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Sample records for cell cycle-dependent localization

  1. Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner

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    Kim, Gwang Su; Kang, Jeeheon; Bang, Sung Woong; Hwang, Deog Su, E-mail: dshwang@snu.ac.kr

    2015-01-16

    Highlights: • Cdc6 protein is a component of the pre-replicative complex required for chromosomal replication initiation. • Cdc6 localized to centrosomes of S and G2 phases in a cell cycle-dependent manner. • The centrosomal localization was governed by centrosomal localization signal sequences of Cdc6. • Deletions or substitution mutations on the centrosomal localization signal interfered with centrosomal localization of the Cdc6 proteins. - Abstract: The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomes during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311–366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311–366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.

  2. Cell cycle-dependent gene networks relevant to cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

  3. Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells.

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    Cobb, Melanie M; Austin, Daniel C; Sack, Jon T; Trimmer, James S

    2015-12-04

    The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.

  4. Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

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    Frederiks, Wilma M; van Marle, Jan; van Oven, Carel; Comin-Anduix, Begonya; Cascante, Marta

    2006-01-01

    Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.

  5. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

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    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.

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    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 SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

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    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.

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

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    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.

  9. Effects of cell-cycle-dependent expression on random fluctuations in protein levels.

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    Soltani, Mohammad; Singh, Abhyudai

    2016-12-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

  10. Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

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    Olga Wiens

    Full Text Available The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1, are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr, serine (p-Ser and threonine-proline (p-Thr-Pro epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

  11. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology

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    Wu, Pei-Hsun; Hung, Shen-Hsiu; Ren, Tina; Shih, Ie-Ming; Tseng, Yiider

    2011-02-01

    NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G0/G1 and G2 phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis.

  12. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.

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    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

    2009-07-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP.

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

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

    2015-07-01

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

  14. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

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    2005-01-01

    sequencing or hybridization array capillary chromatography. After a 6-min-long preliminary technologies.30,31 separation, fractions from the first...characterize single cells. These tools include mass cating cells contain diploid, S-phase and tetraploid frac- spectrometry, electrochemistry and capillary...separation tions; and some advanced tumors contain tetraploid and methods. This review focuses on the use of capillary aneuploid cells [2

  15. Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba.

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    Polit, Justyna Teresa; Kaźmierczak, Andrzej; Walczak-Drzewiecka, Aurelia

    2012-01-01

    The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition.

  16. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells

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    Gersende Caron

    2015-11-01

    Full Text Available Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxymethylation, and cell fate determination.

  17. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

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    Caron, Gersende; Hussein, Mourad; Kulis, Marta; Delaloy, Céline; Chatonnet, Fabrice; Pignarre, Amandine; Avner, Stéphane; Lemarié, Maud; Mahé, Elise A; Verdaguer-Dot, Núria; Queirós, Ana C; Tarte, Karin; Martín-Subero, José I; Salbert, Gilles; Fest, Thierry

    2015-11-03

    Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

  18. Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yan JIN; Yong WEI; Lei XIONG; Ying YANG; Jia Rui WU

    2005-01-01

    Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations.Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expression of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.

  19. Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry.

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    Christian Renz

    Full Text Available The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.

  20. 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...

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

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    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.

  2. Differential repair of UV damage in Saccharomyces cerevisiae is cell cycle dependent.

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    Terleth, C; Waters, R; Brouwer, J; van de Putte, P

    1990-09-01

    In the yeast Saccharomyces cerevisiae the transcriptionally active MAT alpha locus is repaired preferentially to the inactive HML alpha locus after UV irradiation. Here we analysed the repair of both loci after irradiating yeast cells at different stages of the mitotic cell cycle. In all stages repair of the active MAT alpha locus occurs at a rate of 30% removal of dimers per hour after a UV dose of 60 J/m2. The inactive HML alpha is repaired as efficiently as MAT alpha following irradiation in G2 whereas repair of HML alpha is less efficient in the other stages. Thus differential repair is observed in G1 and S but not in G2. Apparently, in G2 a chromatin structure exists in which repair does not discriminate between transcriptionally active and inactive DNA or, alternatively, an additional repair mechanism might exist which is only operational during G2.

  3. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

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    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

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

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    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.

  5. 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...

  6. Cell-Cycle-Dependent Variations in the FTIR Spectroscopy of HeLa Cells Treated with Trichostatin A

    Institute of Scientific and Technical Information of China (English)

    ZHANG Feng-qiu; QI Jian; YANG Zhan-guo

    2011-01-01

    It is quite complex to evaluate the mechanism of action for antitumor drugs on cancer cells.Studies have pointed out that there is an unique advantage of Fourier transform infrared spectrum to obtain a fingerprint of all molecules present in the cells when cancer cells were exposed to anti-cancer drugs.Trichostatin A (TSA) is a most potent reversible inhibitor of mammalian histone deacetylases.It can inhibit cancer cell growth in vitro and in vivo.In the present study,HeLa cells were exposed to 0,50,100,200,300 and 400 nmol · L-1 TSA,and FTIR spectra were applied to evaluate the effect of TSA on cancer cells.Results show that there is some significant relationship between the changes in FTIR absorption and cell cycle arresting.On the other hand,this investigation shows that the concentration of TSA had to be more than 200 nmol · L-1 in order to ensure A1080 cm-1/A1540cm-1 ≥1 for inhibiting cell proliferation.

  7. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  8. 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.

  9. 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

  10. 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

    at the protein level exhibit non-optimal codon preferences. Remarkably, cell cycle-regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non......-optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl-, threonyl-, and glutamyl-prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose...

  11. Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus.

    OpenAIRE

    1990-01-01

    Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence c...

  12. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

    Science.gov (United States)

    Smith, Stephen C; Joshi, Kamal K; Zik, Justin J; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R

    2014-09-30

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

  13. p53 and cell cycle dependent transcription of kinesin family member 23 (KIF23 is controlled via a CHR promoter element bound by DREAM and MMB complexes.

    Directory of Open Access Journals (Sweden)

    Martin Fischer

    Full Text Available The microtubule-dependent molecular motor KIF23 (Kinesin family member 23 is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1 was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.

  14. p53 and cell cycle dependent transcription of kinesin family member 23 (KIF23) is controlled via a CHR promoter element bound by DREAM and MMB complexes.

    Science.gov (United States)

    Fischer, Martin; Grundke, Inga; Sohr, Sindy; Quaas, Marianne; Hoffmann, Saskia; Knörck, Arne; Gumhold, Catalina; Rother, Karen

    2013-01-01

    The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1) was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.

  15. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.

  16. Estrogen Receptor Beta Displays Cell Cycle-Dependent Expression and Regulates the G1 Phase through a Non-Genomic Mechanism in Prostate Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Antoni Hurtado

    2008-01-01

    Full Text Available Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ remain elusive.

  17. Use of 5-ethynyl-2'-deoxyuridine labelling and flow cytometry to study cell cycle-dependent regulation of human cytomegalovirus gene expression.

    Science.gov (United States)

    Wiebusch, Lüder; Hagemeier, Christian

    2014-01-01

    The cell cycle position at the time of infection has a profound influence on human cytomegalovirus (HCMV) gene expression and therefore needs consideration in the design and control of HCMV experiments. While G0/G1 cells support the immediate onset of viral transcription, cells progressing through the S and G2 cell cycle phases prevent HCMV from entering the lytic replication cycle. Here, we provide two fast and reliable protocols that allow one to determine the cell cycle distribution of the designated host cells and monitor viral protein expression as a function of the cell cycle state. Both protocols make use of the thymidine analogue 5-ethynyl-2'-deoxyuridine and "click" chemistry to label HCMV-non-permissive S phase cells in a gentle and sensitive way.

  18. 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.

  19. CtIP is regulated by the APC/C-Cdh1 to mediate cell cycle-dependent control of DNA repair

    NARCIS (Netherlands)

    de Boer, Harmen R.; Lafranchi, Lorenzo; Neugebauer, Christine; Fehrmann, Rudolf; de Vries, Elisabeth G. E.; Sartori, Alessandro A.; van Vugt, Marcel

    2014-01-01

    Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C-Cdh1 ubiquitin ligase regulates mitotic exit but is also implic

  20. Cell-cycle-dependent variation in UV absorption spectrum of Hela cells treated with Trichostatin A%HeLa细胞经曲古菌素A处理后紫外光谱的细胞周期依赖性变化

    Institute of Scientific and Technical Information of China (English)

    Fengqiu Zhang; Xiaoxia Wang; Zhanguo Yang

    2012-01-01

    Objective: The aim of our study was to discovery the different cell cycle arrest effect after different densities HeLa cells treated with Trichostatin A (TSA). In addition, this study would find some important relationship between cycle arrest effect and UV absorption spectrum of cell. Methods: 0.2 μM TSA was applied to act on HeLa cells of different density. Then, the cycle arrest effect and UV absorption spectrum of cells were investigated, which provide support to analyze the effect of TSA on cancer cells. Results: Cell cycle arrest effect in G0/G1 of the lower density cells was more obvious than that in other groups. The other discovery in this work was that the cellular UV absorption value was higher when the density of cultured cell was lower. Conclusion: This experiment would guide the clinical study on early or late stage cancer patients in the future. On the other hand, this work indicates when cells were arrested in G0/G1 phase, the cellular absorption value increased at the same time, so UV absorption spectrum could characterize the change of cell cycle.

  1. Imprinting localized plasmons for enhanced solar cells.

    Science.gov (United States)

    Dunbar, Ricky B; Pfadler, Thomas; Lal, Niraj N; Baumberg, Jeremy J; Schmidt-Mende, Lukas

    2012-09-28

    Imprinted silver nanovoid arrays are investigated via angle-resolved reflectometry to demonstrate their suitability for plasmonic light trapping. Both wavelength- and subwavelength-scale nanovoids are imprinted into standard solar cell architectures to achieve nanostructured metallic electrodes which provide enhanced absorption for improving solar cell performance. The technique is versatile, low-cost and scalable and can be applied to a wide range of organic semiconductors. Absorption features which are independent of incident polarization and weakly dependent on incident angle reveal localized plasmonic modes at the structured interface. Metallic nanostructure-PCPDTBT:PCBM samples demonstrate absorption enhancements of up to 40%. The structured interface provides light trapping, which boosts absorption at wavelengths where the semiconductors absorb poorly.

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

    Institute of Scientific and Technical Information of China (English)

    LU Zhi-hua; WU Mei

    2011-01-01

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

  3. Immunoelectron microscopic localization of calmodulin in corn root cells

    Institute of Scientific and Technical Information of China (English)

    LIJIAXU; JIEWENLIU; DAYESUN

    1993-01-01

    Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.

  4. 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...... of the distortionary effect (on job creation) from providing unemployment insurance, whereas countercyclical benefits facilitate consumption smoothing....

  5. 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.

  6. Local resolved electrochemical impedance spectroscopy of PEFC single cells

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, M.; Gulzow, E. [German Aerospace Center, Inst. of Technical Thermodynamics, Stuttgart (Germany)

    2009-07-01

    Experimental data on a spatial resolved level is needed to understand the integral behaviour of fuel cells as well as to validate models describing fuel cell behaviour. This paper described a new tool developed to increase the accuracy of current density measurements. Based on a printed circuit board, the tool integrated local electrochemical impedance spectroscopy techniques in order to determine local membrane resistance, electrochemical reactions, and transport processes. Solutions for locally resolved impedance spectroscopy measurements were presented. It was concluded that the tool will help to provide a more detailed understanding of fuel cell behaviour.

  7. 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.

  8. Localization of ANP-synthesizing cells in rat stomach

    Institute of Scientific and Technical Information of China (English)

    Chun-Hui Li; Li-Hui Pan; Chun-Yu Li; Chang-Lin Zhu; Wen-Xie Xu

    2006-01-01

    AIM: To study the morphological positive expression of antrial natriuretic peptide (ANP)-synthesizing cells and ultrastructural localization and the relationship between ANP-synthesizing cells and microvessel density in the stomach of rats and to analyze the distribution of the three histologically distinct regions of ANP-synthesizing cells.METHODS: Using immunohistochemical techniques, we studied positive expression of ANP-synthesizing cells in rat stomach. A postembedding immunogold microscopy technique was used for ultrastructural localization of ANP-synthesizing cells. Microvessel density in the rat stomach was estimated using tannic acid-ferric chloride (TAFC) method staining. Distribution of ANP-synthesizing cells were studied in different regions of rat stomach histochemically.RESULTS: Positive expression of ANP-synthesizing cells were localized in the gastric mucosa of rats. Localization of ANP-synthesizing cells identified them to be enterochrochromaffin cells (EC) by using a postembedding immunogold electron microscopy technique. EC cells were in the basal third of the cardiac mucosa region.ANP-synthesizing cells existed in different regions of rat stomach and its density was largest in the gastric cardiac region, and the distribution order of ANP-synthesizing cells in density was cardiac region, pyloric region and fundic region in mucosa layer. We have also found a close relationship between ANP-synthesizing cells and microvessel density in gastric mucosa of rats using TAFC staining.CONCLUSION: ANP-synthesizing cells are expressed in the gastric mucosa. EC synthesize ANP. There is a close relationship between ANP-synthesizing cells and microvessel density in gastric mucosa of rats.The distribution density of ANP-synthesizing cells is largest in the gastric cardiac region.

  9. 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

  10. Local Helioseismology

    Directory of Open Access Journals (Sweden)

    Gizon Laurent

    2005-11-01

    Full Text Available We review the current status of local helioseismology, covering both theoretical and observational results. After a brief introduction to solar oscillations and wave propagation through inhomogeneous media, we describe the main techniques of local helioseismology: Fourier-Hankel decomposition, ring-diagram analysis, time-distance helioseismology, helioseismic holography, and direct modeling. We discuss local helioseismology of large-scale flows, the solar-cycle dependence of these flows, perturbations associated with regions of magnetic activity, and solar supergranulation.

  11. Recognition of local anesthetics by alphabeta+ T cells.

    Science.gov (United States)

    Zanni, M P; von Greyerz, S; Hari, Y; Schnyder, B; Pichler, W J

    1999-02-01

    Patients with drug allergy show a specific immune response to drugs. Chemically nonreactive drugs like, for example, local anesthetics are directly recognized by alphabeta+ T cells in an HLA-DR restricted way, as neither drug metabolism nor protein processing is required for T cell stimulation. In this study we identified some of the structural requirements that determine cross-reactivity of T cells to local anesthetics, with the aim to improve the molecular basis for the selection of alternatives in individuals sensitized to a certain local anesthetic and to better understand presentation and T cell recognition of these drugs. Fifty-five clones (52 lidocaine specific, three mepivacaine specific from two allergic donors) were analyzed. Stimulatory compounds induced a down-regulation of the T cell receptor, demonstrating that these non-peptide antigens are recognized by the T cell receptor itself. A consistent cross-reactivity between lidocaine and mepivacaine was found, as all except one lidocaine specific clone proliferated to both drugs tested. Sixteen chemically related local anesthetics (including ester local anesthetics, OH- and desalkylated metabolites) were used to identify structural requirements for T cell recognition. Each of the four clones examined in detail was uniquely sensitive to changes in the structures of the local anesthetic: clone SFT24, i.e., did not recognize any of the tested OH- or desalkylated metabolites, while the clone OFB2 proliferated to all OH-metabolites and other differently modified molecules. The broadly reactive clone OFB2 allowed us to propose a model, suggesting that the structure of the amine side chain of local anesthetics is essential for recognition by the T cell receptor.

  12. 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.

  13. Combined modality therapy for locally advanced penile squamous cell carcinoma.

    Science.gov (United States)

    Pedrick, T J; Wheeler, W; Riemenschneider, H

    1993-12-01

    We report here a patient who presented with locally advanced Jackson Stage IV penile squamous cell carcinoma who was managed with preoperative 5-fluorouracil, mitomycin C chemotherapy, and concurrent radiation therapy. He experienced an excellent partial response which allowed more limited surgery than would otherwise be indicated. He is still alive and well 5 years after completion of his treatment without side effects, local recurrence, or distant metastatic disease.

  14. Localization of muscarinic acetylcholine receptor in plant guard cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.

  15. Localization and biosynthesis of polyamines in insulin-producing cells

    DEFF Research Database (Denmark)

    Hougaard, D M; Larsson, L I; Nielsen, Jens Høiriis

    1986-01-01

    Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine...

  16. Spatial distributions of red blood cells significantly alter local haemodynamics.

    Directory of Open Access Journals (Sweden)

    Joseph M Sherwood

    Full Text Available Although bulk changes in red blood cell concentration between vessels have been well characterised, local distributions are generally overlooked. Red blood cells aggregate, deform and migrate within vessels, forming heterogeneous distributions which have considerable effect on local haemodynamics. The present study reports data on the local distribution of human red blood cells in a sequentially bifurcating microchannel, representing the branching geometry of the microvasculature. Imaging methodologies with simple extrapolations are used to infer three dimensional, time-averaged velocity and haematocrit distributions under a range of flow conditions. Strong correlation between the bluntness of the velocity and haematocrit profiles in the parent branch of the geometry is observed and red blood cell aggregation has a notable effect on the observed trends. The two branches of the first bifurcation show similar characteristics in terms of the shapes of the profiles and the extent of plasma skimming, despite the difference in geometric configuration. In the second bifurcation, considerable asymmetry between the branches in the plasma skimming relationship is observed, and elucidated by considering individual haematocrit profiles. The results of the study highlight the importance of considering local haematocrit distributions in the analysis of blood flow and could lead to more accurate computational models of blood flow in microvascular networks. The experimental approaches developed in this work provide a foundation for further examining the characteristics of microhaemodynamics.

  17. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    Science.gov (United States)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  18. Spectroscopic investigation of local mechanical impedance of living cells

    CERN Document Server

    Costa, Luca; Benseny-Cases, Núria; Mayeaux, Véronique; Chevrier, Joël; Comin, Fabio

    2013-01-01

    The mechanical properties of PC12 living cells have been studied at the nanoscale with a Force Feedback Microscope using two experimental approaches. Firstly, the local mechanical impedance of the cell membrane has been mapped simultaneously to the cell morphology at constant force. As the force of the interaction is gradually increased, we observed the appearance of the sub-membrane cytoskeleton. We shall compare the results obtained with this method with the measurement of other existing techniques. Secondly, a spectroscopic investigation has been performed varying the indentation of the tip in the cell membrane and consequently the force applied on it. In contrast with conventional dynamic atomic force microscopy techniques, here the small oscillation amplitude of the tip is not necessarily imposed at the cantilever first eigenmode. This allows the user to arbitrarily choose the excitation frequency in developing spectroscopic AFM techniques. The mechanical response of the PC12 cell membrane is found to be...

