveness.Material and methods: We used a line of stem cells HEK-293 GFP,labeled with magnetic particles. The study was made on 84 eyes of 42 chinchilla rabbits 6 months of age, the weight were from 2.5 to 3.5 kg. All right eyes were experimental (42 eyes and all left eyes (42 eyes were the control group. In the experimental group we used original complex of polymer elastic magnetic implant (PEMI with laser probe and fixed it to the sclera, then we made a median vitrectomy and injected HEK-293 GFP under the retina using a specially designed dispenser. In the control group PEMI was not fixed. We examined animals using biomicroscopy, ophthalmoscopy, ultrasound scanning, optical coherence tomography  OCT, computer tomography (CT, morphological study (cryohistological sections in 1, 3, 5, 7, 14 day and 1 month after surgery.Results: According the results of biomicroscopy in observation periods up to 3 days the vascular injection was visualized in the area operation. According the results of ophthalmoscopy and ultrasound scanning in 1 day the local retinal detachment was visualized in the area of local injection of the stem cells, which was not visualized in terms of further observations. CT helped us to confirm the local place of PEMI fixation. The morphological study results showed that cells were located in the subretinal space up to 14 days in the experimental group, and only up 3 days in the control group.Conclusion: The suggested surgical technique enables to control the injection of cells into the subretinal space, reduces the risk of tissue damage and exit cells in the vitreous space. The suggested methodology allows the fixing of the cellular material in the local place of the injection and enables to predict cells`s movement.

  12. Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells.

    Science.gov (United States)

    Robert, Damien; Fayol, Delphine; Le Visage, Catherine; Frasca, Guillaume; Brulé, Séverine; Ménager, Christine; Gazeau, Florence; Letourneur, Didier; Wilhelm, Claire

    2010-03-01

    The in vitro generation of engineered tissue constructs involves the seeding of cells into porous scaffolds. Ongoing challenges are to design scaffolds to meet biochemical and mechanical requirements and to optimize cell seeding in the constructs. In this context, we have developed a simple method based on a magnetic tweezer set-up to manipulate, probe, and position magnetic objects inside a porous scaffold. The magnetic force acting on magnetic objects of various sizes serves as a control parameter to retrieve the local viscosity of the scaffolds internal channels as well as the stiffness of the scaffolds pores. Labeling of human stem cells with iron oxide magnetic nanoparticles makes it possible to perform the same type of measurement with cells as probes and evaluate their own microenvironment. For 18 microm diameter magnetic beads or magnetically labeled stem cells of similar diameter, the viscosity was equivalently equal to 20 mPa s in average. This apparent viscosity was then found to increase with the magnetic probes sizes. The stiffness probed with 100 microm magnetic beads was found in the 50 Pa range, and was lowered by a factor 5 when probed with cells aggregates. The magnetic forces were also successfully applied to the stem cells to enhance the cell seeding process and impose a well defined spatial organization into the scaffold. (c) 2009 Elsevier Ltd. All rights reserved.

  13. Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

    DEFF Research Database (Denmark)

    Nassini, Romina; Pedretti, Pamela; Moretto, Nadia

    2012-01-01

    The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic...... inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express...... functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells...

  14. Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

    Science.gov (United States)

    Suzuki, Hirohumi; Yamamoto, Toshiharu

    2014-02-01

    In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

    Science.gov (United States)

    Okubo, Shinya; Uto, Takuhiro; Goto, Aya; Tanaka, Hiroyuki; Nishioku, Tsuyoshi; Yamada, Katsushi; Shoyama, Yukihiro

    2017-01-01

    Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

  16. Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

    Science.gov (United States)

    Kermani, Pouneh; Rafii, Dahlia; Jin, David K.; Whitlock, Paul; Schaffer, Wendy; Chiang, Anne; Vincent, Loic; Friedrich, Matthias; Shido, Koji; Hackett, Neil R.; Crystal, Ronald G.; Rafii, Shahin; Hempstead, Barbara L.

    2005-01-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels. PMID:15765148

  17. Locally-regionally advanced tonsillar squamous cell carcinoma treated with concurrent chemoradiotherapy

    International Nuclear Information System (INIS)

    Krstevska, Valentina; Stojkovski, Igor

    2013-01-01

    Purpose: To perform a retrospective review of stage III-IV squamous cell carcinoma of the tonsil managed by definitive concurrent chemoradiotherapy (CCRT) in order to analyze the patients’ outcome and to evaluate the acute and late toxic effects of this treatment modality. Material and methods: Between January 2005 and December 2010, 36 patients with locally and/or regionally advanced tonsillar cancer underwent three dimensional conformal radiotherapy (3DCRT) with concurrent platinum-based chemotherapy. The dose prescription of the planning target volume for gross tumor and low-risk subclinical disease was 70 Gy and 50 Gy, respectively. Conventional fractionation with a daily dose of 2.0 Gy, 5 times per week was used. Concurrent chemotherapy consisted of cisplatin 30 mg/m2 given on a weekly basis. Acute and late radiotherapy-related toxicities were recorded using European Organization for Research and Treatment of Cancer/Radiation Therapy Oncology Group (EORTC/RTOG) grading system. The 3-year locoregional relapse-free survival (LRRFS), disease-free survival (DFS), and overall survival (OS) rates were calculated using the Kaplan-Meier method. Results: The median follow-up of all patients was 20.5 months (range, 5 to 90 months). The median followup of living patients was 59 months (range, 30 to 90 months). Complete response rates of the primary tumor and of the nodal disease were 72.2% and 64.0%, respectively. A complete composite response was present in 25 patients (69.4%). Treatment failure occurred in 15 out of 25 patients who achieved complete composite response following CCRT. The 3-year LRRFS, DFS, and OS rate was 38.8%, 27.8%, and 27.3%, respectively. Grade 3 mucositis occurred in 58.3% of patients. Xerostomia grade 2 was revealed in 72.2% of patients. Conclusion: Taking into account the low 3-year survival rates observed in our study and the high percentage of grade 2 xerostomia, it can be concluded that in the future, instead of 3DCRT with concurrent

  18. Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Therese Featherston

    2017-07-01

    Full Text Available AimWe have previously demonstrated the putative presence of two cancer stem cell (CSC subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC, which express components of the renin–angiotensin system (RAS. In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC.Methods3,3-Diaminobenzidine (DAB and immunofluorescent (IF immunohistochemical (IHC staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC.ResultsIHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G.ConclusionThe presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

  19. Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis.

    Directory of Open Access Journals (Sweden)

    Dragana Antic

    2010-02-01

    Full Text Available Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell's apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2 in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

  20. Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor

    Directory of Open Access Journals (Sweden)

    Veronika Y. Sysoeva

    2017-12-01

    Full Text Available Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS. Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs. We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII receptor type 1 (AT1. Using the analysis of Ca2+ mobilization in single cells we found that only 5.2 ± 2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2, which was responsible for increased adipose competency of this ADSC subpopulation.

  1. Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

    Science.gov (United States)

    Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-07-13

    Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Hydrogen sulfide detection based on reflection: from a poison test approach of ancient China to single-cell accurate localization.