  19. A numerical model of localized convection cells of Euglena suspensions

    Science.gov (United States)

    Iima, Makoto; Shoji, Erika; Yamaguchi, Takayuki

    2014-11-01

    Suspension of Euglena gracilis shows localized convection cells when it is illuminated form below with strong light intensity. Experiments in an annular container shows that there are two elementary localized structures. One consists of a pair of convection cells and a single region where number density of Euglena is high. The other consists a localized traveling wave. Based on the measurements of the flux of number density, we propose a model of bioconvection incorporating lateral phototaxis effect proportional to the light intensity gradient. Using pseudo spectral method, we performed numerical simulation of this model. We succeed in reproducing one of the localized structures, a convection pair with single region of high number density. Also, when the aspect ratio is large, there are a parameter region where the localized structure and conductive state are both stable, which is suggested by experiments. Spatial distribution of the number density implies that the accumulation of microorganism due to the convective flow causes such bistability. CREST(PJ74100011) and KAKENHI(26400396).

  20. The exocyst localizes to the primary cilium in MDCK cells.

    Science.gov (United States)

    Rogers, Katherine K; Wilson, Patricia D; Snyder, Richard W; Zhang, Xiaoyu; Guo, Wei; Burrow, Christopher R; Lipschutz, Joshua H

    2004-06-18

    Primary cilia play a role in the maintenance of tubular epithelial differentiation and ciliary dysfunction can result in abnormal cyst formation, such as occurs in autosomal dominant polycystic kidney disease (ADPKD). We previously showed that the exocyst, an eight-protein complex involved in the biogenesis of polarity from yeast to mammals, is centrally involved in cyst formation [Mol. Biol. Cell. 11 (2000) 4259]. Here we show that the exocyst complex localizes to the primary cilium in Madin-Darby canine kidney (MDCK) tubular epithelial cells. We further show that the exocyst is overexpressed in both cell lines and primary cell cultures of ADPKD origin, suggesting that the exocyst may be involved in the pathogenesis of ADPKD.

  1. Algal cell disruption using microbubbles to localize ultrasonic energy.

    Science.gov (United States)

    Krehbiel, Joel D; Schideman, Lance C; King, Daniel A; Freund, Jonathan B

    2014-12-01

    Microbubbles were added to an algal solution with the goal of improving cell disruption efficiency and the net energy balance for algal biofuel production. Experimental results showed that disruption increases with increasing peak rarefaction ultrasound pressure over the range studied: 1.90 to 3.07 MPa. Additionally, ultrasound cell disruption increased by up to 58% by adding microbubbles, with peak disruption occurring in the range of 10(8)microbubbles/ml. The localization of energy in space and time provided by the bubbles improve efficiency: energy requirements for such a process were estimated to be one-fourth of the available heat of combustion of algal biomass and one-fifth of currently used cell disruption methods. This increase in energy efficiency could make microbubble enhanced ultrasound viable for bioenergy applications and is expected to integrate well with current cell harvesting methods based upon dissolved air flotation.

  2. Schelling model of cell segregation based only on local information

    Science.gov (United States)

    Nielsen, Alexander Valentin; Gade, Annika Lund; Juul, Jeppe; Strandkvist, Charlotte

    2015-11-01

    While biological studies suggest that motility of cells is involved in cell segregation, few computational models have investigated this mechanism. We apply a simple Schelling model, modified to reflect biological conditions, demonstrating how differences in cell motility arising exclusively from differences in the composition of the local environment can be sufficient to drive segregation. The work presented here demonstrates that the segregation behavior observed in the original Schelling model is robust to a relaxation of the requirement for global information and that the Schelling model may yield insight in the context of biological systems. In the model, the time course of cell segregation follows a power law in accord with experimental observations and previous work.

  3. 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.

  4. Quantification and Localization of Mast Cells in Periapical Lesions

    Science.gov (United States)

    Mahita, VN; Manjunatha, BS; Shah, R; Astekar, M; Purohit, S; Kovvuru, S

    2015-01-01

    Background: 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. Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: 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. PMID:25861530

  5. A non-local evolution equation model of cell-cell adhesion in higher dimensional space.

    Science.gov (United States)

    Dyson, Janet; Gourley, Stephen A; Webb, Glenn F

    2013-01-01

    A model for cell-cell adhesion, based on an equation originally proposed by Armstrong et al. [A continuum approach to modelling cell-cell adhesion, J. Theor. Biol. 243 (2006), pp. 98-113], is considered. The model consists of a nonlinear partial differential equation for the cell density in an N-dimensional infinite domain. It has a non-local flux term which models the component of cell motion attributable to cells having formed bonds with other nearby cells. Using the theory of fractional powers of analytic semigroup generators and working in spaces with bounded uniformly continuous derivatives, the local existence of classical solutions is proved. Positivity and boundedness of solutions is then established, leading to global existence of solutions. Finally, the asymptotic behaviour of solutions about the spatially uniform state is considered. The model is illustrated by simulations that can be applied to in vitro wound closure experiments.

  6. Vismodegib: in locally advanced or metastatic basal cell carcinoma.

    Science.gov (United States)

    Keating, Gillian M

    2012-07-30

    Vismodegib is the first Hedgehog pathway inhibitor to be approved in the US, where it is indicated for the treatment of adults with metastatic basal cell carcinoma (BCC), or with locally advanced BCC that has recurred following surgery or who are not candidates for surgery, and who are not candidates for radiation. Vismodegib selectively and potently inhibits the Hedgehog signalling pathway by binding to Smoothened, thereby inhibiting the activation of Hedgehog target genes. Oral vismodegib was effective in the treatment of patients with locally advanced (n = 63) or metastatic (n = 33) BCC, according to the results of an ongoing, noncomparative, multinational, pivotal, phase II trial (ERIVANCE BCC). In this trial (using a clinical cutoff date of 26 November 2010), the independent review facility overall response rate was 42.9% in patients with locally advanced BCC and 30.3% in patients with metastatic BCC. In both patients with locally advanced BCC and those with metastatic BCC, the median duration of response was 7.6 months and median progression-free survival was 9.5 months. Oral vismodegib had an acceptable tolerability profile in patients with advanced BCC.

  7. A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Giulia Oliva

    2017-01-01

    Full Text Available Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria.

  8. A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

    Science.gov (United States)

    Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike

    2017-01-01

    ABSTRACT Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. PMID:28074027

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

    Science.gov (United States)

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

    2016-08-03

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

  10. 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.

  11. Arterial identity of endothelial cells is controlled by local cues.

    Science.gov (United States)

    Othman-Hassan, K; Patel, K; Papoutsi, M; Rodriguez-Niedenführ, M; Christ, B; Wilting, J

    2001-09-15

    The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

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

    Science.gov (United States)

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

    2004-12-01

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

  13. Screen for localized proteins in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Jay H Russell

    Full Text Available Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species.

  14. Ultrastructural localization of active genes in Allium cepa cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By using the anti-RNA/DNA hybrid antibody as the probe, we labeled and analyzed the precise transcriptional sites of active genes in Allium cepa cells in situ. The results showed that the location of labeled signals appeared in mitochondrion was the same as that in chloroplast, generally concentrated at the central matrix space where there were no cristae and thylakoids. In the extranucleolar regions of nucleus, the labeled signals of transcriptional sites were situated at the perichromatin fibrils, which decondensed and stretched out from the chromosome territories. Our results also displayed the concentrations of labeled signals in a cer-tain region of nucleus, and this means that the gene tran-scription rich region actually existed in Allium cepa cells. In nucleolus, the synthetic sites of rRNA were localized not only to the periphery of fibrillar centers but also to the DFC near FC.

  15. Geometric localization of thermal fluctuations in red blood cells

    Science.gov (United States)

    Evans, Arthur A.; Bhaduri, Basanta; Popescu, Gabriel; Levine, Alex J.

    2017-01-01

    The thermal fluctuations of membranes and nanoscale shells affect their mechanical characteristics. Whereas these fluctuations are well understood for flat membranes, curved shells show anomalous behavior due to the geometric coupling between in-plane elasticity and out-of-plane bending. Using conventional shallow shell theory in combination with equilibrium statistical physics we theoretically demonstrate that thermalized shells containing regions of negative Gaussian curvature naturally develop anomalously large fluctuations. Moreover, the existence of special curves, “singular lines,” leads to a breakdown of linear membrane theory. As a result, these geometric curves effectively partition the cell into regions whose fluctuations are only weakly coupled. We validate these predictions using high-resolution microscopy of human red blood cells (RBCs) as a case study. Our observations show geometry-dependent localization of thermal fluctuations consistent with our theoretical modeling, demonstrating the efficacy in combining shell theory with equilibrium statistical physics for describing the thermalized morphology of cellular membranes. PMID:28242681

  16. Geometric localization of thermal fluctuations in red blood cells.

    Science.gov (United States)

    Evans, Arthur A; Bhaduri, Basanta; Popescu, Gabriel; Levine, Alex J

    2017-02-27

    The thermal fluctuations of membranes and nanoscale shells affect their mechanical characteristics. Whereas these fluctuations are well understood for flat membranes, curved shells show anomalous behavior due to the geometric coupling between in-plane elasticity and out-of-plane bending. Using conventional shallow shell theory in combination with equilibrium statistical physics we theoretically demonstrate that thermalized shells containing regions of negative Gaussian curvature naturally develop anomalously large fluctuations. Moreover, the existence of special curves, "singular lines," leads to a breakdown of linear membrane theory. As a result, these geometric curves effectively partition the cell into regions whose fluctuations are only weakly coupled. We validate these predictions using high-resolution microscopy of human red blood cells (RBCs) as a case study. Our observations show geometry-dependent localization of thermal fluctuations consistent with our theoretical modeling, demonstrating the efficacy in combining shell theory with equilibrium statistical physics for describing the thermalized morphology of cellular membranes.

  17. 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.

  18. Interplanetary magnetic field and solar cycle dependence of Northern Hemisphere F region joule heating

    Science.gov (United States)

    Bjoland, L. M.; Chen, X.; Jin, Y.; Reimer, A. S.; Skjæveland, Å.; Wessel, M. R.; Burchill, J. K.; Clausen, L. B. N.; Haaland, S. E.; McWilliams, K. A.

    2015-02-01

    Joule heating in the ionosphere takes place through collisions between ions and neutrals. Statistical maps of F region Joule heating in the Northern Hemisphere polar ionosphere are derived from satellite measurements of thermospheric wind and radar measurements of ionospheric ion convection. Persistent mesoscale heating is observed near postnoon and postmidnight magnetic local time and centered around 70° magnetic latitude in regions of strong relative ion and neutral drift. The magnitude of the Joule heating is found to be largest during solar maximum and for a southeast oriented interplanetary magnetic field. These conditions are consistent with stronger ion convection producing a larger relative flow between ions and neutrals. The global-scale Joule heating maps quantify persistent (in location) regions of heating that may be used to provide a broader context compared to small-scale studies of the coupling between the thermosphere and ionosphere.

  19. A kinetic mechanism for cell sorting based on local variations in cell motility

    Science.gov (United States)

    Strandkvist, Charlotte; Juul, Jeppe; Baum, Buzz; Kabla, Alexandre J.; Duke, Tom

    2014-01-01

    Our current understanding of cell sorting relies on physical difference, either in the interfacial properties or motile force, between cell types. But is such asymmetry a prerequisite for cell sorting? We test this using a minimal model in which the two cell populations are identical with respect to their physical properties and differences in motility arise solely from how cells interact with their surroundings. The model resembles the Schelling model used in social sciences to study segregation phenomena at the scale of societies. Our results demonstrate that segregation can emerge solely from cell motility being a dynamic property that changes in response to the local environment of the cell, but that additional mechanisms are necessary to reproduce the envelopment behaviour observed in vitro. The time course of segregation follows a power law, in agreement with the scaling reported from experiment and in other models of motility-driven segregation. PMID:25485079

  20. Crypt region localization of intestinal stem cells in adults

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The intestinal epithelial lining plays a central role in the digestion and absorption of nutrients, but exists in a harsh luminal environment that necessitates continual renewal. This renewal process involves epithelial cell proliferation in the crypt base and later cell migration from the crypt base to the luminal surface. This process is dependent on multi-potent progenitor cells, or stem cells, located in each crypt. There are about 4 to 6 stem cells per crypt, and these stem cells are believed to generate distinct end-differentiated epithelial cell types, including absorptive cells, goblet cells, enteroendocrine cells and Paneth cells, while also maintaining their own progenitor cell state. Earlier studies suggested that intestinal stem cells were located either in the crypt base interspersed between the Paneth cells [i.e. Crypt base columnar (CBC) cell model] or at an average position of 4 cells from the crypt base [I.e. Label-retaining cells (LRC +4) model]. Recent studies have employed biomarkers in the in vivo mammalian state to more precisely evaluate the location of these progenitor cells in the intestinal crypt. Most notable of these novel markers are Lgr5, a gene that encodes a G-protein-coupled receptor with expression restricted to CBC cells, and Bmi 1, which encodes a chromatin remodeling protein expressed by LRC. These studies raise the possibility that there may be separate stem cell lines or different states of stem cell activation involved in the renewal of normal mammalian intestinal tract.

  1. Localization of a new serine protease, ingobsin, in goblet cells in rat, pig and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1985-01-01

    A serine protease, ingobsin, that cleaves Lys-x and Arg-x, has been purified from rat duodenal tissue. By immunohistochemical methods, the enzyme was localized in goblet cells in the small intestine of rat, pig, and man. The immunoreactive cells were most numerous in the proximal part...... of the intestine. In the electron microscope, the immunoreaction was localized mainly to the rough endoplasmic reticulum of the goblet cells and to the secretion being extruded from the cells....

  2. 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.

  3. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    Science.gov (United States)

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  4. Sex-Specific and Estrous Cycle-Dependent Antidepressant-Like Effects and Hippocampal Akt Signaling of Leptin.

    Science.gov (United States)

    Carrier, Nicole; Wang, Xuezhen; Sun, Linshan; Lu, Xin-Yun

    2015-10-01

    Sex differences in the incidence of depression and antidepressant treatment responses are well documented. Depression is twice as common in women as in men. Recent studies indicate that low levels of leptin, an adipocyte-derived hormone, are associated with increased symptoms of depression in women. Leptin has been shown to produce antidepressant-like effects in male rodents. In the present study, we examined sex differences and estrous cycle variations in antidepressant-like responses to leptin. Leptin administration significantly reduced immobility, a putative measure of behavioral despair, in the forced swim test in intact female mice in the proestrus phase but not in the diestrus phase of the estrous cycle. Moreover, leptin administration stimulated Akt phosphorylation in the hippocampus of female mice in proestrus but not in diestrus, in correlation with its differential behavioral effects in these two phases of the cycle. Leptin-induced behavioral responses and stimulation of hippocampal Akt phosphorylation in female mice were abolished by ovariectomy. By contrast, the antidepressant-like effect of leptin in male mice was not affected by gonadectomy (castration). Pretreatment with 17β-estradiol restored sensitivity to the effects of leptin on behavior and hippocampal Akt phosphorylation in ovariectomized female mice. These results suggest leptin regulates depression-like behavior and hippocampal Akt signaling in a sex-specific and estrous cycle-dependent manner.

  5. Smart interface materials integrated with microfluidics for on-demand local capture and release of cells.

    Science.gov (United States)

    Gurkan, Umut Atakan; Tasoglu, Savas; Akkaynak, Derya; Avci, Oguzhan; Unluisler, Sebnem; Canikyan, Serli; Maccallum, Noah; Demirci, Utkan

    2012-09-01

    Stimuli responsive, smart interface materials are integrated with microfluidic technologies creating new functions for a broad range of biological and clinical applications by controlling the material and cell interactions. Local capture and on-demand local release of cells are demonstrated with spatial and temporal control in a microfluidic system.

  6. Local Structure Analysis of Materials for Solar Cell Absorber Layer

    OpenAIRE

    Jewell, Leila Elizabeth

    2016-01-01

    This dissertation examines solar cell absorber materials that have the potential to replace silicon in solar cells, including several copper-based sulfides and perovskites. Earth-abundant absorbers such as these become even more cost-effective when used in a nanostructured solar cell. Atomic layer deposition (ALD) and chemical vapor deposition (CVD) deposit highly conformal films and hence are important tools for developing extremely thin absorber solar cells with scalability. Thus, the prima...

  7. 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....

  8. The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis.

    Science.gov (United States)

    Wang, Jing; Kirby, Celeste E; Herbst, Ronald

    2002-11-29

    PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics.

  9. Interaction of localized convection cells in the bioconvection of Euglena gracilis

    Science.gov (United States)

    Iima, Makoto; Yamaguchi, Takayuki

    2016-11-01

    Euglena gracilis is a unicellular flagellated photosynthetic alga. The suspension of Euglena has behavioral responses to light, which causes a macroscopic localized bioconvection pattern when illuminated from below. One of the fundamental structures of this is a pair of convection cells, and high cell density region exists in the middle of the pair. Experimental studies show various types of interaction in the localized convection cells; bound state, collision, etc. We performed numerical simulation of a hydrodynamic model of this system, and show results of the interactions. Long-range interaction due to the conservation of cell number and merging process of two localized structures will be discussed. KAKENHI.

  10. Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane.

    Science.gov (United States)

    Witzel, Sabine; Zimyanin, Vitaly; Carreira-Barbosa, Filipa; Tada, Masazumi; Heisenberg, Carl-Philipp

    2006-12-04

    Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.

  11. Octreotide scintigraphy localizes somatostatin receptor-positive islet cell carcinomas

    NARCIS (Netherlands)

    W. Becker (W.); J. Marienhagen (J.); R. Scheubel (R.); A. Saptogino (A.); W.H. Bakker (Willem); W.A.P. Breeman (Wouter); F. Wolf (F.)

    1991-01-01

    textabstractTyr-3-Octreotide is a synthetic derivative of somatostatin and a somatostatin-receptor analogue. The iodine-123-labelled compound localizes somatostatin-receptor-positive tumours. In this paper two patients are reported in whom somatostatin receptors were demonstrated in vitro. In a 60-y

  12. Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells.

    Directory of Open Access Journals (Sweden)

    Nurdan Ozkucur

    Full Text Available BACKGROUND: Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10-15 V/cm and weak (< or = 5 V/cm dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs are involved in dcEF-induced intracellular calcium elevation. CONCLUSION/SIGNIFICANCE: Taken together, these data form a time scale of the morphological and physiological

  13. The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila.

    Science.gov (United States)

    Bellaïche, Yohanns; Beaudoin-Massiani, Olivia; Stuttem, Isabella; Schweisguth, François

    2004-01-01

    Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.