    Science.gov (United States)

    Kong, Hao; Ma, Zhuoran; Wang, Song; Gong, Xiaoyun; Zhang, Sichun; Zhang, Xinrong

    2014-08-05

    With the inspiration of an ancient Chinese poison test approach, we report a rapid hydrogen sulfide detection strategy in specific areas of live cells using silver needles with good spatial resolution of 2 × 2 μm(2). Besides the accurate-localization ability, this reflection-based strategy also has attractive merits of convenience and robust response when free pretreatment and short detection time are concerned. The success of endogenous H2S level evaluation in cellular cytoplasm and nuclear of human A549 cells promises the application potential of our strategy in scientific research and medical diagnosis.

  3. Changes in T cell populations due to local irradiation of a portion of the maxilla in mice

    International Nuclear Information System (INIS)

    Satoh, Daigo

    1997-01-01

    The aim of this study was to investigate the changes in the immune organs after head and neck irradiation. The numbers of lymphocytes in peripheral blood, the spleen and the thymus following local irradiation of a portion of the maxilla in mice were studied using three-color fluorometry and were compared with a non-irradiation group. In the peripheral blood, the absolute numbers of T cells, CD4 + SP cells and CD8 + SP cells decreased after irradiation, and the period of the decrease was longer than the decreases in number of leukocytes and lymphocytes. The ratio of CD4 + SP cells showed a significant decrease, and the ratio of CD8+ S P cells showed a significant increase 1 day after irradiation. In the spleen, the absolute number of T cells, the radio of CD4 + SP and CD8 + SP cell subsets showed a decrease, and the period of the decrease was longer than the decrease of the wet-weight of the spleen, and also longer than the decrease of the number of leukocytes. The number of CD4 + SP cells showed a signigicant increase, and CD8 + SP cells showed a significant decrease 21 days after irradiation. In the thymus, the absolute number of TCR αβ-thymocytes did not show a significant decrease. However, the number of DN thymocytes showed a marked decrease. These results indicate that the numbers of T cells in peripheral blood, the spleen and the thymus change immediately after irradiation, and the numbers of lymphocytes and the T cells in the spleen recover more slowly than that in the peripheral blood. As lymphoid tissues showed the suppression of immunological response for a long period, it was suggested that lymphoid tissues have to be observed carefully after irradiation to prevent cancer metastasis. (K.H.)

  4. Intensity-Modulated Radiotherapy for Oral Cavity Squamous Cell Carcinoma: Patterns of Failure and Predictors of Local Control

    International Nuclear Information System (INIS)

    Daly, Megan E.; Le, Quynh-Thu; Kozak, Margaret M.; Maxim, Peter G.; Murphy, James D.; Hsu, Annie; Loo, Billy W.; Kaplan, Michael J.; Fischbein, Nancy J.; Chang, Daniel T.

    2011-01-01

    Purpose: Few studies have evaluated the use of intensity-modulated radiotherapy (IMRT) for squamous cell carcinoma (SCC) of the oral cavity (OC). We report clinical outcomes and failure patterns for these patients. Methods and Materials: Between October 2002 and June 2009, 37 patients with newly diagnosed SCC of the OC underwent postoperative (30) or definitive (7) IMRT. Twenty-five patients (66%) received systemic therapy. The median follow-up was 38 months (range, 10-87 months). The median interval from surgery to RT was 5.9 weeks (range, 2.1-10.7 weeks). Results: Thirteen patients experienced local-regional failure at a median of 8.1 months (range, 2.4-31.9 months), and 2 additional patients experienced local recurrence between surgery and RT. Seven local failures occurred in-field (one with simultaneous nodal and distant disease) and two at the margin. Four regional failures occurred, two in-field and two out-of-field, one with synchronous metastases. Six patients experienced distant failure. The 3-year actuarial estimates of local control, local-regional control, freedom from distant metastasis, and overall survival were 67%, 53%, 81%, and 60% among postoperative patients, respectively, and 60%, 60%, 71%, and 57% among definitive patients. Four patients developed Grade ≥2 chronic toxicity. Increased surgery to RT interval predicted for decreased LRC (p = 0.04). Conclusions: Local-regional control for SCC of the OC treated with IMRT with or without surgery remains unsatisfactory. Definitive and postoperative IMRT have favorable toxicity profiles. A surgery-to-RT interval of <6 weeks improves local-regional control. The predominant failure pattern was local, suggesting that both improvements in target delineation and radiosensitization and/or dose escalation are needed.

  5. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  6. Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines.

    Science.gov (United States)

    Ma, H; Zhu, J; Maronski, M; Kotzbauer, P T; Lee, V M-Y; Dichter, M A; Diamond, S L

    2002-01-01

    Gene transfer into CNS is critical for potential therapeutic applications as well as for the study of the genetic basis of neural development and nerve function. Unfortunately, lipid-based gene transfer to CNS cells is extremely inefficient since the nucleus of these post-mitotic cells presents a significant barrier to transfection. We report the development of a simple and highly efficient lipofection method for primary embryonic rat hippocampal neurons (up to 25% transfection) that exploits the M9 sequence of the non-classical nuclear localization signal of heterogeneous nuclear ribonucleoprotein A1 for targeting beta(2)-karyopherin (transportin-1). M9-assistant lipofection resulted in 20-100-fold enhancement of transfection over lipofection alone for embryonic-derived retinal ganglion cells, rat pheochromocytoma (PC12) cells, embryonic rat ventral mesencephalon neurons, as well as the clinically relevant human NT2 cells or retinoic acid-differentiated NT2 neurons. This technique can facilitate the implementation of promoter construct experiments in post-mitotic cells, stable transformant generation, and dominant-negative mutant expression techniques in CNS cells.

  7. Photodynamic therapy in Pythium insidiosum - an in vitro study of the correlation of sensitizer localization and cell death.

    Directory of Open Access Journals (Sweden)

    Layla Pires

    Full Text Available Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen's cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.

  8. Receptors for corticotropin-releasing hormone in human pituitary: Binding characteristics and autoradiographic localization to immunocytochemically defined proopiomelanocortin cells

    Energy Technology Data Exchange (ETDEWEB)

    Smets, G.; Vauquelin, G.; Moons, L.; Smitz, J.; Kloeppel, G. (Department of Experimental Pathology, Vrije Universiteit Brussel (Belgium))

    1991-08-01

    Using autoradiography combined with immunocytochemistry, the authors demonstrated that the target cells of CRH in the human pituitary were proopiomelanocortin cells. Scatchard analysis of (125I)Tyr0-oCRH saturation binding revealed the presence of one class of saturable, high affinity sites on pituitary tissue homogenate. The equilibrium dissociation constant (Kd) for (125I)Tyr0-oCRH ranged from 1.1-1.6 nM, and the receptor density was between 200-350 fmol/mg protein. Fixation of cryostat sections with 4% paraformaldehyde before tracer incubation improved both tissue preservation and localization of the CRH receptor at the cellular level. Additional postfixation with 1% glutaraldehyde inhibited tracer diffusion during subsequent immunocytochemistry and autoradiography. (125I)Tyr0-oCRH was found in cytoplasmic inclusions or at the cell periphery of ACTH/beta-endorphin cells in the anterior pituitary. Single cells of the posterior pituitary were also CRH receptor positive. Cells staining for PRL or GH were CRH receptor negative. They conclude that CRH binds only to high affinity receptors on ACTH/{beta}-endorphin cells in the human pituitary.

  9. Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells.

    Science.gov (United States)

    Baird, Michelle A; Billington, Neil; Wang, Aibing; Adelstein, Robert S; Sellers, James R; Fischer, Robert S; Waterman, Clare M

    2017-01-15

    The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. © 2017 Baird et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. Localization of calcium in the sensory cells of the Dionaea trigger hair by laser micro-mass analysis (LAMMA)

    International Nuclear Information System (INIS)

    Buchen, B.; Schröder, W.H.

    1986-01-01

    In Dionaea, mechanical bending of the trigger hair induces action potentials which spread over the trap lobes to the motor cells (review Bentrup 1979). The perception of the stimulus and its transformation into a physiological signal occurs in a ring of specialized epidermal cells in the indentation zone of the trigger hair. These sensory cells (Haberlandt 1906) are characterized by a highly evolved ER complex at the apical and the basal cell pole. The ER surrounds several vacuoles containing poly phenols (Buchen et al. 1983). In order to study the function of these cell structures in sensory transduction, we examined the development of the trigger hair (Casser et al. 1985). During its development, a change in the membrane potential could be measured for the first time when the structural polarity of the sensory cell was established. Yet the short action potentials which are necessary for trap closure were fired only if the typical ER complex in the cell poles was visible. Since membrane potential changes are mediated by ions, we tried to identify and to localize ions possibly involved in these processes. Here we present the first results

  11. Predictors of Individual Tumor Local Control After Stereotactic Radiosurgery for Non-Small Cell Lung Cancer Brain Metastases

    International Nuclear Information System (INIS)

    Garsa, Adam A.; Badiyan, Shahed N.; DeWees, Todd; Simpson, Joseph R.; Huang, Jiayi; Drzymala, Robert E.; Barani, Igor J.; Dowling, Joshua L.; Rich, Keith M.; Chicoine, Michael R.; Kim, Albert H.; Leuthardt, Eric C.; Robinson, Clifford G.

    2014-01-01

    Purpose: To evaluate local control rates and predictors of individual tumor local control for brain metastases from non-small cell lung cancer (NSCLC) treated with stereotactic radiosurgery (SRS). Methods and Materials: Between June 1998 and May 2011, 401 brain metastases in 228 patients were treated with Gamma Knife single-fraction SRS. Local failure was defined as an increase in lesion size after SRS. Local control was estimated using the Kaplan-Meier method. The Cox proportional hazards model was used for univariate and multivariate analysis. Receiver operating characteristic analysis was used to identify an optimal cutpoint for conformality index relative to local control. A P value <.05 was considered statistically significant. Results: Median age was 60 years (range, 27-84 years). There were 66 cerebellar metastases (16%) and 335 supratentorial metastases (84%). The median prescription dose was 20 Gy (range, 14-24 Gy). Median overall survival from time of SRS was 12.1 months. The estimated local control at 12 months was 74%. On multivariate analysis, cerebellar location (hazard ratio [HR] 1.94, P=.009), larger tumor volume (HR 1.09, P<.001), and lower conformality (HR 0.700, P=.044) were significant independent predictors of local failure. Conformality index cutpoints of 1.4-1.9 were predictive of local control, whereas a cutpoint of 1.75 was the most predictive (P=.001). The adjusted Kaplan-Meier 1-year local control for conformality index ≥1.75 was 84% versus 69% for conformality index <1.75, controlling for tumor volume and location. The 1-year adjusted local control for cerebellar lesions was 60%, compared with 77% for supratentorial lesions, controlling for tumor volume and conformality index. Conclusions: Cerebellar tumor location, lower conformality index, and larger tumor volume were significant independent predictors of local failure after SRS for brain metastases from NSCLC. These results warrant further investigation in a prospective

  12. Peculiarities in the CT findings of germ cell tumors in various tumor localizations

    International Nuclear Information System (INIS)

    Tazoe, Makoto; Miyagami, Mitsusuke; Tsubokawa, Takashi

    1991-01-01

    The CT findings of 17 germ cell tumors were studied in relation to the locations of the tumor, the pathological diagnoses, and the tumor markers (AFP and HCG). Generally, the CT findings of germ cell tumors depended on the pathological diagnoses more strongly than on the location of the tumors. On plain CT of 7 germ cell tumors in the pineal region, all of them demonstrated heterogeneous findings. Hydrocephalus was seen in 6 cases (86%) and calcification in 6 cases (86%) of the germ cell tumors in the pineal region. Calcification and hydrocephalus that appeared more often than in other regions were characteristic of germ cell tumors of the pineal region. The germ cell tumors in the basal ganglia had a slightly homogenous high density, with small cysts and calcification in most of them on plain CT. On enhanced CT, the tumors were moderately enhanced in all cases located in the basal ganglia. Four cases of germ cell tumors located in the basal ganglia revealed the dilatation of lateral ventricle due to hemispheric atrophy in the tumor side. The germ cell tumors showing an increase in the tumor markers such as AFP and HCG, which were usually malignant germ cell tumors, were strongly enhanced on enhanced CT. (author)

  13. Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression.

    Science.gov (United States)

    Pryzhkova, Marina V; Jordan, Philip W

    2016-04-15

    Correct duplication of stem cell genetic material and its appropriate segregation into daughter cells are requisites for tissue, organ and organism homeostasis. Disruption of stem cell genomic integrity can lead to developmental abnormalities and cancer. Roles of the Smc5/6 structural maintenance of chromosomes complex in pluripotent stem cell genome maintenance have not been investigated, despite its important roles in DNA synthesis, DNA repair and chromosome segregation as evaluated in other model systems. Using mouse embryonic stem cells (mESCs) with a conditional knockout allele of Smc5, we showed that Smc5 protein depletion resulted in destabilization of the Smc5/6 complex, accumulation of cells in G2 phase of the cell cycle and apoptosis. Detailed assessment of mitotic mESCs revealed abnormal condensin distribution and perturbed chromosome segregation, accompanied by irregular spindle morphology, lagging chromosomes and DNA bridges. Mutation of Smc5 resulted in retention of Aurora B kinase and enrichment of condensin on chromosome arms. Furthermore, we observed reduced levels of Polo-like kinase 1 at kinetochores during mitosis. Our study reveals crucial requirements of the Smc5/6 complex during cell cycle progression and for stem cell genome maintenance. © 2016. Published by The Company of Biologists Ltd.

  14. Estrogen action in the mouse uterus: differential nuclear localization of estradiol in uterine cell types

    International Nuclear Information System (INIS)

    Korach, K.S.; Lamb, J.C.

    1981-01-01

    Autoradiographic studies of labeled steroid uptake in mouse uterine tissue indicated that labeled estradiol was predominantly sequestered in the nuclei of stromal and glandular epithelial cells at 1 h. Luminal epithelial cells did not show appreciable nuclear accumulation of labeled estradiol until 7-8 h after hormone injection. Studies using non-target tissues and unlabeled steroids indicated that the nuclear uptake events were tissue and estrogen steroid specific. The temporal pattern of steroid hormone uptake in the uterus would suggest that an initial interaction in stromal and glandular epithelial cells may be required prior to nuclear stimulation in the luminal epithelial target cell

  15. Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

    Directory of Open Access Journals (Sweden)

    Merly Saare

    Full Text Available Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8, a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

  16. Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler.

    Directory of Open Access Journals (Sweden)

    Hans J Rolf

    2008-04-01

    Full Text Available The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama. In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1(+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process.