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

    Science.gov (United States)

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

    2006-04-01

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

  15. Purinergic Receptors in Quiescence and Localization of Leukemic Stem Cells

    Science.gov (United States)

    2012-05-01

    LSK and SLAM LSK cells following radiation (6 Gy, TBI). NAC treatment almost completely abolished the p38 M APK activation. While we were unable to...triggered the activation of both p38 M APK and JNK pathway in KG-1 leukemia cells starting as early as 5 min after UDP-Glc treatment. For JNK proteins...investigate whether UDP-Glc induces the activation of p38 M APK and JNK in primary human leukemia cells, we transplanted human acute leukemia samples

  16. Local homogeneity of cell cycle length in developing mouse cortex

    Science.gov (United States)

    Cai, L.; Hayes, N. L.; Nowakowski, R. S.

    1997-01-01

    We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the

  17. 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.

  18. Solar-cycle dependence of a model turbulence spectrum using IMP and ACE observations over 38 years

    Science.gov (United States)

    Burger, R. A.; Nel, A. E.; Engelbrecht, N. E.

    2014-12-01

    Ab initio modulation models require a number of turbulence quantities as input for any reasonable diffusion tensor. While turbulence transport models describe the radial evolution of such quantities, they in turn require observations in the inner heliosphere as input values. So far we have concentrated on solar minimum conditions (e.g. Engelbrecht and Burger 2013, ApJ), but are now looking at long-term modulation which requires turbulence data over at a least a solar magnetic cycle. As a start we analyzed 1-minute resolution data for the N-component of the magnetic field, from 1974 to 2012, covering about two solar magnetic cycles (initially using IMP and then ACE data). We assume a very simple three-stage power-law frequency spectrum, calculate the integral from the highest to the lowest frequency, and fit it to variances calculated with lags from 5 minutes to 80 hours. From the fit we then obtain not only the asymptotic variance at large lags, but also the spectral index of the inertial and the energy, as well as the breakpoint between the inertial and energy range (bendover scale) and between the energy and cutoff range (cutoff scale). All values given here are preliminary. The cutoff range is a constraint imposed in order to ensure a finite energy density; the spectrum is forced to be either flat or to decrease with decreasing frequency in this range. Given that cosmic rays sample magnetic fluctuations over long periods in their transport through the heliosphere, we average the spectra over at least 27 days. We find that the variance of the N-component has a clear solar cycle dependence, with smaller values (~6 nT2) during solar minimum and larger during solar maximum periods (~17 nT2), well correlated with the magnetic field magnitude (e.g. Smith et al. 2006, ApJ). Whereas the inertial range spectral index (-1.65 ± 0.06) does not show a significant solar cycle variation, the energy range index (-1.1 ± 0.3) seems to be anti-correlated with the variance

  19. Surgical Management of Local Recurrences of Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Ömer Acar

    2016-01-01

    Full Text Available Surgical resection either in the form of radical nephrectomy or in the form of partial nephrectomy represents the mainstay options in the treatment of kidney cancer. In most instances, resecting the tumor bearing kidney or the tumor itself provides durable cancer specific survival rates. However, recurrences may rarely develop in the renal fossa or remnant kidney. Despite its rarity, locally recurrent RCC is a challenging condition in terms of the possible management options and relatively poor prognosis. If technically feasible, wide surgical excision and ensuring negative surgical margins are the most effective treatment options. Repeat surgeries (completion nephrectomy, excision of locally recurrent tumor, or repeat partial nephrectomy may often be complicated, and perioperative morbidity is a major concern. Open approach has been extensively applied in this context and 5-year cancer specific survival rates have been reported to be around 50%. The roles of minimally invasive surgical options (laparoscopic and robotic approach and nonsurgical alternatives (cryoablation, radiofrequency ablation have yet to be described. In selected patients, surgical resection may have to be complemented with (neoadjuvant radiotherapy or medical treatment.

  20. 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

    PURPOSE: We have previously demonstrated a significant negative impact of intratumoral neutrophils in metastatic renal cell carcinoma. This study assessed intratumoral neutrophils in localized clear cell renal cell carcinoma (RCC). PATIENTS AND METHODS: The study comprised 121 consecutive patients...... neutrophils was also an independent prognostic factor for cancer-specific survival (HR, 3.5; 95% CI, 1.9 to 6.4; P .... CONCLUSION: The presence of intratumoral neutrophils is a new, strong, independent prognostic factor for short recurrence-free, cancer-specific, and overall survival in localized clear cell RCC....

  1. "Cell therapy for stroke: use of local astrocytes"

    Directory of Open Access Journals (Sweden)

    Melek eChouchane

    2012-10-01

    Full Text Available Stroke refers to a variety of conditions caused by the occlusion or hemorrhage of blood vessels supplying the brain, which is one of the main causes of death and the leading cause of disability worldwide. In the last years, cell-based therapies have been proposed as a new approach to ameliorate post stroke deficits. However, the most appropriate type of cell to be used in such therapies, as well as their sources, remains a matter of intense research. A good candidate cell should, in principle, display high plasticity to generate diverse types of neurons and, at the same type, low risk to cause undesired outcomes, such as malignant transformation. Recently, a new approach grounded on the reprogramming of endogenous astrocytes towards neuronal fates emerged as an alternative to restore neurological functions in several central nervous system diseases. In this perspective, we review data about the potential of astrocytes to become functional neurons following expression of neurogenic genes and discuss the potential benefits and risks of reprogramming astrocytes in the glial scar to replace neurons lost after stroke.

  2. Keratins mediate localization of hemidesmosomes and repress cell motility.

    Science.gov (United States)

    Seltmann, Kristin; Roth, Wera; Kröger, Cornelia; Loschke, Fanny; Lederer, Marcell; Hüttelmaier, Stefan; Magin, Thomas M

    2013-01-01

    The keratin (K)-hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells.

  3. Delivery of molecules into cells using localized single cell electroporation on ITO micro-electrode based transparent chip.

    Science.gov (United States)

    Chen, Sheng-Chiech; Santra, Tuhin Subhra; Chang, Chia-Jung; Chen, Tsung-Ju; Wang, Pen-Cheng; Tseng, Fan-Gang

    2012-10-01

    Single cell electroporation is one of the nonviral method which successfully allows transfection of exogenous macromolecules into individual living cell. We present localized cell membrane electroporation at single-cell level by using indium tin oxide (ITO) based transparent micro-electrodes chip with inverted microscope. A focused ion beam (FIB) technique has been successfully deployed to fabricate transparent ITO micro-electrodes with submicron gaps, which can generate more intense electric field to produce very localized cell membrane electroporation. In our approach, we have successfully achieved 0.93 μm or smaller electroporation region on the cell surface to inject PI (Propidium Iodide) dye into the cell with 60 % cell viability. This experiments successfully demonstrate the cell self-recover process from the injected PI dye intensity variation. Our localized cell membrane electroporation technique (LSCMEP) not only generates reversible electroporation process but also it provides a clear optical path for potentially monitoring/tracking of drugs to deliver in single cell level.

  4. Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

    Science.gov (United States)

    Wang, Xiu-Ling; Gao, Xin-Qi; Wang, Xue-Chen

    2011-08-01

    Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

  5. 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

  6. Judging diatoms by their cover: variability in local elasticity of Lithodesmium undulatum undergoing cell division.

    Directory of Open Access Journals (Sweden)

    Lee Karp-Boss

    Full Text Available Unique features of diatoms are their intricate cell covers (frustules made out of hydrated, amorphous silica. The frustule defines and maintains cell shape and protects cells against grazers and pathogens, yet it must allow for cell expansion during growth and division. Other siliceous structures have also evolved in some chain-forming species as means for holding neighboring cells together. Characterization and quantification of mechanical properties of these structures are crucial for the understanding of the relationship between form and function in diatoms, but thus far only a handful of studies have addressed this issue. We conducted micro-indentation experiments, using atomic force microscopy (AFM, to examine local variations in elastic (Young's moduli of cells and linking structures in the marine, chain-forming diatom Lithodesmium undulatum. Using a fluorescent tracer that is incorporated into new cell wall components we tested the hypothesis that new siliceous structures differ in elastic modulus from their older counterparts. Results show that the local elastic modulus is a highly dynamic property. Elastic modulus of stained regions was significantly lower than that of unstained regions, suggesting that newly formed cell wall components are generally softer than the ones inherited from the parent cells. This study provides the first evidence of differentiation in local elastic properties in the course of the cell cycle. Hardening of newly formed regions may involve incorporation of additional, possibly organic, material but further studies are needed to elucidate the processes that regulate mechanical properties of the frustule during the cell cycle.

  7. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    Science.gov (United States)

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko

    2010-12-15

    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

  8. Vaccine-specific local T cell reactivity in immunotherapy-associated vitiligo in melanoma patients.

    NARCIS (Netherlands)

    Jacobs, J.F.M.; Aarntzen, E.H.J.G.; Sibelt, L.A.G.; Blokx, W.A.M.; Boullart, A.C.I.; Gerritsen, M.J.P.; Hoogerbrugge, P.M.; Figdor, C.G.; Adema, G.J.; Punt, C.J.A.; Vries, I.J.M. de

    2009-01-01

    The occurrence of vitiligo in patients with melanoma is especially reported for patients undergoing immunotherapy. While vitiligo in these patients is thought to be related to an immune response directed against melanoma cells, solid evidence is lacking. Here we report local cytotoxic T cell reactiv

  9. Cells cultured on microgrooves with or without surface coating: Correlation between cell alignment, spreading and local membrane deformation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Xiongtu [Ecole Normale Superieure, CNRS-ENS-UPMC UMR 8640, 24 rue Lhomond, 75231 Paris (France); College of physics and information engineering, Fuzhou University, 350002 Fuzhou (China); Shi, Jian; Hu, Jie [Ecole Normale Superieure, CNRS-ENS-UPMC UMR 8640, 24 rue Lhomond, 75231 Paris (France); Chen, Yong, E-mail: yong.chen@ens.fr [Ecole Normale Superieure, CNRS-ENS-UPMC UMR 8640, 24 rue Lhomond, 75231 Paris (France); Institute for Integrated Cell-Material Science, Kyoto University, Kyoto 606-8507 (Japan)

    2013-03-01

    The behaviors of cells cultured on patterned substrates vary with the material stiffness, the geometry and the biochemical properties of the pattern. By using a reversed cell imprinting (RCI) technique, together with phase contrast microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM), we have exploited reversed side cellular morphology on patterned microgrooves of different geometries with or without surface coating of adhesion molecules. We have shown a close correlation between the effect of contact guidance and penetration of cellular membrane. Without surface coating, roughly 80% of HeLa cells were aligned along the groove direction regardless of the groove spacing. When the microgrooves were coated with fibronectin, the area of cell spreading was increased but the percentage of aligned cells was significantly decreased. In both cases, the deformation of cell membrane at the cell-pattern interfaces could be measured. We found that the local penetration of the cellular membrane into the grooves was correlated to the cellular alignment for both HeLa and NIH 3T3 cells, and that such a correlation was cell-type dependent. - Highlights: Black-Right-Pointing-Pointer Quantitatively assessment of cell deformation was obtained using RCI technique. Black-Right-Pointing-Pointer Cell alignment is correlated to the cell penetration into microgrooves. Black-Right-Pointing-Pointer Cell spreading is also correlated to the cell penetration into microgrooves. Black-Right-Pointing-Pointer The cell penetration and the cell alignment are cell-type dependent.

  10. 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.

  11. Establishing and maintaining cell polarity with mRNA localization in Drosophila.

    Science.gov (United States)

    Barr, Justinn; Yakovlev, Konstantin V; Shidlovskii, Yulii; Schedl, Paul

    2016-03-01

    How cell polarity is established and maintained is an important question in diverse biological contexts. Molecular mechanisms used to localize polarity proteins to distinct domains are likely context-dependent and provide a feedback loop in order to maintain polarity. One such mechanism is the localized translation of mRNAs encoding polarity proteins, which will be the focus of this review and may play a more important role in the establishment and maintenance of polarity than is currently known. Localized translation of mRNAs encoding polarity proteins can be used to establish polarity in response to an external signal, and to maintain polarity by local production of polarity determinants. The importance of this mechanism is illustrated by recent findings, including orb2-dependent localized translation of aPKC mRNA at the apical end of elongating spermatid tails in the Drosophila testis, and the apical localization of stardust A mRNA in Drosophila follicle and embryonic epithelia.

  12. Subcellular Localization and Differentiation-Induced Redistribution of the Protein Tyrosine Phosphatase PTP-BL in Neuroblastoma Cells.

    NARCIS (Netherlands)

    Ham, M. van der; Kemperman, L.; Wijers-Rouw, M.J.P.; Fransen, J.; Hendriks, W.J.A.J.

    2005-01-01

    1.In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells uti

  13. Local modulation of chemoattractant concentrations by single cells: dissection using a bulk-surface computational model

    Science.gov (United States)

    Nolan, M.

    2016-01-01

    Chemoattractant gradients are usually considered in terms of sources and sinks that are independent of the chemotactic cell. However, recent interest has focused on ‘self-generated’ gradients, in which cell populations create their own local gradients as they move. Here, we consider the interplay between chemoattractants and single cells. To achieve this, we extend a recently developed computational model to incorporate breakdown of extracellular attractants by membrane-bound enzymes. Model equations are parametrized, using the published estimates from Dictyostelium cells chemotaxing towards cyclic AMP. We find that individual cells can substantially modulate their local attractant field under physiologically appropriate conditions of attractant and enzymes. This means the attractant concentration perceived by receptors can be a small fraction of the ambient concentration. This allows efficient chemotaxis in chemoattractant concentrations that would be saturating without local breakdown. Similar interactions in which cells locally mould a stimulus could function in many types of directed cell motility, including haptotaxis, durotaxis and even electrotaxis. PMID:27708760

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

    Energy Technology Data Exchange (ETDEWEB)

    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 (/sup 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 ..beta..-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-/sup 3/H. MCPs are examined in flagellated and non-flagellated vesicles prepared from cells by immunoaffinity chromatography.

  15. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  16. Preliminary Study of Local Immunotherapy with Autologous Cytokine-Induced Killer Cells for Glioma Patients

    Institute of Scientific and Technical Information of China (English)

    Li Lin; Yonggao Mu; Zhongping Chen

    2008-01-01

    OBJECTIVE Cytokine-induced killer (CIK) cells are T-cells that display effective anti-tumor activity. In this study, we investigated the anti-tumor activity of CIK cells in vitro, and conducted a preliminary investigation using autologous CIK cells to treat glioma patients through local administration.METHODS The CIK cells were derived from peripheral blood monocytes (PBMCs) of the glioma patients. The anti-tumor activity of the CIK cells against human T98-G glioma cell was tested In vitro. In addition, the autologous CIK cells were locally administrated into the tumor cavity in the malignant glioma patients through an Ommaya reservoir which was pre-inserted during tumor resection. The 4×108 CIK cells in a 5 ml suspension were injected once a week 2 times per cycle. Five hundreds KU of IL-2 was injected every other day.RESULTS (I) With incubation, the CIK cells showed dual staining of CD3+CD56+ with a positive rate of 3.45% on day 10 and 55.2% on day 30. In vitro anti-tumor activity (againstT98-G cells) of the CIK cells reached the highest level after 18 days of incubation with different effector/target (E:T) ratios. (ii)Six patients received autologous CIK cell treatment (10 cycles).Two patients showed no recurrence and are still alive (24 and 10 months), while 4 cases had a recurrence 3 of which have died. The mean survival time from the first CIK cell treatment to the end of follow-up was 12.5 months. The main side-effects of the local CIK cell treatment was brain edema, which was controlled by mannitol in most of the cases. However for one patient injection of CIK cells and IL-2 had to be discontinued.CONCLUSION In vitro CIK cells are effective anti-glioma T-cells. Local therapy with CIK cells has potential anti-glioma efficacy and tolerable side-effects.

  17. Local Mechanical Stimulation of Mardin-Darby Canine Kidney Cell Sheets on Temperature-Responsive Hydrogel

    Directory of Open Access Journals (Sweden)

    Toshihiro Akaike

    2012-01-01

    Full Text Available Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide (PNIPAAm hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.

  18. Interleukin-8 derived from local tissue-resident stromal cells promotes tumor cell invasion.

    Science.gov (United States)

    Welte, Gabriel; Alt, Eckhard; Devarajan, Eswaran; Krishnappa, Srinivasalu; Jotzu, Constantin; Song, Yao-Hua

    2012-11-01

    The aim of this study is to evaluate the role of adipose tissue resident stromal cells on tumor cell invasion. Our data show that a subpopulation of adipose tissue derived stromal cells expressing Nestin, NG2, α-smooth muscle actin and PDGFR-α migrate toward the cancer cells. Microarray analysis revealed the upregulation of IL-8 in the migrated cells. We demonstrated that stromal cell derived IL-8 promote the invasion and the anchorage-independent growth of cancer cells. We conclude that human breast cancer cells attract a subpopulation of stromal cells that secrete IL-8 to promote tumor cell invasion in a paracrine fashion.

  19. Local 3D matrix confinement determines division axis through cell shape.

    Science.gov (United States)

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-02-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

  20. Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

    Directory of Open Access Journals (Sweden)

    Patrick C. Baer

    2012-01-01

    Full Text Available Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods.

  1. TREATMENT OF RAT HEPATOMA BY LOCALLY INJECTION OF MURINE IL-12 RETROVIRUS PACKAGING CELL

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the therapeutic effects of the murine IL-12 (mIL-12) retrovirus packaging cell line on hepatoma injected locally. Methods: The retrovirus vector encoding mIL-12 gene was constructed and transfected into packaging cell line PA317. The cells were then used to treat the rats with experimental orthotopic hepatoma at different time. The therapeutic effects, immune functions of the hosts, pathological and toxicological responses were documented. Results: the results showed that the mIL-12 retrovirus packaging cell line could significantly inhibit the growth of the hepatoma cells injected locally to the hepatoma. The early treatment made the rats survive long, while the medium or late stage treatment could prolong the life time of the rats compared with the bland control group or bland vector control group, though the rats did not survive. The number of NK cells and T cells increased significantly in the treatment group. The effects of the early treatment were superior to those of the medium and late stage treatment. Moreover, the transfection of IL-12 gene locally in the hepatoma tissue could make the hepatoma disappear from other liver lobe. This phenomenon demonstrated that IL-12 could activate the immune cells of the host to kill the untransfected tumor cells. This is very important for IL-12 to be used in gene therapy clinically. Meanwhile, the hepatoma would not recur in the rats that had survived more than 2 months from the early treatment after being re-challenged with tumor cells. Conclusion: the results showed that IL-12 gene injected locally in the hepatoma tissue could enhance the anti-tumor immunity of the host.