  17. Nuclear localization of the CK2α-subunit correlates with poor prognosis in Clear Cell Renal Cell Carcinoma

    DEFF Research Database (Denmark)

    Rabjerg, Maj; Guerra, Barbara; Oliván-Viguera, Aida

    2017-01-01

    Protein kinase CK2a, one of the two catalytic isoforms of the protein kinase CK2 has been shown to contribute to tumor development, tumor proliferation and suppression of apoptosis in various malignancies. We conducted this study to investigate CK2 expression in different subtypes of Renal Cell...... Carcinoma (RCC) and in the benign oncocytoma. qRT-PCR, immunohistochemistry and Western blot analyses revealed that CK2a expression was significantly increased at the mRNA and protein levels in clear cell RCC (ccRCC). Also the kinase activity of CK2 was significantly increased in ccRCC compared to normal...... renal cortex. Nuclear protein expression of CK2a correlated in univariate analysis with poor Progression Free Survival (HR = 8.11, p = 0.016). Functional analyses (cell proliferation assay) revealed an inhibitory effect of Caki-2 cell growth following CK2 inhibition with CX-4945. Our results suggest...

  18. New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status.

    Science.gov (United States)

    Telechea-Fernández, Marcelino; Rodríguez-Fernández, Lucia; García, Concha; Zaragozá, Rosa; Viña, Juan; Cervantes, Andrés; García-Trevijano, Elena R

    2018-02-06

    Calpain-2 belongs to a family of pleiotropic Cys-proteases with modulatory rather than degradative functions. Calpain (CAPN) overexpression has been controversially correlated with poor prognosis in several cancer types, including colorectal carcinoma (CRC). However, the mechanisms of substrate-recognition, calpain-2 regulation/deregulation and specific functions in CRC remain elusive. Herein, calpain subcellular distribution was studied as a key event for substrate-recognition and consequently, for calpain-mediated function. We describe a new localization for calpain-2 in the nucleoli of CRC cells. Calpain-2 nucleolar distribution resulted dependent on its enzymatic activity and on the mutational status of KRAS. In KRASWT/- cells serum-starvation induced CAPN2 expression, nucleolar accumulation and increased binding to the rDNA-core promoter and intergenic spacer (IGS), concomitant with a reduction in pre-rRNA levels. Depletion of calpain-2 by specific siRNA prevented pre-rRNA down-regulation after serum removal. Conversely, ribosomal biogenesis proceeded in the absence of serum in unresponsive KRASG13D/- cells whose CAPN2 expression, nucleolar localization and rDNA-occupancy remained unchanged during the time-course of serum starvation. We propose here that nucleolar calpain-2 might be a KRAS-dependent sensor to repress ribosomal biogenesis in growth limiting conditions. Under constitutive activation of the pathway commonly found in CRC, calpain-2 is deregulated and tumor cells become insensitive to the extracellular microenvironment.

  19. Effect of local UV irradiation of generative nuclei of Paramecium caudatum on sexual functions of the cells

    International Nuclear Information System (INIS)

    Skoblo, I.I.; Borkhsenius, O.N.

    1979-01-01

    The functions of micronucleus (MI) were studied in the period of the sexual process in Paramecium caudatum-infusoria the descendants of the cells, whose micronucleus was locally irradiated with UV rays at the dose of 3060 erg/mm 2 . It has been found that the descendants of the irradiated cells (the cells of UV clones) can be involved into the sexual process irrespoctive of the morphological type of their MI. Four out of forteen UV clones have changed the initial mating type for the opposite one. It is suggested that the change of the mating type is related to the changes of MI genome resulted from UV irradiation. The pathway of nuclear transformations in the course of sexual process was traced in cells of three UV clones. It has been found, that local UV irradiation of MI causes a number of deviations in the behaviour of MI derivatives in the metagamic part of the sexual process in the clones, whose MI have undergone substantial morphological changes. The exconjugants produced by crossing of UV clones with such changed MI and the normal test-clone were characterized by low viability

  20. Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells

    Directory of Open Access Journals (Sweden)

    Tessa Bergsbaken

    2017-04-01

    Full Text Available Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8+ T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103−CD69+ Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN-β and interleukin-12 (IL-12, which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103−CD69+ Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-β and IL-12 during infection, and deletion of CCR2+ IL-12-producing cells reduced the size of the CD103− Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity.

  1. Localization and movement of newly synthesized cholesterol in rat ovarian granulosa cells

    International Nuclear Information System (INIS)

    Lange, Y.; Schmit, V.M.; Schreiber, J.R.

    1988-01-01

    The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newly synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered [3H]acetate and [14C]acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol

  2. BIM-EL localization: The key to understanding anoikis resistance in inflammatory breast cancer cells.

    Science.gov (United States)

    Buchheit, Cassandra L; Schafer, Zachary T

    2016-01-01

    Inflammatory breast cancer (IBC) is a highly metastatic and rare type of breast cancer, accounting for 2-6% of newly diagnosed breast cancer cases each year. The highly metastatic nature of IBC cells remains poorly understood. Here we describe our recent data regarding the ability of IBC cells to overcome anoikis.

  3. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel

    2006-01-01

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  4. Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

    Science.gov (United States)

    Wollman, Adam J M; Leake, Mark C

    2015-01-01

    We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of

  5. Long-term changes in cell population kinetics of skin tissue after local beta-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, T [National Inst. of Radiological Sciences, Chiba (Japan)

    1975-06-01

    Using /sup 3/H-thymidine autoradiography, long-term alterations in cell kinetics were studied in guinea pig skin after ..beta..irradiation with 3000 rads. After complete depopulation, epidermal basal cells at the radiation margin became proliferative 10 days postirradiation and spread over the depopulated area. When epithelization (20 days) was complete the cell cycle time of the basal cells reverted to normal, but the differentiation rate was much slower than that in unirradiated skin. This appeared to be a cause of the persistent (acanthotic) hyperplasia. Similar but slower changes were found in dermal tissue. Reparative proliferation of fibroblasts and capillary endothelial cells began at 20 and 30 days, respectively. Active fibroblastic proliferation was found as late as 110 days. This, along with the abortive nature of the reparative angiogenesis, seemed to be a cause of the later fibrosis (150 to 400 days).

  6. Characterization of the Distance Relationship Between Localized Serotonin Receptors and Glia Cells on Fluorescence Microscopy Images of Brain Tissue.

    Science.gov (United States)

    Jacak, Jaroslaw; Schaller, Susanne; Borgmann, Daniela; Winkler, Stephan M

    2015-08-01

    We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.

  7. Prediction of the local power factor in BWR fuel cells by means of a multilayer neural network

    International Nuclear Information System (INIS)

    Montes, J.L.; Ortiz, J.J.; Perusquia C, R.; Francois, J.L.; Martin del Campo M, C.

    2007-01-01

    To the beginning of a new operation cycle in a BWR reactor the reactivity of this it increases by means of the introduction of fresh fuel, the one denominated reload fuel. The problem of the definition of the characteristics of this reload fuel represents a combinatory optimization problem that requires significantly a great quantity of CPU time for their determination. This situation has motivated to study the possibility to substitute the Helios code, the one which is used to generate the new cells of the reload fuel parameters, by an artificial neuronal network, with the purpose of predicting the parameters of the fuel reload cell of a BWR reactor. In this work the results of the one training of a multilayer neuronal net that can predict the local power factor (LPPF) in such fuel cells are presented. The prediction of the LPPF is carried out in those condition of beginning of the life of the cell (0.0 MWD/T, to 40% of holes in the one moderator, temperature of 793 K in the fuel and a moderator temperature of 560 K. The cells considered in the present study consist of an arrangement of 10x10 bars, of those which 92 contains U 235 , some of these bars also contain a concentration of Gd 2 O 3 and 8 of them contain only water. The axial location inside the one assembles of recharge of these cells it is exactly up of the cells that contain natural uranium in the base of the reactor core. The training of the neuronal net is carried out by means of a retro-propagation algorithm that uses a space of training formed starting from previous evaluations of cells by means of the Helios code. They are also presented the results of the application of the neuronal net found for the prediction of the LPPF of some cells used in the real operation of the Unit One of the Laguna Verde Nuclear Power station. (Author)

  8. The cell wall-localized atypical β-1,3 glucanase ZERZAUST controls tissue morphogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Vaddepalli, Prasad; Fulton, Lynette; Wieland, Jennifer; Wassmer, Katrin; Schaeffer, Milena; Ranf, Stefanie; Schneitz, Kay

    2017-06-15

    Orchestration of cellular behavior in plant organogenesis requires integration of intercellular communication and cell wall dynamics. The underlying signaling mechanisms are poorly understood. Tissue morphogenesis in Arabidopsis depends on the receptor-like kinase STRUBBELIG. Mutations in ZERZAUST were previously shown to result in a strubbelig -like mutant phenotype. Here, we report on the molecular identification and functional characterization of ZERZAUST We show that ZERZAUST encodes a putative GPI-anchored β-1,3 glucanase suggested to degrade the cell wall polymer callose. However, a combination of in vitro , cell biological and genetic experiments indicate that ZERZAUST is not involved in the regulation of callose accumulation. Nonetheless, Fourier-transformed infrared-spectroscopy revealed that zerzaust mutants show defects in cell wall composition. Furthermore, the results indicate that ZERZAUST represents a mobile apoplastic protein, and that its carbohydrate-binding module family 43 domain is required for proper subcellular localization and function whereas its GPI anchor is dispensable. Our collective data reveal that the atypical β-1,3 glucanase ZERZAUST acts in a non-cell-autonomous manner and is required for cell wall organization during tissue morphogenesis. © 2017. Published by The Company of Biologists Ltd.

  9. Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

    Science.gov (United States)

    Szewczyk, Maciej; Fedoryszak-Kuśka, Natalia; Tkaczuk, Katarzyna; Dobrucki, Jurek; Waligórska, Agnieszka; Stępień, Piotr P

    2017-01-01

    The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

  10. Localization of tritiated vitamin A in lymph nodes of the mouse: an autoradiographic study of vitamin A-storing cells

    International Nuclear Information System (INIS)

    Hirosawa, K.; Yamada, E.

    1981-01-01

    Localization of tritiated vitamin A in lymph nodes of the mouse was investigated by the use of light- and electron-microscopic autoradiography. Young male mice were fed a diet containing 3H-vitamin A acetate for a week. Lymph nodes were removed and prepared for autoradiography. Lipid droplets in fibroblast-like cells showed high concentrations of radioactivity. These cells were distributed around lymphatic sinuses and blood vessels. The cells can, therefore, be classified as ''vitamin A-storing cells'' according to criteria proposed earlier by Yamada and Hirosawa (1976). Control animals from the same litter were maintained on ordinary laboratory food for the same period and examined by electron microscopy. Lipid-droplet-containing cells were found in the same areas as in the experimental animals but in fewer numbers. This suggests that the increased number of vitamin A-containing lipid droplets is due to hypervitaminosis A in the experimental animals. The presence of some cells containing these droplets in the control animals would imply that even under normal feeding conditions the animals ingested excess amounts of vitamin A, which was retained in lipid droplets. The stored vitamin A probably participates in metabolic processes such as the formation of glycoproteins in ground substance

  11. Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

    KAUST Repository

    Ferraro, Daniela; Tredici, Ilenia G.; Ghigna, Paolo; Castillio-Michel, Hiram; Falqui, Andrea; Di Benedetto, Cristiano; Alberti, Giancarla; Ricci, Vittorio; Anselmi-Tamburini, Umberto; Sommi, Patrizia

    2017-01-01

    CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

  12. Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

    KAUST Repository

    Ferraro, Daniela

    2017-01-09

    CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

  13. Dynein and EFF-1 control dendrite morphology by regulating the localization pattern of SAX-7 in epidermal cells.

    Science.gov (United States)

    Zhu, Ting; Liang, Xing; Wang, Xiang-Ming; Shen, Kang

    2017-12-01

    Our previous work showed that the cell adhesion molecule SAX-7 forms an elaborate pattern in Caenorhabditis elegans epidermal cells, which instructs PVD dendrite branching. However, the molecular mechanism forming the SAX-7 pattern in the epidermis is not fully understood. Here, we report that the dynein light intermediate chain DLI-1 and the fusogen EFF-1 are required in epidermal cells to pattern SAX-7. While previous reports suggest that these two molecules act cell-autonomously in the PVD, our results show that the disorganized PVD dendritic arbors in these mutants are due to the abnormal SAX-7 localization patterns in epidermal cells. Three lines of evidence support this notion. First, the epidermal SAX-7 pattern was severely affected in dli-1 and eff-1 mutants. Second, the abnormal SAX-7 pattern was predictive of the ectopic PVD dendrites. Third, expression of DLI-1 or EFF-1 in the epidermis rescued both the SAX-7 pattern and the disorganized PVD dendrite phenotypes, whereas expression of these molecules in the PVD did not. We also show that DLI-1 functions cell-autonomously in the PVD to promote distal branch formation. These results demonstrate the unexpected roles of DLI-1 and EFF-1 in the epidermis in the control of PVD dendrite morphogenesis. © 2017. Published by The Company of Biologists Ltd.

  14. Pannexin2 oligomers localize into endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    Directory of Open Access Journals (Sweden)

    Daniela eBoassa

    2015-02-01

    Full Text Available Pannexin2 (Panx2 is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS have been documented. Whereas Pannexin1 (Panx1 is fairly ubiquitous and Pannexin3 (Panx3 is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa and HEK293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the

  15. Radiological response and survival in locally advanced non-small-cell lung cancer patients treated with three-drug induction chemotherapy followed by radical local treatment

    Directory of Open Access Journals (Sweden)

    Bonanno L

    2016-06-01

    Full Text Available Laura Bonanno,1 Giulia Zago,1 Giuseppe Marulli,2 Paola Del Bianco,3 Marco Schiavon,2 Giulia Pasello,1 Valentina Polo,1,4 Fabio Canova,1 Fabrizio Tonetto,5 Lucio Loreggian,5 Federico Rea,2 PierFranco Conte,1,4 Adolfo Favaretto1 1Medical Oncology Unit 2, Veneto Institute of Oncology IOV-IRCCS, 2Thoracic Surgery Department, University of Padova, 3Clinical Trials and Biostatistics Unit, Veneto Institute of Oncology IOV-IRCCS, 4Department of Surgery, Oncology and Gastroenterology, University of Padova, 5Radiotherapy Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy Objectives: If concurrent chemoradiotherapy cannot be performed, induction chemotherapy followed by radical-intent surgical treatment is an acceptable option for non primarily resectable non-small-cell lung cancers (NSCLCs. No markers are available to predict which patients may benefit from local treatment after induction. This exploratory study aims to assess the feasibility and the activity of multimodality treatment, including triple-agent chemotherapy followed by radical surgery and/or radiotherapy in locally advanced NSCLCs. Methods: We retrospectively collected data from locally advanced NSCLCs treated with induction chemotherapy with carboplatin (area under the curve 6, d [day]1, paclitaxel (200 mg/m2, d1, and gemcitabine (1,000 mg/m2 d1, 8 for three to four courses, followed by radical surgery and/or radiotherapy. We analyzed radiological response and toxicity. Estimated progression-free survival (PFS and overall survival (OS were correlated to response, surgery, and clinical features. Results: In all, 58 NSCLCs were included in the study: 40 staged as IIIA, 18 as IIIB (according to TNM Classification of Malignant Tumors–7th edition staging system. A total of 36 (62% patients achieved partial response (PR, and six (10% progressions were recorded. Grade 3–4 hematological toxicity was observed in 36 (62% cases. After chemotherapy, 37 (64% patients underwent surgery