  2. Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

    Science.gov (United States)

    Chang, Hsiang-Hua D; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J; Pan, Yu; Zhou, Emily; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

  3. Pemetrexed disodium in recurrent locally advanced or metastatic squamous cell carcinoma of the head and neck

    OpenAIRE

    Pivot, X; Raymond, E; Laguerre, B.; Degardin, M; Cals, L; Armand, J P; Lefebvre, J L; Gedouin, D; Ripoche, V; Kayitalire, L; Niyikiza, C; Johnson, R.; Latz, J.; Schneider, M.

    2001-01-01

    This phase II study determined response rate of patients with locally advanced or metastatic head and neck cancer treated with pemetrexed disodium, a new multitargeted antifolate that inhibits thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase. 35 patients with local or metastatic relapse of squamous cell carcinoma of the head and neck (31 male, 4 female; median age 53 years) were treated with pemetrexed 500 mg m2 administered as a 10-minute infusi...

  4. Vismodegib: a guide to its use in locally advanced or metastatic basal cell carcinoma.

    Science.gov (United States)

    Lyseng-Williamson, Katherine A; Keating, Gillian M

    2013-02-01

    Vismodegib is the first Hedgehog pathway inhibitor to be approved in the USA, where it is indicated for the treatment of adults with metastatic basal cell carcinoma (BCC), or with locally advanced BCC that has recurred following surgery or who are not candidates for surgery, and who are not candidates for radiation. In an ongoing, noncomparative, phase II trial, oral vismodegib was effective in and had an acceptable tolerability profile in the treatment of patients with locally advanced or metastatic BCC.

  5. 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...

  6. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

    Science.gov (United States)

    Liapis, Evangelos; Sandiford, Simone; Wang, Qiang; Gaidosh, Gabriel; Motti, Dario; Levay, Konstantin; Slepak, Vladlen Z

    2012-08-01

    The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

  7. [The Langerhans cell histiocytosis with thymic localization as initial and exclusive place].

    Science.gov (United States)

    Hernández Pérez, J M; Franquet Casas, T; Rodríguez, S; Giménez, A

    2007-10-01

    The Langerhans' cell histiocytosis (LCH), also known as Histiocitosis X it is an illness not very frequent granulomatosus etiology not clarified yet, that it can have different manifestations and localizations, however the thymic localization as initial and exclusive place gives presentation HCL it is quite unusual. The present case is presented a patient that debuted with a clinical unspecific, where the tests give image they put she gives apparent a mass in previous mediastinum and that after the pathologic and immunohistochemical analysis they evidenced a proliferation Langerhans s cells and eosinophils it being positive for CD1a and S-100 confirming the diagnosis of the LCH.

  8. 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.

  9. Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

    OpenAIRE

    Susanne Zibek; Britta Hagmeyer; Alfred Stett; Martin Stelzle

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses.In an experimental setup micro-droplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporou...

  10. Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.

    Science.gov (United States)

    Sato, Mayumi; Amemiya, Kana; Hayakawa, Sumio; Munakata, Hiroshi

    2008-08-01

    Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT-PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS-7 cells this variant did not show any heparanase activity. Full-length heparanase and the exon 5-deleted splice variant were expressed in COS-7 cells and examined by confocal laser scanning microscopy. Both proteins co-localized with calnexin, a marker protein for the endoplasmic reticulum, and they co-immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS-PAGE analyses. Treatment with endoglycosidases revealed that all potential N-glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS-7 cells inhibited N-glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS-7 cells.

  11. Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Steven L. K. Ching; Satinder K. Gidda; Amanda Rochon; Owen R. van Cauwenberghe; Barry J. Shelp; Robert T. Mullen

    2012-01-01

    Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye,an intermediate of the γ-aminobutyrate (GABA) pathway.Two isoforms of GLYR exist in plants,GLYR1 and GLYR2,and while GLYR2 is known to be localized in plastids,GLYR1 has been reported to be localized in either peroxisomes or the cytosol.Here,we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L.Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants,in combination with fluorescence microscopy.The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species,tissue and/or cell type,or exposure of plants to environmental stresses that would increase flux through the GABA pathway.Moreover,the C-terminal tripeptide sequence of GLYR1,-SRE,despite its resemblance to a type 1 peroxisomal targeting signal,is not sufficient for targeting to peroxisomes.Collectively,these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells,but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

  12. Experimental and numerical studies of local current mapping on a PEM fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Hwnag, J.J. [Department of Environment and Energy, National University of Tainan, Tainan 700 (China); Chang, W.R. [Department of Landscape Architecture, Chung-Hua University, Hsinchu 300 (China); Peng, R.G. [Department of Mechanical Engineering, National Chiao Tong University, Hsinchu 300 (China); Chen, P.Y. [Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300 (China); Su, A. [Department of Mechanical Engineering and Fuel Cell Center, Yuan Ze University, Taoyuan (China)

    2008-10-15

    Local current distribution on a PEM fuel cell has been mapped experimentally by using a special-designed single cell fixture. It is composed of a composite cathodic flow-field plate, a membrane electrode assembly (MEA) and a stainless-steel anodic flow-field plate. An array of 16 individual conductive segments was distributed on the composite plate. A self-made MEA is in direct contact with the segmented current collectors. Regional-averaged current through each segment is determined by using the Hall-effect sensor. To ensure the data reliability, a comparison of polarization curves was made between the composite flow-field plate and the conventional flow-field plate. Then, the effects of flow-field patterns, dew points of the cathodic feedings and cathodic stoichiometrics on the local current distribution were examined. The transient variation of the local current distribution on the cathode under supersaturated conditions was further visualized to illustrate the flooding phenomena in different flow patterns. This technique developed by the present work has contributed to knowledge and understanding the local current distributions in a PEM fuel cell that is helpful in designing the fuel-cell components. (author)

  13. Differential and cell development-dependent localization of myelin mRNAs in oligodendrocytes

    NARCIS (Netherlands)

    deVries, H; deJonge, JC; Schrage, C; vanderHaar, ME; Hoekstra, D

    1997-01-01

    In oligodendrocytes (OLG), the mRNAs for the various myelin proteins localize to different intracellular sites, Whereas the confinement of myelin basic protein (MBP) mRNA to the processes of the cell has been well established, we demonstrate that most other myelin mRNA species are mainly present in

  14. 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.

  15. Localization of type I interferon receptor limits interferon-induced TLR-3 in epithelial cells

    Science.gov (United States)

    This study aimed to expand on the role of type I IFNs in the influenza-induced upregulation of TLR3 and determine whether and how the localization of the IFN-alpha/beta receptor (IFNAR) in respiratory epithelial cells could modify IFN-induced responses. Using differentiated prima...

  16. Subcellular Expression of Mammary Serine Proteinase Inhibitor (MASPIN in Locally Advance Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Samina Zaheer

    2014-08-01

    Conclusions: MASPIN expression was observed in the majority of OSCC. However, it was localized to the cytoplasm of tumor cells in all cases. Loss of MASPIN expression was observed more frequently in poorly differentiated cancers. [J Interdiscipl Histopathol 2014; 2(4.000: 213-216

  17. 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 suggest

  18. Systematic Review of Adrenalectomy and Lymph Node Dissection in Locally Advanced Renal Cell Carcinoma

    NARCIS (Netherlands)

    Bekema, Hendrika J.; MacLennan, Steven; Imamura, Mari; Lam, Thomas B. L.; Stewart, Fiona; Scott, Neil; MacLennan, Graeme; McClinton, Sam; Griffiths, T. R. Leyshon; Skolarikos, Andreas; MacLennan, Sara J.; Sylvester, Richard; Ljungberg, Borje; N'Dow, James

    2013-01-01

    Context: Controversy remains over whether adrenalectomy and lymph node dissection (LND) should be performed concomitantly with radical nephrectomy (RN) for locally advanced renal cell carcinoma (RCC) cT3-T4N0M0. Objective: To systematically review all relevant literature comparing oncologic, periope

  19. 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.

    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

  20. 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

    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

  1. Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells.

    NARCIS (Netherlands)

    Sinko, E; Ilias, A; Ujhelly, O; Homolya, L; Scheffer, G.L.; Bergen, AA; Sarkadi, B; Varadi, A

    2003-01-01

    Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the bas

  2. Challenges in optimizing chemoradiation in locally advanced non small-cell lung cancers in India

    Directory of Open Access Journals (Sweden)

    Sushma Agrawal

    2013-01-01

    Full Text Available Data supporting use of concurrent chemoradiation in locally advanced lung cancers comes from clinical trials from developed countries. Applicability and outcomes of such schedules in developing countries is not widely reported. There are various challenges in delivering chemoradiation in locally advanced non small cell lung cancer in developing countries which is highlighted by an audit of patients treated with chemoradiation in our center. This article deals with the challenges in the context of a developing country. We conclude that sequential chemoradiotherapy is better tolerated than concurrent chemoradiation in Indian patients with locally advanced non-small cell lung cancers. Patients with stage IIIa, normal weight or overweight, and adequate baseline pulmonary function should be offered concurrent chemoradiation.

  3. Digital Holographic Tomography and Fluorescence Used to Localize sGC in HEK293 Cells

    Science.gov (United States)

    Sheldrake, Eric; Mann, Christopher; Gage, Matthew

    2014-03-01

    Digital Holographic Tomography (DHT) is used to analyze and localize the intracellular protein soluble guanylate cyclase (sGC) in human embryonic kidney 293 (HEK293) cells. DHT is a non-invasive phase microscopy technique that provides three dimensional quantitative information of HEK293 cells including variance of index of refraction or physical thickness. A fluorescence component will be added to the microscope to further studies of sGC localization. The signaling pathway including nitric oxide (NO) and sGC is studied and has been linked to various cardiovascular diseases, platelet aggregation, and variations in blood pressure via vasodilation. sGC will be labeled using a fluorescent antibody and analyzed using the DHT microscope. DHT will be used to analyze changes in sGC localization in its natural environment and when stimulated by NO. An understanding of how sGC interacts with its surroundings is vital to further research in cardiovascular disease.

  4. Application of Local Activity Theory of Cellular Neural Network with Two Ports to the Coupled Lorenz-Cell Model

    Institute of Scientific and Technical Information of China (English)

    MIN LeQuan; YU Na

    2002-01-01

    Some criteria for the local activity theory in two-port cellular neural network cells with three local state variables are applied to a coupled Lorenz-cell model. The numerical simulation exhibited that emergence may exist if the selected cell parameters are nearby or on the edge of chaos domain. The local activity theory has provided a new tool of studying the complexity of high dimensional coupled nonlinear physical systems.

  5. 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.

  6. Localized plasmonic losses at metal back contacts of thin-film silicon solar cells

    Science.gov (United States)

    Paetzold, U. W.; Hallermann, F.; Pieters, B. E.; Rau, U.; Carius, R.; von Plessen, G.

    2010-05-01

    Investigations of optical losses induced by localized plasmons in protrusions on silver back contacts of thin-film silicon solar cells are presented. The interaction of electromagnetic waves with nanoprotrusions on flat silver layers is simulated with a three-dimensional numerical solver of Maxwell's equations. Spatial absorption profiles and spatial electric field profiles as well as the absorption inside the protrusions are calculated. The results presented here show that the absorption of irradiated light at nanorough silver layers can be strongly enhanced by localized plasmonic resonances in Ag nanoprotrusions. Especially, localized plasmons in protrusions with a radius below 60 nm induce strong absorption, which can be several times the energy irradiated on the protrusion's cross section. The localized plasmonic resonances in single protrusions on Ag layers are observed to shift to longer wavelengths with increasing refractive index of the surrounding material. At wavelengths above 500 nm localized plasmonic resonances will increase the absorption of nanorough μc-Si:H/Ag interfaces. The localized plasmon induced absorption at nanorough ZnO/Ag interfaces lies at shorter wavelengths due to the lower refractive index of ZnO. For wavelengths above 500 nm, a high reflectivity of the silver back contacts is essential for the light-trapping of thin-film silicon solar cells. Localized-plasmon induced losses at silver back contacts can explain the experimentally observed increase of the solar cell performance when applying a ZnO/Ag back contact in comparison to a μc-Si:H/Ag back contact.

  7. Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

    Science.gov (United States)

    Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

    2015-05-01

    Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.

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

    Directory of Open Access Journals (Sweden)

    Jessamine P Winer

    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.

  9. A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity.

    Science.gov (United States)

    Perez, Adam M; Mann, Thomas H; Lasker, Keren; Ahrens, Daniel G; Eckart, Michael R; Shapiro, Lucy

    2017-02-28

    Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment.IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this

  10. Local Augmented Angiotensinogen Secreted from Apoptotic Vascular Endothelial Cells Is a Vital Mediator of Vascular Remodelling.

    Directory of Open Access Journals (Sweden)

    Shyh-Jong Wu

    Full Text Available Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs and vascular smooth muscle cells (VSMCs through serum starvation (SS. After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II were found in a serum-free medium conditioned by HUVECs (SSC. The increased Ang II was suppressed using lisinopril (an ACE inhibitor treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

  11. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

    Science.gov (United States)

    de Renty, Christelle; DePamphilis, Melvin L; Ullah, Zakir

    2014-01-01

    Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

  12. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Christelle de Renty

    Full Text Available Proliferating trophoblast stem cells (TSCs can differentiate into nonproliferating but viable trophoblast giant cells (TGCs that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

  13. Diacylglycerol kinase ε localizes to subsurface cisterns of cerebellar Purkinje cells.

    Science.gov (United States)

    Hozumi, Yasukazu; Fujiwara, Hiroki; Kaneko, Kenya; Fujii, Satoshi; Topham, Matthew K; Watanabe, Masahiko; Goto, Kaoru

    2017-02-13

    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.

  14. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    Science.gov (United States)

    Voldgorn, Yana I; Adilgereeva, Elmira P; Nekrasov, Evgeny D; Lavrov, Alexander V

    2015-01-01

    Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  15. Emergence of Complex-Like Cells in a Temporal Product Network with Local Receptive Fields

    CERN Document Server

    Gregor, Karo

    2010-01-01

    We introduce a new neural architecture and an unsupervised algorithm for learning invariant representations from temporal sequence of images. The system uses two groups of complex cells whose outputs are combined multiplicatively: one that represents the content of the image, constrained to be constant over several consecutive frames, and one that represents the precise location of features, which is allowed to vary over time but constrained to be sparse. The architecture uses an encoder to extract features, and a decoder to reconstruct the input from the features. The method was applied to patches extracted from consecutive movie frames and produces orientation and frequency selective units analogous to the complex cells in V1. An extension of the method is proposed to train a network composed of units with local receptive field spread over a large image of arbitrary size. A layer of complex cells, subject to sparsity constraints, pool feature units over overlapping local neighborhoods, which causes the feat...

  16. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Yana I Voldgorn

    Full Text Available Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  17. 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.

  18. CyDiv, a conserved and novel filamentous Cyanobacteria cell division protein involved in septum localization.

    Directory of Open Access Journals (Sweden)

    Dinka eMandakovic

    2016-02-01

    Full Text Available Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division, encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120.

  19. CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization

    Science.gov (United States)

    Mandakovic, Dinka; Trigo, Carla; Andrade, Derly; Riquelme, Brenda; Gómez-Lillo, Gabriela; Soto-Liebe, Katia; Díez, Beatriz; Vásquez, Mónica

    2016-01-01

    Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120. PMID:26903973

  20. Physiological and pathological role of local and immigrating colonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Ferenc Sipos; Gábor Valcz; Béla Molnár

    2012-01-01

    The latest avenue of research is revealing the existence of and role for the colonic stem cells in the physiological renewal of the mucosa and in pathological circumstances where they have both positive and negative effects. In the case of human colon, different levels of stem cell compartments exist. First, the crypt epithelial stem cells, which have a role in the normal crypt epithelial cell dynamics and in colorectal carcinogenesis. Close to the crypts, the second layer of stem cells can be found; the local subepithelial stem cell niche, including the pericryptic subepithelial myofibroblasts that regulate the epithelial cell differentiation and have a crucial role in cancer progression and chronic inflammation-related fibrosis. The third level of stem cell compartment is the immigrating bone-marrow-derived stem cells, which have an important role in wound healing after severe mucosal inflammation, but are also involved in cancer invasion. This paper focuses on stem cell biology in the context of physiological and pathological processes in the human colon.

  1. Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics.

    Science.gov (United States)

    Pancrazi, Laura; Di Benedetto, Giulietta; Colombaioni, Laura; Della Sala, Grazia; Testa, Giovanna; Olimpico, Francesco; Reyes, Aurelio; Zeviani, Massimo; Pozzan, Tullio; Costa, Mario

    2015-11-10

    Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.

  2. Augmentation of CAR T-cell Trafficking and Antitumor Efficacy by Blocking Protein Kinase A Localization.

    Science.gov (United States)

    Newick, Kheng; O'Brien, Shaun; Sun, Jing; Kapoor, Veena; Maceyko, Steven; Lo, Albert; Puré, Ellen; Moon, Edmund; Albelda, Steven M

    2016-06-01

    Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the "regulatory subunit I anchoring disruptor" (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541-51. ©2016 AACR.

  3. 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.

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

    Science.gov (United States)

    Trombetta, Mark; Packard, Matthew; Velosa, Claudia; Silverman, Jan; Werts, Day; Parda, David

    2011-03-30

    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.

  5. Numerical study of cell performance and local transport phenomena in PEM fuel cells with various flow channel area ratios

    Science.gov (United States)

    Wang, Xiao-Dong; Duan, Yuan-Yuan; Yan, Wei-Mon

    Three-dimensional models of proton exchange membrane fuel cells (PEMFCs) with parallel and interdigitated flow channel designs were developed including the effects of liquid water formation on the reactant gas transport. The models were used to investigate the effects of the flow channel area ratio and the cathode flow rate on the cell performance and local transport characteristics. The results reveal that at high operating voltages, the cell performance is independent of the flow channel designs and operating parameters, while at low operating voltages, both significantly affect cell performance. For the parallel flow channel design, as the flow channel area ratio increases the cell performance improves because fuel is transported into the diffusion layer and the catalyst layer mainly by diffusion. A larger flow channel area ratio increases the contact area between the fuel and the diffusion layer, which allows more fuel to directly diffuse into the porous layers to participate in the electrochemical reaction which enhances the reaction rates. For the interdigitated flow channel design, the baffle forces more fuel to enter the cell and participate in the electrochemical reaction, so the flow channel area ratio has less effect. Forced convection not only increases the fuel transport rates but also enhances the liquid water removal, thus interdigitated flow channel design has higher performance than the parallel flow channel design. The optimal performance for the interdigitated flow channel design occurs for a flow channel area ratio of 0.4. The cell performance also improves as the cathode flow rate increases. The effects of the flow channel area ratio and the cathode flow rate on cell performance are analyzed based on the local current densities, oxygen flow rates and liquid water concentrations inside the cell.