  16. Decorin in Human Colon Cancer: Localization In Vivo and Effect on Cancer Cell Behavior In Vitro.

    Science.gov (United States)

    Nyman, Marie C; Sainio, Annele O; Pennanen, Mirka M; Lund, Riikka J; Vuorikoski, Sanna; Sundström, Jari T T; Järveläinen, Hannu T

    2015-09-01

    Decorin is generally recognized as a tumor suppressing molecule. Nevertheless, although decorin has been shown to be differentially expressed in malignant tissues, it has often remained unclear whether, in addition to non-malignant stromal cells, cancer cells also express it. Here, we first used two publicly available databases to analyze the current information about decorin expression and immunoreactivity in normal and malignant human colorectal tissue samples. The analyses demonstrated that decorin expression and immunoreactivity may vary in cancer cells of human colorectal tissues. Therefore, we next examined decorin expression in normal, premalignant and malignant human colorectal tissues in more detail using both in situ hybridization and immunohistochemistry for decorin. Our results invariably demonstrate that malignant cells within human colorectal cancer tissues are devoid of both decorin mRNA and immunoreactivity. Identical results were obtained for cells of neuroendocrine tumors of human colon. Using RT-qPCR, we showed that human colon cancer cell lines are also decorin negative, in accordance with the above in vivo results. Finally, we demonstrate that decorin transduction of human colon cancer cell lines causes a significant reduction in their colony forming capability. Thus, strategies to develop decorin-based adjuvant therapies for human colorectal malignancies are highly rational. © The Author(s) 2015.

  17. Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

    Science.gov (United States)

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

    2012-01-01

    Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

  18. Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary

    NARCIS (Netherlands)

    Slot, K.A.; Voorendt, M.; Boer-Brouwer, de M.; Vugt, van H.H.; Teerds, K.J.

    2006-01-01

    In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts,

  19. Feasibility of stereotactic body radiotherapy for locally-advanced non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Katrina Woodford

    2017-10-01

    Full Text Available SBRT was feasible for approximately half of the locally-advanced NSCLC patients we assessed and for these patients has the potential to reduce a 30 fraction course to 12 fractions. Using SBRT in this setting requires compromises in techniques and further compromises may allow SBRT in a greater proportion of patients.

  20. Liquid fiducial marker performance during radiotherapy of locally advanced non small cell lung cancer

    DEFF Research Database (Denmark)

    Rydhög, Jonas Scherman; Mortensen, Steen Riisgaard; Larsen, Klaus Richter

    2016-01-01

    We analysed the positional and structural stability of a long-term biodegradable liquid fiducial marker (BioXmark) for radiotherapy in patients with locally advanced lung cancer. Markers were injected via endoscopic- or endobronchial ultrasound in lymph nodes and reachable primary tumours. Marker...

  1. Presence of intratumoral neutrophils is an independent prognostic factor in localized renal cell carcinoma

    DEFF Research Database (Denmark)

    Jensen, Hanne Krogh; Donskov, Frede; Marcussen, Niels

    2009-01-01

    : The intratumoral neutrophils ranged from zero to 289 cells/mm(2) tumor tissue. The presence of intratumoral neutrophils was statistically significantly associated with increasing tumor size, low hemoglobin, high creatinine, and CA IX

  2. Subcellular localization of Cd in the root cells of Allium sativum by ...

    Indian Academy of Sciences (India)

    Unknown

    Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiat- ing and ... The bulbs were germinated, and grown in 3 containers ..... Greger M 1999 Metal availability and bioconcentration in plants ...

  3. Quantitative assessment of CA1 local circuits: knowledge base for interneuron-pyramidal cell connectivity.

    Science.gov (United States)

    Bezaire, Marianne J; Soltesz, Ivan

    2013-09-01

    In this work, through a detailed literature review, data-mining, and extensive calculations, we provide a current, quantitative estimate of the cellular and synaptic constituents of the CA1 region of the rat hippocampus. Beyond estimating the cell numbers of GABAergic interneuron types, we calculate their convergence onto CA1 pyramidal cells and compare it with the known input synapses on CA1 pyramidal cells. The convergence calculation and comparison are also made for excitatory inputs to CA1 pyramidal cells. In addition, we provide a summary of the excitatory and inhibitory convergence onto interneurons. The quantitative knowledge base assembled and synthesized here forms the basis for data-driven, large-scale computational modeling efforts. Additionally, this work highlights specific instances where the available data are incomplete, which should inspire targeted experimental projects toward a more complete quantification of the CA1 neurons and their connectivity. Copyright © 2013 Wiley Periodicals, Inc.

  4. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling.

    Science.gov (United States)

    Monterisi, Stefania; Lobo, Miguel J; Livie, Craig; Castle, John C; Weinberger, Michael; Baillie, George; Surdo, Nicoletta C; Musheshe, Nshunge; Stangherlin, Alessandra; Gottlieb, Eyal; Maizels, Rory; Bortolozzi, Mario; Micaroni, Massimo; Zaccolo, Manuela

    2017-05-02

    cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.

  5. Mapping the Local Organization of Cell Membranes Using Excitation-Polarization-Resolved Confocal Fluorescence Microscopy

    OpenAIRE

    Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

    2013-01-01

    International audience; Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive t...

  6. Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

    OpenAIRE

    Moran, Yehu; Genikhovich, Grigory; Gordon, Dalia; Wienkoop, Stefanie; Zenkert, Claudia; Özbek, Suat; Technau, Ulrich; Gurevitz, Michael

    2011-01-01

    Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficie...

  7. Methods of Cell Propulsion through the Local Stroma in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Kerry J. Davies

    2014-01-01

    Full Text Available In the normal breast, cellular structures change cyclically in response to ovarian hormones. Cell proliferation, apoptosis, invasion, and differentiation are integral processes that are precisely regulated. Normal epithelial cells depend on the formation of intercellular adhesion contacts to form a continuous sheet of stratifying cell layers that are attached to one and other horizontally and vertically. Cells migrate by extending membrane protrusions to explore the extracellular space locating their targets in a chemotactic manner. The formation of cell protrusions is driven by the assembly of actin filaments at the leading edge. Reorganisation is regulated by a highly integrated signalling cascade that transduces extracellular stimuli to the actin filaments. This signalling cascade is governed by GTPases which act as molecular switches leading to actin polymerisation and the formation of filopodia and lamellipodia. This process is linked to downstream molecules known collectively as WASP proteins, which, in the presence of cortactin, form a complex leading to nucleation and formation of branched filaments. In breast cancer, the cortactin is over expressed leading to increased cellular motility and invasiveness. This hugely complex and integrated signalling cascade transduces extracellular stimuli. There are multiple genes related to cell motility which are dysregulated in human breast cancers.