  6. Numerical study of cell performance and local transport phenomena in PEM fuel cells with various flow channel area ratios

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiao-Dong [Department of Thermal Engineering, School of Mechanical Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Duan, Yuan-Yuan [Key Laboratory of Thermal Science and Power Engineering of MOE, Tsinghua University, Beijing 100084 (China); Yan, Wei-Mon [Department of Mechatronic Engineering, Huafan University, Shih-Ting 22305 (China)

    2007-10-11

    Three-dimensional models of proton exchange membrane fuel cells (PEMFCs) with parallel and interdigitated flow channel designs were developed including the effects of liquid water formation on the reactant gas transport. The models were used to investigate the effects of the flow channel area ratio and the cathode flow rate on the cell performance and local transport characteristics. The results reveal that at high operating voltages, the cell performance is independent of the flow channel designs and operating parameters, while at low operating voltages, both significantly affect cell performance. For the parallel flow channel design, as the flow channel area ratio increases the cell performance improves because fuel is transported into the diffusion layer and the catalyst layer mainly by diffusion. A larger flow channel area ratio increases the contact area between the fuel and the diffusion layer, which allows more fuel to directly diffuse into the porous layers to participate in the electrochemical reaction which enhances the reaction rates. For the interdigitated flow channel design, the baffle forces more fuel to enter the cell and participate in the electrochemical reaction, so the flow channel area ratio has less effect. Forced convection not only increases the fuel transport rates but also enhances the liquid water removal, thus interdigitated flow channel design has higher performance than the parallel flow channel design. The optimal performance for the interdigitated flow channel design occurs for a flow channel area ratio of 0.4. The cell performance also improves as the cathode flow rate increases. The effects of the flow channel area ratio and the cathode flow rate on cell performance are analyzed based on the local current densities, oxygen flow rates and liquid water concentrations inside the cell. (author)

  7. FOXP3 Subcellular Localization Predicts Recurrence in Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Weed, Donald T.; Walker, Gail; De La Fuente, Adriana C.; Nazarian, Ronen; Vella, Jennifer L.; Gomez-Fernandez, Carmen R.; Serafini, Paolo

    2013-01-01

    Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4+T cells is generally associated with an intrinsic capacity to suppress tumor immunity. Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4+FOXP3+T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4+T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4+ T cells as a predictor of OSCC recurrence. PMID:23977174

  8. Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells

    Directory of Open Access Journals (Sweden)

    Zarai Zied

    2011-11-01

    Full Text Available Abstract Background Among the digestive enzymes, phospholipase A2 (PLA2 hydrolyzes the essential dietary phospholipids in marine fish and shellfish. However, we know little about the organs that produce PLA2, and the ontogeny of the PLA2-cells. Accordingly, accurate localization of PLA2 in marine snails might afford a better understanding permitting the control of the quality and composition of diets and the mode of digestion of lipid food. Results We have previously producted an antiserum reacting specifically with mSDPLA2. It labeled zymogen granules of the hepatopancreatic acinar cells and the secretory materials of certain epithelial cells in the depths of epithelial crypts in the hepatopancreas of snail. To confirm this localization a laser capture microdissection was performed targeting stained cells of hepatopancreas tissue sections. A Western blot analysis revealed a strong signal at the expected size (30 kDa, probably corresponding to the PLA2. Conclusions The present results support the presence of two hepatopancreatic intracellular and extracellular PLA2 in the prosobranchs gastropods molluscs, Littorina littorea and Buccinum undatum and bring insights on their localizations.

  9. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets.

    Science.gov (United States)

    Stockert, Juan C; Blázquez-Castro, Alfonso; Cañete, Magdalena; Horobin, Richard W; Villanueva, Angeles

    2012-12-01

    Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

  10. 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.

  11. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC

    Science.gov (United States)

    Wang, C.; Jung, D.; Cao, Z.; Chung, C. Y.

    2015-01-01

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC− cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC− cells. racC− cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC− cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

  12. Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts.

    Science.gov (United States)

    Turner, Rachael A; Wauthier, Eliane; Lozoya, Oswaldo; McClelland, Randall; Bowsher, James E; Barbier, Claire; Prestwich, Glenn; Hsu, Edward; Gerber, David A; Reid, Lola M

    2013-02-01

    Cell therapies are potential alternatives to organ transplantation for liver failure or dysfunction but are compromised by inefficient engraftment, cell dispersal to ectopic sites, and emboli formation. Grafting strategies have been devised for transplantation of human hepatic stem cells (hHpSCs) embedded into a mix of soluble signals and extracellular matrix biomaterials (hyaluronans, type III collagen, laminin) found in stem cell niches. The hHpSCs maintain a stable stem cell phenotype under the graft conditions. The grafts were transplanted into the livers of immunocompromised murine hosts with and without carbon tetrachloride treatment to assess the effects of quiescent versus injured liver conditions. Grafted cells remained localized to the livers, resulting in a larger bolus of engrafted cells in the host livers under quiescent conditions and with potential for more rapid expansion under injured liver conditions. By contrast, transplantation by direct injection or via a vascular route resulted in inefficient engraftment and cell dispersal to ectopic sites. Transplantation by grafting is proposed as a preferred strategy for cell therapies for solid organs such as the liver.

  13. Reaction of cells to local, regional, and general low-intensive laser irradiation

    Science.gov (United States)

    Baibekov, Iskander M.; Kasymov, A. S.; Musaev, Erkin S.; Vorojeikin, V. M.; Artikov, S. N.

    1993-07-01

    Local influence of low intensive laser irradiation (LILI) of Helium-Neon (HNL), Copper vapor (CVL), Nitrogen (UVL) and Arsenic Gallium (AGL) lasers cause stimulation of processes of physiological and reparative regeneration in intact skin, and mucous membrane of stomach and duodenum, dermatome wounds and gastroduodenal ulcers. Structural bases of these effects are the acceleration of cell proliferation and differentiation and also the activation of intracellular structures and intensification of cell secretion. Regional influence of the pointed types of LILI on hepar in cirrhosis and hepatitis causes decreasing of the inflammatory and cirrhotic changes. After endo- and exo-vascular laser irradiations of blood the decreasing of the number of pathological forms of erythrocytes and the increasing of their catalase activity, are indicated. General (total) laser irradiation of the organism--laser shower, increases the bone marrow cells proliferation, especially myeloid series. It is accompanied with acceleration of their differentiation and migration in circulation. It was revealed, that HNL to a considerable extent influences the epithelial cells and CVL the connective tissue cells. UVL increases the amount of microorganisms on cell surfaces (membrane bound microorganisms). Regional irradiation of the LILI causes both direct and indirect influence of cells. Structural changes of bone marrow cells and gut mucous membrane cells indicate intersystemic interaction.

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

    Directory of Open Access Journals (Sweden)

    Coulter JA

    2012-06-01

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

  15. 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.

  16. Dachsous-Dependent Asymmetric Localization of Spiny-Legs Determines Planar Cell Polarity Orientation in Drosophila

    Directory of Open Access Journals (Sweden)

    Tomonori Ayukawa

    2014-07-01

    Full Text Available In Drosophila, planar cell polarity (PCP molecules such as Dachsous (Ds may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz. However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk isoforms, Pk and Spiny-legs (Sple. Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.

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

    CERN Document Server

    Abreu, Salvator; Codognet, Philippe

    2009-01-01

    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.

  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. Localization of Sonic hedgehog secreting and receiving cells in the developing and adult rat adrenal cortex.

    Science.gov (United States)

    Guasti, Leonardo; Paul, Alex; Laufer, Ed; King, Peter

    2011-04-10

    Sonic hedgehog signaling was recently demonstrated to play an important role in murine adrenal cortex development. The organization of the rat adrenal differs from that of the mouse, with the zona glomerulosa and zona fasciculata separated by an undifferentiated zone in the rat, but not in the mouse. In the present study we aimed to determine the mRNA expression patterns of Sonic hedgehog and the hedgehog signaling pathway components Patched-1 and Gli1 in the developing and adult rat adrenal. Sonic hedgehog expression was detected at the periphery of the cortex in cells lacking CYP11B1 and CYP11B2 expression, while signal-receiving cells were localized in the overlying capsule mesenchyme. Using combined in situ hybridization and immunohistochemistry we found that the cells expressing Sonic hedgehog lie between the CYP11B2 and CYP11B1 layers, and thus Sonic hedgehog expression defines one cell population of the undifferentiated zone.

  20. 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

    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...... of satellite cells 8 days after exercise. These results suggest that NSAIDs negatively affect satellite cell activity after unaccustomed eccentric exercise.......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...

  1. Localized Surface Plasmons Enhanced Light Transmission into c-Silicon Solar Cells

    Directory of Open Access Journals (Sweden)

    Y. Premkumar Singh

    2013-01-01

    Full Text Available The paper investigates the light incoupling into c-Si solar cells due to the excitation of localized surface plasmon resonances in periodic metallic nanoparticles by finite-difference time-domain (FDTD technique. A significant enhancement of AM1.5G solar radiation transmission has been demonstrated by depositing nanoparticles of various metals on the upper surface of a semi-infinite Si substrate. Plasmonic nanostructures located close to the cell surface can scatter incident light efficiently into the cell. Al nanoparticles were found to be superior to Ag, Cu, and Au nanoparticles due to the improved transmission of light over almost the entire solar spectrum and, thus, can be a potential low-cost plasmonic metal for large-scale implementation of solar cells.

  2. Local Entropy Production Rates in a Polymer Electrolyte Membrane Fuel Cell

    Science.gov (United States)

    Siemer, Marc; Marquardt, Tobias; Huerta, Gerardo Valadez; Kabelac, Stephan

    2017-01-01

    A modeling study on a polymer electrolyte membrane fuel cell by means of non-equilibrium thermodynamics is presented. The developed model considers a one-dimensional cell in steady-state operation. The temperature, concentration and electric potential profiles are calculated for every domain of the cell. While the gas diffusion and the catalyst layers are calculated with established classical modeling approaches, the transport processes in the membrane are calculated with the phenomenological equations as dictated by the non-equilibrium thermodynamics. This approach is especially instructive for the membrane as the coupled transport mechanisms are dominant. The needed phenomenological coefficients are approximated on the base of conventional transport coefficients. Knowing the fluxes and their intrinsic corresponding forces, the local entropy production rate is calculated. Accordingly, the different loss mechanisms can be detected and quantified, which is important for cell and stack optimization.

  3. 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.

  4. 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.

  5. Local circuitry involving parvalbumin-positive basket cells in the CA2 region of the hippocampus.

    Science.gov (United States)

    Mercer, Audrey; Eastlake, Karen; Trigg, Hayley L; Thomson, Alex M

    2012-01-01

    There is a growing recognition that the CA2 region of the hippocampus has its own distinctive properties, inputs, and pathologies. The dendritic and axonal patterns of some interneurons in this region are also strikingly different from those described previously in CA1 and CA3. The local circuitry in this region, however, had yet to be studied in detail. Accordingly, using dual intracellular recordings and biocytin-filling, excitatory and inhibitory connections involving CA2 parvalbumin-positive basket cells were characterized for the first time. CA2 basket cells targeted neighboring pyramidal cells and received excitatory inputs from them. CA2 basket cells that resembled those in CA1 with a fast spiking behavior and dendritic tree confined to the region of origin received depressing excitatory postsynaptic potentials (EPSPs). In contrast, unlike CA1 basket cells but like CA1 Oriens-Lacunosum Moleculare (OLM) cells, the majority of CA2 basket cells had horizontally oriented dendrites in Stratum Oriens (SO), which extended into all three CA subfields, had an adapting firing pattern, presented a "sag" in their voltage responses to hyperpolarizing current injection, and received facilitating EPSPs. The expression of I(h) did not influence the EPSP time courses and paired pulse ratios (PPR). Estimates of the probability of release (p) for the depressing and facilitating EPSPs were correlated with the PPR. Connections with low probabilities of release had higher PPR. Quantal amplitude (q) for the facilitating connections was larger than q at depressing inputs onto fast spiking basket cells.

  6. Expression and cell-specific localization of cholecystokinin receptors in rat lung

    Institute of Scientific and Technical Information of China (English)

    Bin Cong; Shu-Jin Li; Yi-Ling Ling; Yu-Xia Yao; Zhen-Yong Gu; Jun-xia Wang; Hong-Yu You

    2003-01-01

    AIM: To elucidate whether CCK receptors exist in lung tissues and their precise cellular localization in the lung. METHODS: CCK-AR and CCK-BR mRNA expression andcellular distribution in the rat lung were detected by highly sensitive method of in situ reverse transcription-polymerase chain reaction (RT-PCR) and conventional in situ hybridization. RESULTS: CCK-AR and CCK-BR gene positive signals were observed in bronchial epithelial cells, alveolar epithelial cells,pulmonary macrophages and vascular endothelial cells of the rats' lung byin situ RT-PCR. The hybridization signals of CCK-AR were relatively faint. By in situ hybridization,however, only the signals of CCK-BR but not CCK-AR were detected in the lung, and the positive staining was only found in vascular endothelial cells and macrophages. CONCLUSION: CCK-AR and CCK-BR gene were present in pulmonary vascular endothelial cells, macrophages, bronchial epithelial cells and alveolar epithelial cells, which play an important role in mediating the regulatory actions of CCK-8on these cells.

  7. Protein-specific localization of a rhodamine-based calcium-sensor in living cells.

    Science.gov (United States)

    Best, Marcel; Porth, Isabel; Hauke, Sebastian; Braun, Felix; Herten, Dirk-Peter; Wombacher, Richard

    2016-06-28

    A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.

  8. The influence of local environment on the organization of mesenchyme cells.

    Science.gov (United States)

    Fisher, M; Solursh, M

    1979-01-01

    Limb, somite, and neural crest mesenchyme from quail embryos were implanted orthotopically and heterotopically into chick hosts to ascertain the relative importance of the local environment on mesenchyme migration. It was found that mesenchyme behavior is strongly influenced by the environment. Normally non-migratory, limb mesenchyme is capable of spreading like sclerotome when placed in the somite region. A somite placed in the limb acquires an appearance typical of limb mesenchyme. Neural crest placed in the limb migrates only along the co-implanted neural tube or axons growing out from it. The orthotopic transplantations showed that quail mesenchyme behaves normally in chick embryos. Furthermore, it was observed in the orthotopic transplants that there was no intermingling of quail and chick cells even at the edge of the graft. This result indicates that cells within mesenchyme are normally not locomotory; rather, the mesenchyme "migrates" by spreading and expansion of the tissue as a unit in response to local influences.

  9. Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale

    Science.gov (United States)

    Szczurek, Aleksander; Xing, Jun; Birk, Udo J.; Cremer, Christoph

    2016-01-01

    Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50–100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field. PMID:27446198

  10. Localization and function of calmodulin in live-cells of Aspergillus nidulans.

    Science.gov (United States)

    Chen, Shaochun; Song, Yiju; Cao, Jinling; Wang, Gang; Wei, Hua; Xu, Xushi; Lu, Ling

    2010-03-01

    Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca(2+). Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP-CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP-CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP-CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.

  11. Columnar deformation of human red blood cell by highly localized fiber optic Bessel beam stretcher.

    Science.gov (United States)

    Lee, Sungrae; Joo, Boram; Jeon, Pyo Jin; Im, Seongil; Oh, Kyunghwan

    2015-11-01

    A single human red blood cell was optically stretched along two counter-propagating fiber-optic Bessel-like beams in an integrated lab-on-a-chip structure. The beam enabled highly localized stretching of RBC, and it induced a nonlinear mechanical deformation to finally reach an irreversible columnar shape that has not been reported. We characterized and systematically quantified this optically induced mechanical deformation by the geometrical aspect ratio of stretched RBC and the irreversible stretching time. The proposed RBC mechanism can realize a versatile and compact opto-mechanical platform for optical diagnosis of biological substances in the single cell level.

  12. Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

    OpenAIRE

    Antalffy, Géza; Caride, Ariel J.; Pászty, Katalin; Hegedus, Luca; Padanyi, Rita; STREHLER, EMANUEL E.; Enyedi, Ágnes

    2011-01-01

    The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amoun...

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

    DEFF Research Database (Denmark)

    Tormin, Ariane; Li, Ou; Brune, Jan Claas

    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...... 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...

  14. Local positive feedback regulation determines cell shape in root hair cells.

    Science.gov (United States)

    Takeda, Seiji; Gapper, Catherine; Kaya, Hidetaka; Bell, Elizabeth; Kuchitsu, Kazuyuki; Dolan, Liam

    2008-02-29

    The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase-derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.

  15. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    Science.gov (United States)

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  16. Myc localizes to histone locus bodies during replication in Drosophila.

    Directory of Open Access Journals (Sweden)

    Kaveh Daneshvar

    Full Text Available Myc is an important protein at the center of multiple pathways required for growth and proliferation in animals. The absence of Myc is lethal in flies and mice, and its over-production is a potent inducer of over-proliferation and cancer. Myc protein is localized to the nucleus where it executes its many functions, however the specific sub-nuclear localization of Myc has rarely been reported. The work we describe here began with an observation of unexpected, punctate spots of Myc protein in certain regions of Drosophila embryos. We investigated the identity of these puncta and demonstrate that Myc is co-localized with coilin, a marker for sub-nuclear organelles known as Cajal Bodies (CBs, in embryos, larvae and ovaries. Using antibodies specific for U7 snRNP component Lsm11, we show that the majority of Myc and coilin co-localization occurs in Histone Locus Bodies (HLBs, the sites of histone mRNA transcription and processing. Furthermore, Myc localizes to HLBs only during replication in mitotic and endocycling cells, suggesting that its role there relates to replication-dependent canonical histone gene transcription. These results provide evidence that sub-nuclear localization of Myc is cell-cycle dependent and potentially important for histone mRNA production and processing.

  17. Sumoylation regulates nuclear localization of lipin-1alpha in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Guang-Hui Liu

    Full Text Available Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1alpha is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1alpha is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1alpha loses the capacity to coactivate the transcriptional (co- activators PGC-1alpha and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1alpha that we observe in cultured neuronal cells, and suggest that lipin-1alpha may act as a sumoylation-regulated transcriptional coactivator in brain.

  18. Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes.

    Science.gov (United States)

    Das, Gishnu; Jenny, Andreas; Klein, Thomas J; Eaton, Suzanne; Mlodzik, Marek

    2004-09-01

    Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. We provide evidence that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. We show that the initial membrane association of Dgo depends on Fz, and that Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi.

  19. Interneurons are the local targets of hippocampal inhibitory cells which project to the medial septum.

    Science.gov (United States)

    Gulyás, A I; Hájos, N; Katona, I; Freund, T F

    2003-05-01

    A subset of GABAergic neurons projecting to the medial septum has long been described in the hippocampus. However, the lack of information about their local connectivity pattern or their correspondence with any of the well-established hippocampal interneuron types has hampered the understanding of their functional role. Retrograde tracing combined with immunostaining for neurochemical markers in the adult rat hippocampus showed that nearly all hippocampo-septal (HS) neurons express somatostatin (>95%) and, in the hilus and CA3 stratum lucidum, many contain calretinin (>45%). In contrast, in stratum oriens of the CA1 and CA3 subfields, the majority of HS neurons contain somatostatin (>86%) and calbindin (>73%), but not calretinin. Because somatostatin-positive hippocampal interneurons have been most extensively characterized in the stratum oriens of CA1, we focused our further analysis on HS cells found in this region. In 18-20-day-old rats, intracellularly filled CA1-HS cells had extensive local axon collaterals crossing subfield boundaries and innervating the CA3 region and the dentate gyrus. Electron microscopic analysis provided evidence that the axon terminals of CA1-HS cells form symmetrical synapses selectively on GABAergic interneurons, both locally and in the CA3 region. In addition, double retrograde labelling experiments revealed that many CA1-HS neurons of the dorsal hippocampus also have collateral projections to the ventral hippocampus. Thus, CA1-HS cells innervate inhibitory interneurons locally and in remote hippocampal regions, in addition to targeting mostly GABAergic neurons in the medial septum. This dual projection with striking target selectivity for GABAergic neurons may be ideally suited to synchronize neuronal activity along the septo-hippocampal axis.

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

    Directory of Open Access Journals (Sweden)

    Lori B Huberman

    Full Text Available 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.

  1. 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.

  2. 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.

    2016-02-01

    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.

  3. Local circadian clock gates cell cycle progression of transient amplifying cells during regenerative hair cycling.

    Science.gov (United States)

    Plikus, Maksim V; Vollmers, Christopher; de la Cruz, Damon; Chaix, Amandine; Ramos, Raul; Panda, Satchidananda; Chuong, Cheng-Ming

    2013-06-04

    Regenerative cycling of hair follicles offers an unique opportunity to explore the role of circadian clock in physiological tissue regeneration. We focused on the role of circadian clock in actively proliferating transient amplifying cells, as opposed to quiescent stem cells. We identified two key sites of peripheral circadian clock activity specific to regenerating anagen hair follicles, namely epithelial matrix and mesenchymal dermal papilla. We showed that peripheral circadian clock in epithelial matrix cells generates prominent daily mitotic rhythm. As a consequence of this mitotic rhythmicity, hairs grow faster in the morning than in the evening. Because cells are the most susceptible to DNA damage during mitosis, this cycle leads to a remarkable time-of-day-dependent sensitivity of growing hair follicles to genotoxic stress. Same doses of γ-radiation caused dramatic hair loss in wild-type mice when administered in the morning, during mitotic peak, compared with the evening, when hair loss is minimal. This diurnal radioprotective effect becomes lost in circadian mutants, consistent with asynchronous mitoses in their hair follicles. Clock coordinates cell cycle progression with genotoxic stress responses by synchronizing Cdc2/Cyclin B-mediated G2/M checkpoint. Our results uncover diurnal mitotic gating as the essential protective mechanism in highly proliferative hair follicles and offer strategies for minimizing or maximizing cytotoxicity of radiation therapies.

  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.

    2017-01-01

    Abstract Introduction 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. Methods 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. Results 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. Conclusions 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. PMID:28250928

  5. Long-term label retaining cells localize to distinct regions within the female reproductive epithelium.

    Science.gov (United States)

    Patterson, Amanda L; Pru, James K

    2013-09-01

    distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.

  6. 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.

  7. Localization of connexin 30 in the luminal membrane of cells in the distal nephron.

    Science.gov (United States)

    McCulloch, Fiona; Chambrey, Régine; Eladari, Dominique; Peti-Peterdi, János

    2005-12-01

    Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

  8. Localization of Vibrio vulnificus infection in dendritic cells and its effects on the cytoskeleton

    Institute of Scientific and Technical Information of China (English)

    WANG Zhi-gang; XU Shui-ling; SHAO Ping-yang; BAO Yi; CUI Ge; CAI Yu-jie

    2012-01-01

    Background Vibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections.However,little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton.In this study,we aimed to investigate the invasion,internalization,and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection.Methods The study model was the cultured DCs infected by a Vv 1.758 strain.Electron microscopy was used to observe the localization of bacteria at the different time points of infection,cell morphology,and the process of organelles changes.The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope.Results The Vv were pinocytosised into the DC cells through double-sides,and localized at 1-2 μm of the inner side membrane.It took 1.3,1.9,and 3.4 hours to reach the infection ratio of 25%,50%,and 75%,respectively.Using electron microscopy,the DCs had been observed to have developed chromatin aggregation within 4.0 hours,and significant cytoskeleton structure disruption was noted within 6.0 hours.Conclusion The high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

  9. Emergence of large-scale cell morphology and movement from local actin filament growth dynamics.

    Directory of Open Access Journals (Sweden)

    Catherine I Lacayo

    2007-09-01

    Full Text Available Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP, affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged "canoe" shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features--cell shape, leading-edge shape, filamentous actin (F-actin distribution, cell speed, and directional persistence--that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes--which had VASP highly enriched at their leading edges and migrated fast with straight trajectories--to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and

  10. Zona occludens-2 inhibits cyclin D1 expression and cell proliferation and exhibits changes in localization along the cell cycle.

    Science.gov (United States)

    Tapia, Rocio; Huerta, Miriam; Islas, Socorro; Avila-Flores, Antonia; Lopez-Bayghen, Esther; Weiske, Jörg; Huber, Otmar; González-Mariscal, Lorenza

    2009-02-01

    Here, we have studied the effect of the tight junction protein zona occludens (ZO)-2 on cyclin D1 (CD1) protein expression. CD1 is essential for cell progression through the G1 phase of the cell cycle. We have found that in cultures of synchronized Madin-Darby canine kidney cells, ZO-2 inhibits cell proliferation at G0/G1 and decreases CD1 protein level. These effects occur in response to a diminished CD1 translation and an augmented CD1 degradation at the proteosome triggered by ZO-2. ZO-2 overexpression decreases the amount of Glycogen synthase kinase-3beta phosphorylated at Ser9 and represses beta-catenin target gene expression. We have also explored the expression of ZO-2 through the cell cycle and demonstrate that ZO-2 enters the nucleus at the late G1 phase and leaves the nucleus when the cell is in mitosis. These results thus explain why in confluent quiescent epithelia ZO-2 is absent from the nucleus and localizes at the cellular borders, whereas in sparse proliferating cultures ZO-2 is conspicuously present at the nucleus.

  11. 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.

  12. Subcellular localization of Cd in the root cells of Allium sativum by electron energy loss spectroscopy

    Indian Academy of Sciences (India)

    Donghua Liu; Ingrid Kottke

    2003-06-01

    The ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cd (100 M, 1 mM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes – high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.

  13. 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.

  14. ELECTRON MICROSCOPIC AUTORADIOGRAPHIC STUDY ON SUBCELLULAR LOCALIZATION OF FISSION PRODUCT 147Pm IN TISSUE CELLS

    Institute of Scientific and Technical Information of China (English)

    朱寿彭; 汪源长

    1994-01-01

    The early risk of internal contaminated accumualtion of 147Pm is in blood cells and endothelial cells,especially in red blood cells.Then 147Pm is selectively deposited in ultrastructure of liver cells,such as in nucleus,nucleolus,rough endoplasmic reticulum,mitochondria and microbodies,Dense tracks also appear in mitochondria and lysosome of pedal cells within renal corpuscle,and so dose in nucleus as well as in mitochondria and microbodies of epicyte of kidney near-convoluted tubule.With the prolongation of observing time,147Pm is selectively and steadily depostied in subcellular level of organic ocmponent for bone.Substantial amount of 147Pm is taken up into the nuclear fraction of osteoclasts and osteoblasts.Particularly,in organelles 147Pm is mainly accumulated in rough endoplasmic reticulum and in mitochondria.Autoradiographic tracks especially localize in combined point between Golgi complex and transitive vesicle of rough endoplasmic reticulum.In addition,numerous 147Pm deposited in collagenous fibre within interstitial of bone cells is hardly excreted.

  15. Optimization of Rear Local Contacts on High Efficiency PERC Solar Cells Structures

    Directory of Open Access Journals (Sweden)

    Kapila Wijekoon

    2013-01-01

    Full Text Available A local contact formation process and integration scheme have been developed for the fabrication of rear passivated point contact solar cells. Conversion efficiency of 19.6% was achieved using  mm, pseudo square, p-type single crystalline silicon wafers. This is a significant improvement when compared to unpassivated, full area aluminum back surface field solar cells, which exhibit only 18.9% conversion efficiency on the same wafer type. The effect of rear contact formation on cell efficiency was studied as a function of contact area and contact pitch, hence the metallization fraction. Contact shape and the thickness of Al-BSF layer were found to be heavily dependent on the laser ablation pattern and contact area. Simulated cell parameters as a function of metallization showed that there is a tradeoff between open circuit voltage and fill factor gains as the metallization fraction varies. The rear surface was passivated with an Al2O3 layer and a capping layer. The rear surface contact pattern was created by laser ablation and the contact geometry was optimized to obtain voids free contact filling, resulting in a uniform back surface field. The efficiency gain in rear passivated cells over the reference cells is mainly due to improved short circuit current and open circuit voltage.

  16. 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

    NKT cells are a subgroup of T cells, which express a restricted TCR repertoire and are critical for the innate immune responses to viral infections. Activation of NKT cells depends on the major histocompatibility complex-related molecule CD1d, which presents bioactive lipids to NKT cells....... 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...

  17. Retinoic acid as target for local pharmacokinetic interaction with modafinil in neural cells.

    Science.gov (United States)

    Hellmann-Regen, Julian; Gertz, Karen; Uhlemann, Ria; Colla, Michael; Endres, Matthias; Kronenberg, Golo

    2012-12-01

    While the biological importance of the cytochrome P450 system in the liver is well established, much less is known about its role in the brain and drug interactions at the level of brain cells have hardly been investigated. Here, we show that modafinil, a well-known inducer of hepatic CYP enzymes, also increases CYP3A4 expression in human-derived neuron-like SH-SY5Y cells. Upregulation of CYP3A4 by modafinil was associated with increased retinoic acid (RA) degradation, which could be blocked by specific CYP3A4 inhibitor erythromycin. In turn, reduced RA levels in culture medium during modafinil treatment resulted in decreased neuronal differentiation of SH-SY5Y cells as assessed by intracellular neurotransmitter concentrations and proliferative activity. Again, this differentiation-impeding effect of modafinil on SH-SY5Y cells was antagonized by erythromycin. Similarly, modafinil treatment of the murine GL261 glioma cell line resulted in increased proliferative activity. This was associated with upregulation of RA-degrading CYP26A1 in GL261 cells. Taken together, our results indicate that psychopharmacological agents such as modafinil may directly act on CYP enzymes in neural tissue. These kinds of drug effects may become highly relevant especially in the context of biomolecules such as RA whose local metabolism in brain is under tight spatial and temporal control.

  18. Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells.

    Science.gov (United States)

    Robben, Joris H; Fenton, Robert A; Vargas, Sarah L; Schweer, Horst; Peti-Peterdi, Janos; Deen, Peter M T; Milligan, Graeme

    2009-12-01

    When the succinate receptor (SUCNR1) is activated in the afferent arterioles of the glomerulus it increases renin release and induces hypertension. To study its location in other nephron segments and its role in kidney function, we performed immunohistochemical analysis and found that SUCNR1 is located in the luminal membrane of macula densa cells of the juxtaglomerular apparatus in close proximity to renin-producing granular cells, the cortical thick ascending limb, and cortical and inner medullary collecting duct cells. In order to study its signaling, SUCNR1 was stably expressed in Madin-Darby Canine Kidney (MDCK) cells, where it localized to the apical membrane. Activation of the cells by succinate caused Gq and Gi-mediated intracellular calcium mobilization, transient phosphorylation of extracellular regulated kinase (ERK)1/2 and the release of arachidonic acid along with prostaglandins E2 and I2. Signaling was desensitized without receptor internalization but rapidly resensitized upon succinate removal. Immunohistochemical evidence of phosphorylated ERK1/2 was found in cortical collecting duct cells of wild type but not SUCNR1 knockout streptozotocin-induced diabetic mice, indicating in vivo relevance. Since urinary succinate concentrations in health and disease are in the activation range of the SUCNR1, this receptor can sense succinate in the luminal fluid. Our study suggests that changes in the luminal succinate concentration may regulate several aspects of renal function.

  19. TOR signaling regulates planarian stem cells and controls localized and organismal growth.

    Science.gov (United States)

    Peiris, T Harshani; Weckerle, Frank; Ozamoto, Elyse; Ramirez, Daniel; Davidian, Devon; García-Ojeda, Marcos E; Oviedo, Néstor J

    2012-04-01

    Target of Rapamycin (TOR) controls an evolutionarily conserved signaling pathway that modulates cellular growth and division by sensing levels of nutrients, energy and stress. As such, TOR signaling is a crucial component of tissues and organs that translates systemic signals into cellular behavior. The ubiquitous nature of TOR signaling, together with the difficulty of analyzing tissue during cellular turnover and repair, have limited our understanding of how this kinase operates throughout the body. Here, we use the planarian model system to address TOR regulation at the organismal level. The planarian TOR homolog (Smed-TOR) is ubiquitously expressed, including stem cells (neoblasts) and differentiated tissues. Inhibition of TOR with RNA interference severely restricts cell proliferation, allowing the study of neoblasts with restricted proliferative capacity during regeneration and systemic cell turnover. Strikingly, TOR signaling is required for neoblast response to amputation and localized growth (blastema). However, in the absence of TOR signaling, regeneration takes place only within differentiated tissues. In addition, TOR is essential for maintaining the balance between cell division and cell death, and its dysfunction leads to tissue degeneration and lack of organismal growth in the presence of nutrients. Finally, TOR function is likely to be mediated through TOR Complex 1 as its disruption recapitulates signs of the TOR phenotype. Our data reveal novel roles for TOR signaling in controlling adult stem cells at a systemic level and suggest a new paradigm for studying TOR function during physiological turnover and regeneration.

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

    Directory of Open Access Journals (Sweden)

    Rizvi SB

    2014-03-01

    Full Text Available Sarwat B Rizvi,1 Sepideh Rouhi,1 Shohei Taniguchi,2 Shi Yu Yang,1 Mark Green,2 Mo Keshtgar,1,3 Alexander M Seifalian1,3 1UCL Centre for Nanotechnology and Regenerative Medicine, University College London, 2Department of Physics, King's College London, 3Royal Free London NHS Foundation Trust Hospital, London, UK Background: 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. Methods: 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. Results: 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

  1. [Granular cell tumor: report of a complicated vulvar localization of pulmonary metastases].

    Science.gov (United States)

    Tawfiq, N; Sabri, S; Saiss, K; Bouchbika, Z; Benchekroun, N; Jouhadi, H; Sahraoui, S; Benider, A

    2013-11-01

    Granular cell tumour, or Abrikossoff's tumour, is a rare, ubiquitous tumour, with mostly benign evolution. The malignant forms represent 1-3% of the cases. The vulvar localization is exceptional. We report the case of a 28-year-old patient who presented a vulvar Abrikossoff's tumour. Initially considered benign, the tumour was treated by exclusive local surgery. Five months later, the tumour showed a malignant transformation. The tumour size was 14cm in its major axis with the presence of not systematized scattered mitoses, PS 100 was positive at 100% and Ki 67 to 20%. Because of malignant and economic excision of the neoplasm, the patient received adjuvant radiotherapy. After 20 months, the patient had a local recurrence and lung metastasis, supporting the diagnosis of malignancy. She received two chemotherapy regimens of 5-fluoro-uracil/cisplatin and pacliatxel/carboplatin without any response. Following the failure of chemotherapy, the patient received symptomatic medical treatment. This observation leads to advocate an aggressive surgical treatment and strict supervision of all granular cell tumours.

  2. Control of vertebrate core planar cell polarity protein localization and dynamics by Prickle 2.

    Science.gov (United States)

    Butler, Mitchell T; Wallingford, John B

    2015-10-01

    Planar cell polarity (PCP) is a ubiquitous property of animal tissues and is essential for morphogenesis and homeostasis. In most cases, this fundamental property is governed by a deeply conserved set of 'core PCP' proteins, which includes the transmembrane proteins Van Gogh-like (Vangl) and Frizzled (Fzd), as well as the cytoplasmic effectors Prickle (Pk) and Dishevelled (Dvl). Asymmetric localization of these proteins is thought to be central to their function, and understanding the dynamics of these proteins is an important challenge in developmental biology. Among the processes that are organized by the core PCP proteins is the directional beating of cilia, such as those in the vertebrate node, airway and brain. Here, we exploit the live imaging capabilities of Xenopus to chart the progressive asymmetric localization of fluorescent reporters of Dvl1, Pk2 and Vangl1 in a planar polarized ciliated epithelium. Using this system, we also characterize the influence of Pk2 on the asymmetric dynamics of Vangl1 at the cell cortex, and we define regions of Pk2 that control its own localization and those impacting Vangl1. Finally, our data reveal a striking uncoupling of Vangl1 and Dvl1 asymmetry. This study advances our understanding of conserved PCP protein functions and also establishes a rapid, tractable platform to facilitate future in vivo studies of vertebrate PCP protein dynamics.