  8. Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree.

    Directory of Open Access Journals (Sweden)

    Tomoki Yagai

    Full Text Available Cholangiocarcinoma (CC is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC at the peribiliary gland (PBG of large bile ducts and liver stem/progenitor cell (LPC at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5 is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines.SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

  9. The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

    2012-10-01

    Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

  10. Macular Ganglion Cell Inner Plexiform Layer Thickness in Glaucomatous Eyes with Localized Retinal Nerve Fiber Layer Defects.

    Directory of Open Access Journals (Sweden)

    Chunwei Zhang

    Full Text Available To investigate macular ganglion cell-inner plexiform layer (mGCIPL thickness in glaucomatous eyes with visible localized retinal nerve fiber layer (RNFL defects on stereophotographs.112 healthy and 149 glaucomatous eyes from the Diagnostic Innovations in Glaucoma Study (DIGS and the African Descent and Glaucoma Evaluation Study (ADAGES subjects had standard automated perimetry (SAP, optical coherence tomography (OCT imaging of the macula and optic nerve head, and stereoscopic optic disc photography. Masked observers identified localized RNFL defects by grading of stereophotographs.47 eyes had visible localized RNFL defects on stereophotographs. Eyes with visible localized RNFL defects had significantly thinner mGCIPL thickness compared to healthy eyes (68.3 ± 11.4 μm versus 79.2 ± 6.6 μm respectively, P<0.001 and similar mGCIPL thickness to glaucomatous eyes without localized RNFL defects (68.6 ± 11.2 μm, P = 1.000. The average mGCIPL thickness in eyes with RNFL defects was 14% less than similarly aged healthy controls. For 29 eyes with a visible RNFL defect in just one hemiretina (superior or inferior mGCIPL was thinnest in the same hemiretina in 26 eyes (90%. Eyes with inferior-temporal RNFL defects also had significantly thinner inferior-temporal mGCIPL (P<0.001 and inferior mGCIPL (P = 0.030 compared to glaucomatous eyes without a visible RNFL defect.The current study indicates that presence of a localized RNFL defect is likely to indicate significant macular damage, particularly in the region of the macular that topographically corresponds to the location of the RNFL defect.

  11. Mast cells facilitate local VEGF release as an early event in the pathogenesis of postoperative peritoneal adhesions.

    LENUS (Irish Health Repository)

    Cahill, Ronan A

    2012-02-03

    BACKGROUND: Peritoneal injury sustained at laparotomy may evoke local inflammatory responses that result in adhesion formation. Peritoneal mast cells are likely to initiate this process, whereas vascular permeability\\/endothelial growth factor (VEGF) may facilitate the degree to which subsequent adhesion formation occurs. METHODS: Mast cell deficient mice (WBB6F1-\\/-), along with their mast cell sufficient counterparts (WBB6F1+\\/+), underwent a standardized adhesion-inducing operation (AIS) with subsequent sacrifice and adhesion assessment 14 days later in a blinded fashion. Additional CD-1 and WBB6F1+\\/+, and WBB6F1-\\/- mice were killed 2, 6, 12, and 24 hours after operation for measurement of VEGF by ELISA in systemic serum and peritoneal lavage fluid. Two further groups of CD-1 mice underwent AIS and received either a single perioperative dose of anti-VEGF monoclonal antibody (10 mug\\/mouse) or a similar volume of IgG isotypic antibody and adhesion formation 2 weeks later was evaluated. RESULTS: WBB6F1-\\/- mice had less adhesions then did their WBB6F1+\\/+ counterparts (median [interquartile range] adhesion score 3[3-3] vs 1.5[1-2] respectively; P < .003). Local VEGF release peaked 6 hours after AIS in both WBB6F1+\\/+ and CD-1 mice whereas levels remained at baseline in WBB6F1-\\/- mice. CD-1 mice treated with a single dose of anti-VEGF therapy during operation had less adhesions than controls (2[1.25-2] vs 3[2.25-3], P = .0002). CONCLUSIONS: Mast cells and VEGF are central to the formation of postoperative intra-abdominal adhesions with mast cells being responsible, either directly or indirectly, for VEGF release into the peritoneal cavity after operation. In tandem with the recent clinical success of anti-VEGF monoclonal antibodies in oncologic practice, our observations suggest an intriguing avenue for research and development of anti-adhesion strategy.

  12. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    Science.gov (United States)

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  13. Local influence of south-east France topography and land cover on the distribution and characteristics of intense rainfall cells

    Science.gov (United States)

    Renard, Florent

    2017-04-01

    The Greater Lyon area is strongly built up, grouping 58 communes and a population of 1.3 million in approximately 500 km2. The flood risk is high as the territory is crossed by two large watercourses and by streams with torrential flow. Floods may also occur in case of runoff after heavy rain or because of a rise in the groundwater level. The whole territory can therefore be affected, and it is necessary to possess in-depth knowledge of the depths, causes and consequences of rainfall to achieve better management of precipitation in urban areas and to reduce flood risk. This study is thus focused on the effects of topography and land cover on the occurrence, intensity and area of intense rainfall cells. They are identified by local radar meteorology (C-band) combined with a processing algorithm running in a geographic information system (GIS) which identified 109,979 weighted mean centres of them in a sample composed of the five most intense rainfall events from 2001 to 2005. First, analysis of spatial distribution at an overall scale is performed, completed by study at a more detailed scale. The results show that the distribution of high-intensity rainfall cells is spread in cluster form. Subsequently, comparison of intense rainfall cells with the topography shows that cell density is closely linked with land slope but that, above all, urbanised zones feature nearly twice as many rainfall cells as farm land or forest, with more intense intensity.

  14. Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

    Science.gov (United States)

    Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

    1998-01-01

    In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

  15. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Science.gov (United States)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  16. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Energy Technology Data Exchange (ETDEWEB)

    Volland, Stefanie, E-mail: Stefanie.Volland@stud.sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria); Luetz, Cornelius, E-mail: cornelius.luetz@uibk.ac.at [Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck (Austria); Michalke, Bernhard, E-mail: bernhard.michalke@helmholtz-muenchen.de [Helmholtz Zentrum Muenchen, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstaedter Landstrasse 1, 85764 Neuherberg (Germany); Luetz-Meindl, Ursula, E-mail: ursula.luetz-meindl@sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria)

    2012-03-15

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 {mu}M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  17. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    International Nuclear Information System (INIS)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  18. Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

    Science.gov (United States)

    Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

    2015-01-01

    S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

  19. The Experimental Measurement of Local and Bulk Oxygen Transport Resistances in the Catalyst Layer of Proton Exchange Membrane Fuel Cells.

    Science.gov (United States)

    Wang, Chao; Cheng, Xiaojing; Lu, Jiabin; Shen, Shuiyun; Yan, Xiaohui; Yin, Jiewei; Wei, Guanghua; Zhang, Junliang

    2017-12-07

    Remarkable progress has been made in reducing the cathodic Pt loading of PEMFCs; however, a huge performance loss appears at high current densities, indicating the existence of a large oxygen transport resistance associated with the ultralow Pt loading catalyst layer. To reduce the Pt loading without sacrificing cell performance, it is essential to illuminate the oxygen transport mechanism in the catalyst layer. Toward this goal, an experimental approach to measure the oxygen transport resistance in catalyst layers is proposed and realized for the first time in this study. The measuring approach involves a dual-layer catalyst layer design, which consists of a dummy catalyst layer and a practical catalyst layer, followed by changing the thickness of dummy layer to respectively quantify the local and bulk resistances via limiting current measurements combined with linear extrapolation. The experimental results clearly reveal that the local resistance dominates the total resistance in the catalyst layer.