  3. Immunocytochemical identification and localization of APUD cells in the gut of seven stomachless teleost fishes

    Institute of Scientific and Technical Information of China (English)

    Qian Sheng Pan; Zhi Ping Fang; Ya Xin Zhao

    2000-01-01

    AIM To study the cell types, localization,distribution density and morphology of APUD cells in the intestinal mucosa of stomachless teleost fishes.METHOD By using the peroxidaseantiperoxidase complex ( PAP )immunocytochemical staining technique the identification, localization and morphology of immunoreactive (IR) endocrine cells seattered in the intestinal mucosa of grass carp ( Cyenopharyngodon idellus ), black carp (Mylopharyngodon piceus ) and common carp (Cyprinus carpio ) were investigated with 20 kinds of antisera prepared against mammalian peptide hormones of APUD cells, and likewise by using avidin-biotin-peroxidase complex (ABC)method those of silver carp ( Hypophthalmichthys molitrix ), bighead (Aristichthys nobilis ), silver crucian carp (Carassius gibelio ) and bluntnose black bream ( Megalobrama amblyocephala ) were also studied with 5 different antisera. The replacement of the first antiserum by phosphate buffered saline (PBS) was employed as a control. IR endocrine cells were counted with a square-mesh ocular micrometer from 10 fields selected randomly in every section of each part of the intestine specimen. The average number of IR endocrine cells per mm2 was counted to quantify their distribution density.RESULT Gastrin (GAS)-, Gastric inhibitory peptide (GIP)-, glucagon (GLU)-, glucagon-like immunoreactants ( GLI )-, bovine pancreatic polypeptide (BPP)-, leucine-enkephalin (ENK)-and substance P (SP)-IR endocrine cells were found in the gut of grass carp, black carp and common carp, and somatostatin ( SOM )-IR endocrine cells were only seen in common carp.GAS-, GIP- and GLU-IR endocrine cells were found in the intestinal mucosa of silver carp,bighead, silver crucian carp and bluntnose black bream. Most of IR endocrine cells had the higher distribution density in the foregut and midgut,and were longer in shape. They had a long apical cytoplasmic process extended to the gut lumen and a basal process extended to adjacent cells or basement membrane and

  4. 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...

  5. 77 FR 24717 - Scientific Information Request on Local Therapies for the Treatment of Stage I Non-Small Cell...

    Science.gov (United States)

    2012-04-25

    ... Therapies for the Treatment of Stage I Non-Small Cell Lung Cancer and Endobronchial Obstruction Due to... for the Treatment of Stage I Non-Small Cell Lung Cancer and Endobronchial Obstruction Due to Advanced... effectiveness review of the evidence for local therapies for the treatment of stage I non-small cell lung...

  6. 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....

  7. PAT1 (SLC36A1) shows nuclear localization and affect growth of smooth muscle cells from rats

    DEFF Research Database (Denmark)

    Jensen, Anne; Figueiredo-Larsen, Evan Manuel; Holm, René

    2014-01-01

    the localization and function of PAT1 in smooth muscle cells (SMCs). The PAT1 protein was found in smooth muscles from rat intestine and in the embryonic rat aorta cell line A7r5. Immunolocalization and cellular fractionation studies revealed that the majority of the PAT1 protein located within the cell nucleus...

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

    Energy Technology Data Exchange (ETDEWEB)

    Estrela-Lopis, I; Donath, E [Institute of Medical Physics and Biophysics, Leipzig University, Haertelstrasse 16, 04107 Leipzig (Germany); Romero, G; Rojas, E; Moya, S E, E-mail: Irina.Estrela-Lopis@medizin.uni-leipzig.de [CIC biomaGUNE, Paseo Miramon 182 Edificio Empresarial C, 20009 San Sebastian, Gipuzkoa (Spain)

    2011-07-06

    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{sub 2}O{sub 3} NPs were found in the cytoplasm. CeO{sub 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.

  9. 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.

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

    Science.gov (United States)

    Sommer, Felix; Bäckhed, Fredrik

    2016-05-01

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

  11. Identifying locally advanced basal cell carcinoma eligible for treatment with vismodegib: an expert panel consensus.

    Science.gov (United States)

    Peris, Ketty; Licitra, Lisa; Ascierto, Paolo A; Corvò, Renzo; Simonacci, Marco; Picciotto, Franco; Gualdi, Giulio; Pellacani, Giovanni; Santoro, Armando

    2015-01-01

    Basal cell carcinoma (BCC) is the most common skin cancer worldwide. Most occur on the head and neck, where cosmetic and functional outcomes are critical. BCC can be locally destructive if not diagnosed early and treated appropriately. Surgery is the treatment of choice for the majority of high-risk lesions. Aggressive, recurrent or unresectable tumors can be difficult to manage. Until recently, no approved systemic therapy was available for locally advanced or metastatic BCC inappropriate for surgery or radiotherapy. Vismodegib provides a systemic treatment option. However, a consensus definition of advanced BCC is lacking. A multidisciplinary panel with expertise in oncology, dermatology, dermatologic surgery and radiation oncology proposes a consensus definition based on published evidence and clinical experience.

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

    Science.gov (United States)

    Barbarin, Alice; Séité, Paule; Godet, Julie; Bensalma, Souheyla; Muller, Jean-Marc; Chadéneau, Corinne

    2014-11-28

    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.

  13. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof

    Science.gov (United States)

    Linardi, Renata L.; Megee, Susan O.; Mainardi, Sarah R.; Senoo, Makoto; Galantino-Homer, Hannah L.

    2015-01-01

    Background The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. Hypothesis/Objectives To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Methods Indirect immunofluorescence microscopy and immunoblotting were utilized to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14, and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 (pp63, a marker of ESC to transit-amplifying (TA) cell transition) in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). Results K14 expression was restricted to the basal layer of epidermal lamellae, and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. Conclusions This is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses. PMID:25963063

  14. 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 

  15. Progesterone Receptor Subcellular Localization and Gene Expression Profile in Human Astrocytoma Cells Are Modified by Progesterone

    Directory of Open Access Journals (Sweden)

    Aliesha González-Arenas

    2014-11-01

    Full Text Available Intracellular progesterone receptor (PR has been identified in human astrocytomas, the most common and aggressive primary brain tumors in humans. It has been reported that PR cell distribution affects their transcriptional activity and turnover. In this work we studied by immunofluorescence the effects of estradiol and progesterone on the subcellular localization of PR in a grade III human astrocytoma derived cell line (U373. We observed that total PR was mainly distributed in the cytoplasm without hormonal treatment. Estradiol (10 nM increased PR presence in the cytoplasm of U373 cells, whereas progesterone (10 nM and RU486 (PR antagonist, 1 μM blocked this effect. To investigate the role of PR activity in the regulation of gene expression pattern of U373 cells, we evaluated by microarray analysis the profile of genes regulated by progesterone, RU486, or both steroids. We found different genes regulated by steroid treatments that encode for proteins involved in metabolism, transport, cell cycle, proliferation, metastasis, apoptosis, processing of nucleic acids and proteins, adhesion, pathogenesis, immune response, cytoskeleton, and membrane receptors. We determined that 30 genes were regulated by progesterone, 41 genes by RU486 alone, and 13 genes by the cotreatment of progesterone+RU486, suggesting that there are many genes regulated by intracellular PR or through other signaling pathways modulated by progesterone. All these data suggest that PR distribution and activity should modify astrocytomas growth.

  16. Periodic, quasiperiodic, and chaotic potentials generated by electrochemical concentration cells: Local and global dynamics

    Science.gov (United States)

    Zeyer, K.-P.; Münster, A. F.; Hauser, M. J. B.; Schneider, F. W.

    1994-09-01

    We extend previous work describing the passive electrical coupling of two periodic chemical states to include quasiperiodic and chaotic states. Our setup resembles an electrochemical concentration cell (a battery) whose half cells [continuous-flow stirred tank reactors (CSTRs)] each contain the Belousov-Zhabotinsky (BZ) reaction. For a closed electrical circuit the two half cells are weakly coupled by an external variable resistance and by a constant low mass flow. This battery may produce either periodic, quasiperiodic, or chaotic alternating current depending on the dynamic BZ states chosen in the half cells. A lower fractal dimensionality is calculated from the electrical potential of a single chaotic CSTR than from the difference potential (relative potential) of the two chaotic half cell potentials. A similar situation is observed in model calculations of a chaotic spatiotemporal system (the driven Brusselator in one space dimension) where the dimensionality derived from a local time series is lower than the dimensionality of the global trajectory calculated from the Karhunen-Loeve coefficients.

  17. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

    Science.gov (United States)

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forte, Trudy M; Ryan, Robert O

    2015-08-21

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

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

    Directory of Open Access Journals (Sweden)

    Rebai Tarek

    2011-06-01

    Full Text Available Abstract 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.

  19. Allergic rhinitis is a local disease: the role of local IgE production, basophils and mast cells

    NARCIS (Netherlands)

    A. Kleinjan (Alex)

    2002-01-01

    textabstractThe introduction to this thesis summarizes the literature which indicates that there is a discrepancy between sensitisation and allergic disease. Two aspects which might play a role in this discrepancy are the differences between production and funtion of local versus systemic lgE and th

  20. Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

    Directory of Open Access Journals (Sweden)

    Andrew J Muinonen-Martin

    2014-10-01

    Full Text Available The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

  1. In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth

    Science.gov (United States)

    Fedder, C; Beck-Schimmer, B; Aguirre, J; Hasler, M; Roth-Z'graggen, B; Urner, M; Kalberer, S; Schlicker, A; Votta-Velis, G; Bonvini, J M; Graetz, K; Borgeat, A

    2010-01-01

    Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non-fluorescent-2,7′-dichlorofluorescin. Treatment of cells with the three LA showed a concentration-dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine- and bupivacaine-exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration-dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine. PMID:20819090

  2. High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

    Science.gov (United States)

    Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Ma, Chao; Zhao, Lei; Wang, Yaolei; Ouyang, Jia; Pang, Long; Wang, Jinyi

    2015-02-07

    Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PμSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PμS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PμSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PμS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PμS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PμS groups. We demonstrate that PμS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays.

  3. 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.

  4. GnRH receptors in human granulosa cells: Anatomical localization and characterization by autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Latouche, J.; Crumeyrolle-Arias, M.; Jordan, D.; Kopp, N.; Augendre-Ferrante, B.; Cedard, L.; Haour, F. (Institut Pasteur, Paris (France))

    1989-09-01

    The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for {sup 125}I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

  5. 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

  6. Phosphorylated vasodilator-stimulated phosphoprotein is localized on mitotic spindles of the gastric cancer cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    Yan Tao; Yong-Chang Chen; Ying Wang; Zhi-Jian Zhang; Wen-Rong Xu

    2006-01-01

    AIM: To elucidate the localization of vasodilator stimulated phosphoprotein (VASP), a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells.METHODS: Tmmunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP (p-VASP) in interphase of mitotic gastric cancer of the cell line SGC-7901.RESULTS: Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles.CONCLUSION: VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.

  7. Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons

    Science.gov (United States)

    Merianda, Tanuja T.; Jin, Ying

    2017-01-01

    Abstract The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons. PMID:28197547

  8. 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.

  9. Intraarterial chemotherapy with gemcitabine and cisplatin in locally advanced or recurrent penile squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-Ye Liu; Yong-Hong Li; Zhuo-Wei Liu; Zhi-Ling Zhang; Yun-Lin Ye; Kai Yao; Hui Han; Zi-Ke Qin; Fang-Jian Zhou

    2013-01-01

    The prognosis of locally advanced or recurrent squamous cell carcinoma (SCC) of the penis after conventional treatment is dismal. This study aimed to evaluate the therapeutic effects of intraarterial chemotherapy with gemcitabine and cisplatin on local y advanced or recurrent SCC of the penis. Between April 1999 and May 2011, we treated 5 patients with locally advanced penile SCC and 7 patients with recurrent disease with intraarterial chemotherapy. The response rate and toxicity data were analyzed, and survival rates were calculated. After 2 to 6 cycles of intraarterial chemotherapy with gemcitabine and cisplatin, 1 patients with locoregional y advanced disease achieved a complete response, and 4 achieved partial response. Of the 7 patients with recurrent disease, 2 achieved complete response, 3 achieved partial response, 3 had stable disease, and 1 developed progressive disease. An objective tumor response was therefore achieved in 10 of the 12 patients. The median overal survival for the patients was 24 months (range, 10-50 months). Three out of 10 patients who responded were long-term survivors after intraarterial chemotherapy. Intraarterial chemotherapy with gemcitabine and cisplatin may be effective and potential y curative in locoregional y advanced or recurrent penile SCC. The contribution of this therapy in the primary management of advanced or recurrent penile SCC should be prospectively investigated.

  10. Repetitive transarterial chemoembolization (TACE) of liver metastases from renal cell carcinoma: Local control and survival results

    Energy Technology Data Exchange (ETDEWEB)

    Nabil, Mohamed [Johann Wolfgang Goethe University, Institute of Diagnostic and Interventional Radiology, Frankfurt am Main (Germany); Klinikum der Johann Wolfgang Goethe-Universitaet, Institut fuer Diagnostische und Interventionelle Radiologie, Frankfurt am Main (Germany); Gruber, Tatjana; Zangos, Stephan; Vogl, Thomas J. [Johann Wolfgang Goethe University, Institute of Diagnostic and Interventional Radiology, Frankfurt am Main (Germany); Yakoub, Danny [Imperial College London, St Mary' s Hospital, Department of Biosurgery and Surgical Technology, London (United Kingdom); Ackermann, Hanns [Johann Wolfgang Goethe University, Department of Biostatistics and Medical Information, Frankfurt am Main (Germany)

    2008-07-15

    The purpose was to evaluate the effectiveness of transarterial chemoembolization (TACE) in local tumor control and survival in patients with hepatic metastases from renal cell carcinoma (RCC). Prospective evaluation of TACE treatment outcome in 22 patients recruited from 1999 and 2005 was performed. The chemotherapeutic agent used was mitomycin only in 45% of the patients and mitomycin together with gemcitabine in the other 55%. The embolizing materials used in all of the patients were iodized oil (lipiodol) and degradable starch microspheres. Local response was evaluated by MRI and judged according to Response Evaluation Criteria in Solid Tumors (RECIST). Mean and median survival and survival probability after diagnosis and treatment were both calculated by Kaplan-Meier method. Partial response was achieved in 13.7%, stable disease in 59% and progressive disease in 27.3% of patients. Survival time from the diagnosis of metastases ranged from 18 to 307 months and from 2.2 to 35 months from the start of TACE treatment. The median and mean survival times from the date of diagnosis were 68.6 and 102.9 months, respectively. The median and mean survival times from the start of TACE were 8.2 and 11.7 months, respectively. Survival probability from the start of treatment was 31% after 1 year and 6% after 2 years. TACE can result in a favorable local tumor response in patients with hepatic metastases from RCC, but survival results are still limited. (orig.)

  11. Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues.

    Science.gov (United States)

    Aleku, Manuela; Fisch, Gerald; Möpert, Kristin; Keil, Oliver; Arnold, Wolfgang; Kaufmann, Jörg; Santel, Ansgar

    2008-05-01

    Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.

  12. Dyskerin localizes to the mitotic apparatus and is required for orderly mitosis in human cells.

    Directory of Open Access Journals (Sweden)

    Faizan Alawi

    Full Text Available Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G1 and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G2/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G2/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.

  13. Dyskerin localizes to the mitotic apparatus and is required for orderly mitosis in human cells.

    Science.gov (United States)

    Alawi, Faizan; Lin, Ping

    2013-01-01

    Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G1 and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G2/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G2/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.

  14. Optogenetics in the cerebellum: Purkinje cell-specific approaches for understanding local cerebellar functions.

    Science.gov (United States)

    Tsubota, Tadashi; Ohashi, Yohei; Tamura, Keita

    2013-10-15

    The cerebellum consists of the cerebellar cortex and the cerebellar nuclei. Although the basic neuronal circuitry of the cerebellar cortex is uniform everywhere, anatomical data demonstrate that the input and output relationships of the cortex are spatially segregated between different cortical areas, which suggests that there are functional distinctions between these different areas. Perturbation of cerebellar cortical functions in a spatially restricted fashion is thus essential for investigating the distinctions among different cortical areas. In the cerebellar cortex, Purkinje cells are the sole output neurons that send information to downstream cerebellar and vestibular nuclei. Therefore, selective manipulation of Purkinje cell activities, without disturbing other neuronal types and passing fibers within the cortex, is a direct approach to spatially restrict the effects of perturbations. Although this type of approach has for many years been technically difficult, recent advances in optogenetics now enable selective activation or inhibition of Purkinje cell activities, with high temporal resolution. Here we discuss the effectiveness of using Purkinje cell-specific optogenetic approaches to elucidate the functions of local cerebellar cortex regions. We also discuss what improvements to current methods are necessary for future investigations of cerebellar functions to provide further advances.

  15. Local Langerhans cell histiocytosis (eosinophilic granuloma in a six-month baby: a case report

    Directory of Open Access Journals (Sweden)

    Bahador M

    2008-12-01

    Full Text Available "nBackground: Langerhans cell histiocytosis (LCH is a group of idiopathic disorders characterized by the proliferation of specialized bone marrow-derived Langerhans cells and mature eosinophils. The estimated annual incidence ranges from 0.5-2 cases per 100,000 persons per year. The pathogenesis of LCH is unknown. The prevalence of LCH seems to be higher among whites and males. The most common complaints at presentation are those related to bone lesions. Treatment consists of surgery, chemotherapy and radiotherapy alone or in combination. The age of onset varies according to the variety of LCH. Solitary lesions may occur in bones or skin. Cutaneous lesions present with firm, painless papulonodules or vesicles. "nCase report: This six-month-old baby presented with firm papulonodules on her temporal skin, but fortunately her other organs were healthy. She underwent two surgeries, separated by a one-month interval. Due to local recurrence after a short period of time, she underwent a 10-Gy dose of radiation. Her response proved good during follow-up. "nConclusion: Radiotherapy is good for controlling local recurrence in LCH, with few sequelae related to treatment.

  16. 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.

  17. Alterations in expression, proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hong-Zhi He; Xiao-Hang Zhao; Zhen-Mei Song; Kun Wang; Liang-Hong Teng; Fang Liu; You-Sheng Mao; Ning Lu; Shang-Zhong Zhang; Min Wu

    2004-01-01

    AIM: To investigate biogenesis and intracellular localizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa.METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular localizations of clusterin were carried out in both tissues and cell lines of ESCC.RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform,were the major alterations in cancer cells of esophagus.Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia.CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

  18. 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.