  20. Local effect of bisphenol A on the estradiol synthesis of ovarian granulosa cells from PCOS.

    Science.gov (United States)

    Wang, Yuan; Zhu, Qinling; Dang, Xuan; He, Yaqiong; Li, Xiaoxue; Sun, Yun

    2017-01-01

    Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50 ± 63.70 pg/ml) was significantly higher than that from non-PCOS patients (338.00 ± 57.88 pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1 μM for 24 h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.

  1. Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

    Directory of Open Access Journals (Sweden)

    Yoshinari Ando

    Full Text Available Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

  2. A Numerical Analysis on the Local Deformation of a Spacer Grid Structure for Nuclear Fuel Cells

    International Nuclear Information System (INIS)

    Jang, Myung-Geun; Na, Geum Ju; Kim, Jong-Bong; Shin, Hyunho

    2016-01-01

    The result of a preliminary numerical investigation on local deformation characteristics of a multi-layered spacer-grid structure with five guide tubes is reported based on implicit finite element analysis. For the numerical analysis, displacements of top and bottom cross sections of each guide tube in a single-layer model were constrained while a lateral displacement was imposed on the single layer. Unlike the impact hammer test that is generally employed to characterize the deformation characteristics of the space-grid structure, the buckling phenomenon occurs locally in this study; it takes place at the inner grids around each tube and the degree of bucking is more apparent for tubes near the lateral surface where the lateral displacement was imposed. (paper)

  3. Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells.

    Directory of Open Access Journals (Sweden)

    Borja Belda-Palazón

    Full Text Available Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS and thermospermine synthase (TSPMS use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.

  4. Locally advanced oral cavity squamous cell carcinoma: Barriers related to effective treatment

    Directory of Open Access Journals (Sweden)

    K C Lakshmaiah

    2015-01-01

    Full Text Available Background: Oral cavity cancer is a significant health problem in India. Majority of patients present with locally advanced disease requiring multimodality treatment. Compliance to recommended treatment is an important factor affecting outcome. Aims: The aim was to evaluate the outcome of locally advanced oral cavity cancer patients with regards to treatment adherence and to assess reasons of noncompliance. Materials and Methods: This was a prospective observational study. We included patients referred to Department of Medical Oncology for induction chemotherapy in view of locally advanced oral cavity cancer. Results: Only 15 (26% patients completed planned treatment schedule. Their 1 year overall survival was 93%. The remaining 43 patients who received inadequate treatment had a dismal 21% 1 year overall survival. Illiteracy, poverty, long waiting list for surgery, prolonged delay for health scheme treatment plan approval and dissatisfaction with attitude of hospital staffs are major barriers related to effective treatment of these patients. Conclusions: A detailed discussion with patient and their relatives regarding recommended treatment, proper implementation of health schemes, increasing trained manpower to avoid long waiting list for surgery, provision of additional financial support for family member accompanying the patient and a sympathetic approach toward patients are needed to help these patients overcome the battle.

  5. Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

    Science.gov (United States)

    Koh, Ai Leen; Shachaf, Catherine M; Elchuri, Sailaja; Nolan, Garry P; Sinclair, Robert

    2008-12-01

    We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

  6. Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

    International Nuclear Information System (INIS)

    Koh, Ai Leen; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P.; Sinclair, Robert

    2008-01-01

    We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

  7. Changes in the neuroglial cell populations of the rat spinal cord after local X-irradiation

    International Nuclear Information System (INIS)

    Hubbard, B.M.; Hopewell, J.W.

    1979-01-01

    A 16 mm length of cervical spinal cord of young adult female rats was irradiated with 4000 rad of 250 kV X-rays. Counts of astrocyte and oligodendrocyte nuclei were made in the dorsal columns of both irradiated and control cervical cords during the latent period before the onset of radionecrosis. The numbers of both astrocyte and oligodendrocyte nuclei were reduced one month after exposure to radiation. Both cell populations showed an apparent recovery but this was subsequently followed by a rapid loss of cells prior to the development of white-matter necrosis. The oligodendrocyte population in unirradiated spinal cords increased with age, and mitotic figures were observed among the neuroglia of both irradiated and control cervical spinal cords. A slow, natural turnover of neuroglial cells in the cervical spinal cord is proposed and the relevance of this to the manifestation of delayed white matter necrosis is discussed. (author)

  8. Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

    Science.gov (United States)

    Béland, Kathie; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

    2012-01-01

    The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C) in presence or absence of activated liver-specific autoreactive CD8(+) T cells. Regardless of autoreactive CD8(+) T cells, mice injected with CpG and Poly(I:C) showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C) and autoreactive CD8(+)T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+) T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+) T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

  9. Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

    Directory of Open Access Journals (Sweden)

    Kathie Béland

    Full Text Available The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C in presence or absence of activated liver-specific autoreactive CD8(+ T cells. Regardless of autoreactive CD8(+ T cells, mice injected with CpG and Poly(I:C showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C and autoreactive CD8(+T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+ T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+ T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

  10. Preferential localization of Lactococcus lactis cells entrapped in a caseinate/alginate phase separated system.

    Science.gov (United States)

    Léonard, Lucie; Gharsallaoui, Adem; Ouaali, Fahima; Degraeve, Pascal; Waché, Yves; Saurel, Rémi; Oulahal, Nadia

    2013-09-01

    This study aimed to entrap bioprotective lactic acid bacteria in a sodium caseinate/sodium alginate aqueous two-phase system. Phase diagram at pH=7 showed that sodium alginate and sodium caseinate were not miscible when their concentrations exceeded 1% (w/w) and 6% (w/w), respectively. The stability of the caseinate/alginate two-phase system was also checked at pH values of 6.0 and 5.5. Lactococcus lactis subsp. lactis LAB3 cells were added in a 4% (w/w) caseinate/1.5% (w/w) alginate two-phase system at pH=7. Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. lactis LAB3 cells stained with Live/Dead(®) Baclight kit™ were located exclusively in the protein phase. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems. Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Caseinate/alginate phase separated systems appeared thus well suited for Lc. lactis LAB3 cells entrapment. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    Directory of Open Access Journals (Sweden)

    Wagner Shin Nishitani

    Full Text Available A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7 expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  12. Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

    Science.gov (United States)

    Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

    2011-04-01

    Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

  13. Low dose combined chemotherapy/radiotherapy in the management of locally advanced urethral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Johnson, D.W.; Kessler, J.F.; Ferrigni, R.G.; Anderson, J.D.

    1989-01-01

    The successful treatment of a patient with bulky squamous cell carcinoma of the urethra using low dose preoperative radiation therapy and concurrent chemotherapy is described. Dramatic rapid tumor response facilitated surgical resection of the remaining microscopic disease. This clinical behavior is remarkably similar to that seen with squamous cell carcinoma of the anal canal and esophagus when a similar regimen is used. At the latter tumor sites the successful use of combination radiotherapy and chemotherapy has reduced the morbidity of subsequent surgery, and in selected cases has obviated the need for a radical operation. Further investigation of such combination treatment is warranted for urethral carcinoma

  14. Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

    Directory of Open Access Journals (Sweden)

    C. Soldani

    2010-05-01

    Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.