  19. APOPTOSIS AND PROLIFERATION OF TUMOR CELLS IN LOCALLY ADVANCED CERVICAL CANCER AFTER NEOADJUVANT INTRAARTERIAL CHEMOTHERAPY

    Institute of Scientific and Technical Information of China (English)

    朱雪琼; 岳天孚; 惠京; 张颖; 王德华

    2003-01-01

    Objective: Through observing the clinical response to neoadjuvant intraarterial chemotherapy in locally advanced cervical cancer and investigating the changes of p53 protein expression, proliferation and apoptosis of tumor cells after chemotherapy, to study the relationship between biological markers and chemotherapeutic response. Methods: 20 women with locally advanced squamous cervical cancer received consecutive infusion chemotherapy of five days of cisplatin and adriamycin via the superselective uterine artery. The response to chemotherapy was evaluated by gynecologic examination and ultrasonography 3 weeks after chemotherapy. The changes of apoptotic index (AI), proliferation index (PI) and p53 expression of tumor cells were detected by immunohistochemical technique. Results: The clinical response rate of locally advanced squamous cervical cancer to uterine artery infusion chemotherapy was 70%. No change of PI was found 3 weeks after treatment, but AI significantly increased from 2.79±0.76 to 4.29±1.13 (P<0.01), and AI/PI from 5.68±1.21 to 9.00±1.95 (P<0.05). On the contrary, the expression of p53 was significantly decreased (P<0.05). Patients who responded to chemotherapy showed higher PI before chemotherapy and significantly increased AI and AI/PI after chemotherapy than non-responders (P<0.05). Conclusion: Higher PI was an indication for neoadjuvant intraarterial chemotherapy. One more cycle of chemotherapy should be given to those who have significantly increased AI or AI/PI after chemotherapy, while definite treatment such as surgery or/and radiotherapy should be immediately given to those patients without increased AI or AI/PI.

  20. 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

    by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents......-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.......BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced...

  1. A dynamic complex of signaling proteins uses polar localization to regulate cell-fate asymmetry in Caulobacter crescentus.

    Science.gov (United States)

    Tsokos, Christos G; Perchuk, Barrett S; Laub, Michael T

    2011-03-15

    Cellular asymmetry is critical to metazoan development and the life cycle of many microbes. In Caulobacter, cell cycle progression and the formation of asymmetric daughter cells depend on the polarly-localized histidine kinase CckA. How CckA is regulated and why activity depends on localization are unknown. Here, we demonstrate that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Early in the cell cycle, CckA is activated by the dephosphorylation of DivK throughout the cell. However, in later stages, when phosphorylated DivK levels are high, CckA activation relies on polar localization with a DivK phosphatase. Localization thus creates a protected zone for CckA within the cell, without the use of membrane-enclosed compartments. Our results reveal the mechanisms by which CckA is regulated in a cell-type-dependent manner. More generally, our findings reveal how cells exploit subcellular localization to orchestrate sophisticated regulatory processes.

  2. NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine.

    Science.gov (United States)

    Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M; Anjuère, Fabienne

    2015-01-01

    Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses.

  3. NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

    Science.gov (United States)

    Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M.; Anjuère, Fabienne

    2015-01-01

    Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses. PMID:26630176

  4. Microbubble moving in blood flow in microchannels: effect on the cell-free layer and cell local concentration.

    Science.gov (United States)

    Bento, David; Sousa, Lúcia; Yaginuma, Tomoko; Garcia, Valdemar; Lima, Rui; Miranda, João M

    2017-03-01

    Gas embolisms can hinder blood flow and lead to occlusion of the vessels and ischemia. Bubbles in microvessels circulate as tubular bubbles (Taylor bubbles) and can be trapped, blocking the normal flow of blood. To understand how Taylor bubbles flow in microcirculation, in particular, how bubbles disturb the blood flow at the scale of blood cells, experiments were performed in microchannels at a low Capillary number. Bubbles moving with a stream of in vitro blood were filmed with the help of a high-speed camera. Cell-free layers (CFLs) were observed downstream of the bubble, near the microchannel walls and along the centerline, and their thicknesses were quantified. Upstream to the bubble, the cell concentration is higher and CFLs are less clear. While just upstream of the bubble the maximum RBC concentration happens at positions closest to the wall, downstream the maximum is in an intermediate region between the centerline and the wall. Bubbles within microchannels promote complex spatio-temporal variations of the CFL thickness along the microchannel with significant relevance for local rheology and transport processes. The phenomenon is explained by the flow pattern characteristic of low Capillary number flows. Spatio-temporal variations of blood rheology may have an important role in bubble trapping and dislodging.

  5. DGCR8 Localizes to the Nucleus as well as Cytoplasmic Structures in Mammalian Spermatogenic Cells and Epididymal Sperm

    Directory of Open Access Journals (Sweden)

    Akane Nakano

    2013-01-01

    Full Text Available The localization of DGCR8 in spermatogenic cells and sperm from rat and mouse was studied by immunofluorescence and immunoelectron microscopy. Spermatogenic cells from these species yielded similar DGCR8 localization pattern. Immunofluorescence microscopy results showed that DGCR8 localized to both the cytoplasm and nucleus. In the cytoplasm, diffuse cytosolic and discrete granular staining was observed. Dual staining showed that DGCR8 colocalized to the granules with MAEL (a nuage marker. In the nucleus of spermatocytes, both the nucleoli and nucleoplasm were stained, whereas in the nucleus of early spermatids small spots were stained. In late spermatids, DGCR8 localized to the tip of their head and to small granules (neck granules of the neck cytoplasm. The neck granules were also observed in the neck of epididymal sperm. Immunoelectron microscopy results showed that DGCR8 localized to nuage structures. Moreover, DGCR8 localized to nonnuage structures in late spermatids. DGCR8 also localized to the nucleolus and euchromatin in spermatocytes and round spermatids and to small granules in the nucleus of late spermatids. The results suggest that in spermatogenic cells DGCR8 localizes not only to the nuclei but also to the cytoplasmic structures such as nuage and nonnuage structures. Furthermore, DGCR8 seems to be imported into the egg with neck granules in sperm during fertilization.

  6. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    Science.gov (United States)

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  7. 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.

  8. 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.

  9. Nuclear localization of TEF3-1 promotes cell cycle progression and angiogenesis in cancer.

    Science.gov (United States)

    Teng, Kaixuan; Deng, Cuilan; Xu, Jie; Men, Qiuxu; Lei, Tao; Di, Da; Liu, Ting; Li, Wenhua; Liu, Xin

    2016-03-22

    TEF3-1 (transcriptional enhancer factor 3 isoform 1), also known as TEAD4 (TEA domain family member 4), was recently revealed as an oncogenic character in cancer development. However, the underlying molecular pathogenic mechanisms remain undefined. In this paper, we investigated nuclear TEF3-1 could promote G1/S transition in HUVECs, and the expression levels of cyclins and CDKs were upregulated. Additionally, if TEF3-1 was knocked down, the expression of cyclins and CDKs was downregulated while the expression of P21, a negative regulator of the cell cycle, was upregulated. A microarray analysis also confirmed that TEF3-1 overexpression upregulates genes that are related to cell cycle progression and the promotion of angiogenesis. Moreover, we observed that nuclear TEF3-1 was highly expressed during the formation of vascular structures in gastric cancer (GC). Finally, tumor xenograft experiments indicated that, when TEF3-1 was knocked down, tumor growth and angiogenesis were also suppressed. Taken together, these results demonstrate for the first time that TEF3-1 localization to the nucleus stimulates the cell cycle progression in HUVECs and specifically contributes to tumor angiogenesis. Nuclear TEF3-1 in HUVECs may serve as an oncogenic biomarker, and the suppression of TEF3-1 may be a potential target in anti-tumor therapy.

  10. Localization of human immunodeficiency virus antigens in infected cells by scanning/transmission-immunogold techniques

    Energy Technology Data Exchange (ETDEWEB)

    Herrera, M.I.; Santa Maria, I.; de Andres, R.; Najera, R.

    1988-01-01

    An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labelling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies.

  11. Chemical stimulation of adherent cells by localized application of acetylcholine from a microfluidic system

    Directory of Open Access Journals (Sweden)

    Susanne Zibek

    2010-11-01

    Full Text Available Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses.In an experimental setup micro-droplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution travelled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively.Numerical modelling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 µm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 µM acetylcholine independent of pore size were determined.

  12. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

    Science.gov (United States)

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  13. Targeting the tumor-draining area : local immunotherapy and its effect on the systemic T cell response

    NARCIS (Netherlands)

    Herbert-Fransen, Marieke Fernande

    2012-01-01

    This dissertation deals with the role of local immune stimulation in the lymph node and tumor microenvironment and its effect on systemic CD8+ T cell responses, in particular the anti-tumor CD8+ T cell responses. In chapter 2 the use of a slow-release system is described to deliver the immune-acti

  14. Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gomà A

    2014-12-01

    Full Text Available Alba Gomà,1,* Roser Mir,1–3,* Fina Martínez-Soler,1,4 Avelina Tortosa,4 August Vidal,5,6 Enric Condom,5,6 Ricardo Pérez–Tomás,6 Pepita Giménez-Bonafé1 1Departament de Ciències Fisiològiques II, Faculty of Medicine, Campus of Health Sciences of Bellvitge, Universitat de Barcelona, IDIBELL, Barcelona, Spain; 2División de Investigación Básica, Instituto Nacional de Cancerología, México DF, Mexico; 3Instituto de Física, Universidad Nacional Autónoma de México (UNAM, México DF, Mexico; 4Department of Basic Nursing, School of Nursing of the Health Campus of Bellvitge, Universitat de Barcelona, 5Department of Pathology, Hospital Universitari de Bellvitge, 6Department of Pathology and Experimental Therapeutics, Universitat de Barcelona, IDIBELL, Barcelona, Spain*These authors contributed equally to this work Background: One of the problems in prostate cancer (CaP treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1 play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype.Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent in order to understand its possible role in CaP chemoresistance.Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy.Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59

  15. Hair-cycle-dependent expression of parathyroid hormone-related protein and its type I receptor: evidence for regulation at the anagen to catagen transition.

    Science.gov (United States)

    Cho, Yong Mee; Woodard, Grant L; Dunbar, Maureen; Gocken, Todd; Jimènez, Juan A; Foley, John

    2003-05-01

    The humoral hypercalcemia factor parathyroid hormone-related protein is a paracrine-signaling molecule that regulates the development of several organ systems, including the skin. In pathologic circumstances such as hypercalcemia and in development, parathyroid hormone-related protein signaling appears to be mediated by the type I parathyroid hormone/parathyroid hormone-related protein receptor. In order to clarify the role of the ligand and receptor pair in cutaneous biology, gene expression was monitored in a series of murine skin samples ranging from embryonic day 14 to 2 y with in situ hybridization and RNase protection. In all samples, high levels of parathyroid hormone-related protein transcripts were exclusively expressed in the developing and adult hair follicle but were not observed in the interfollicular epidermis. In the adult, parathyroid hormone-related protein mRNA expression was dynamically regulated as a function of the murine hair cycle in a way similar to other signaling molecules that regulate the anagen to catagen transition. PTH receptor transcripts were abundantly expressed in the developing dermis. In the adult skin, PTH receptor mRNA was markedly reduced, but again demonstrated hair-cycle-dependent expression. The dorsal skin of the keratin 14-parathyroid hormone-related protein mouse was used to evaluate the impact of overexpression of the peptide on the murine hair cycle. All types of hair were 30-40% shorter in adult keratin 14-parathyroid hormone-related protein mice as compared with wild-type littermates. This appeared to result from a premature entry into the catagen phase of the hair cycle. Finally, the relationship between parathyroid hormone-related protein signaling and other growth factors that regulate the hair cycle was examined by cross-breeding experiments employing keratin 14-parathyroid hormone-related protein mice and fibroblast growth factor-5-knockout mice. It appears that parathyroid hormone-related protein and

  16. 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)

  17. Primary squamous cell carcinoma of thyroid gland with local recurrence: ultrasonographic and computed tomographic findings

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Ja Yoon; Kwon, Kye Won; Kim, Sang Wook [Bundang Jesaeng General Hospital, Seongnam (Korea, Republic of); Youn, In Young [Dept. of Radiology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2014-04-15

    Primary squamous cell carcinoma of the thyroid gland (PSCCT) is a rare malignancy that presents with advanced disease and poor prognosis. It is difficult to diagnose PSCCT in its early stage because of its rarity and lack of typical imaging findings. We experienced an elderly woman with PSCCT confirmed by surgery. Although preoperative fine-needle aspiration revealed no malignancy, surgical resection was performed because the ultrasonogram showed diffuse microcalcifications, which suggested malignancy, and clinically, the mass grew rapidly to compress the trachea. Local tumor recurrence was noted at 3 months after surgery. Surgical resection or repeat biopsy should be considered if a cytologically benign thyroid mass shows imaging or clinical features of malignancy.

  18. The magnetic field of Betelgeuse: a local dynamo from giant convection cells?

    CERN Document Server

    Auriere, M; Konstantinova-Antova, R; Perrin, G; Petit, P; Roudier, T

    2010-01-01

    Betelgeuse is an M supergiant with a complex and extended atmosphere, which also harbors spots and giant granules at its surface. A possible magnetic field could contribute to the mass loss and to the heating of the outer atmosphere. We observed Betelgeuse, to directly study and infer the nature of its magnetic field. We used the new-generation spectropolarimeter NARVAL and the least square deconvolution (LSD) method to detect circular polarization within the photospheric absorption lines of Betelgeuse. We have unambiguously detected a weak Stokes V signal in the spectral lines of Betelgeuse, and measured the related surface-averaged longitudinal magnetic field Bl at 6 different epochs over one month. The detected longitudinal field is about one Gauss and is apparently increasing on the time scale of our observations. This work presents the first direct detection of the magnetic field of Betelgeuse. This magnetic field may be associated to the giant convection cells that could enable a "local dynamo:.

  19. AWireless Local Area Network Soft Cell Phone (SCP System with Multimedia and Data Services

    Directory of Open Access Journals (Sweden)

    Olakanmi O.Oladayo

    2014-01-01

    Full Text Available Most organizations concurrently maintain private Automatic Branch Exchange (PABX and Local Area Network (LAN for information interchange within their organization. This is obviously a waste of resources and avoidable duplication of communication systems. The existing LAN can be used as a communication backbone for the in house telephone operations with no extra cost and resources. In view of this, a portable and a platform independent Software-Based Cell Phone (SCP was proposed for the existing LANs infrastructure in the organizations. The SPC is a telephony application with a user friendly interface which is capable of handling voice, video and text messages without compromising the Quality of Service (QoS of the existing LAN.

  20. Preoperative Erythrocyte Sedimentation Rate Independently Predicts Overall Survival in Localized Renal Cell Carcinoma following Radical Nephrectomy

    Directory of Open Access Journals (Sweden)

    Brian W. Cross

    2012-01-01

    Full Text Available Objectives. To determine the relationship between preoperative erythrocyte sedimentation rate (ESR and overall survival in localized renal cell carcinoma (RCC following nephrectomy. Methods. 167 patients undergoing nephrectomy for localized RCC had ESR levels measured preoperatively. Receiver Operating Characteristics curves were used to determine Area Under the Curve and relative sensitivity and specificity of preoperative ESR in predicting overall survival. Cut-offs for low (0.0–20.0 mm/hr, intermediate (20.1–50.0 mm/hr, and high risk (>50.0 mm/hr groups were created. Kaplan-Meier analysis was conducted to assess the univariate impact of these ESR-based groups on overall survival. Univariate and multivariate Cox regression analysis was conducted to assess the potential of these groups to predict overall survival, adjusting for other patient and tumor characteristics. Results. Overall, 55.2% were low risk, while 27.0% and 17.8% were intermediate and high risk, respectively. Median (95% CI survival was 44.1 (42.6–45.5 months, 35.5 (32.3–38.8 months, and 32.1 (25.5–38.6 months, respectively. After controlling for other patient and tumor characteristics, intermediate and high risk groups experienced a 4.5-fold (HR: 4.509, 95% CI: 0.735–27.649 and 18.5-fold (HR: 18.531, 95% CI: 2.117–162.228 increased risk of overall mortality, respectively. Conclusion. Preoperative ESR values represent a robust predictor of overall survival following nephrectomy in localized RCC.

  1. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

    Science.gov (United States)

    Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (ptissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  2. 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

  3. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    Science.gov (United States)

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

  4. Polyunsaturated fatty acids affect the localization and signaling of PIP3/AKT in prostate cancer cells.

    Science.gov (United States)

    Gu, Zhennan; Wu, Jiansheng; Wang, Shihua; Suburu, Janel; Chen, Haiqin; Thomas, Michael J; Shi, Lihong; Edwards, Iris J; Berquin, Isabelle M; Chen, Yong Q

    2013-09-01

    AKT is a serine-threonine protein kinase that plays important roles in cell growth, proliferation and apoptosis. It is activated after binding to phosphatidylinositol phosphates (PIPs) with phosphate groups at positions 3,4 and 3,4,5 on the inositol ring. In spite of extensive research on AKT, one aspect has been largely overlooked, namely the role of the fatty acid chains on PIPs. PIPs are phospholipids composed of a glycerol backbone with fatty acids at the sn-1 and sn-2 position and inositol at the sn-3 position. Here, we show that polyunsaturated fatty acids (PUFAs) modify phospholipid content. Docosahexaenoic acid (DHA), an ω3 PUFA, can replace the fatty acid at the sn-2 position of the glycerol backbone, thereby changing the species of phospholipids. DHA also inhibits AKT(T308) but not AKT(S473) phosphorylation, alters PI(3,4,5)P3 (PIP3) and phospho-AKT(S473) protein localization, decreases pPDPK1(S241)-AKT and AKT-BAD interaction and suppresses prostate tumor growth. Our study highlights a potential novel mechanism of cancer inhibition by ω3 PUFA through alteration of PIP3 and AKT localization and affecting the AKT signaling pathway.

  5. Mast cell-derived heparin proteoglycans as a model for a local antithrombotic.

    Science.gov (United States)

    Lassila, Riitta; Jouppila, Annukka

    2014-11-01

    Mast cell-derived heparin proteoglycans (HEP-PG) reside in vascular tissue and serve as a local antithrombotic. Heparin, as used clinically, is isolated from its protein backbone from porcine and bovine gut mucosa. The isolated heparin is an anticoagulant; however, when bound to a protein carrier the heparin conjugates will become antiplatelet agents by inhibiting collagen-induced platelet aggregation and procoagulant activity, as distinct from soluble heparin. HEP-PG, whether soluble or immobilized to a surface, inhibit platelet deposition on the collagen surface in flowing blood. Mimics of HEP-PG, can be tailored to molecules carrying both antiplatelet and anticoagulant (APAC) properties. These molecules can target the vascular injury site and take residence there. Inhibition of thrombus growth using these APACs under these conditions has been demonstrated in several animal models. Although efficacious for antiplatelet and anticoagulant effects, the bleeding time is shorter with APACs than with unfractionated heparin, suggestive of beneficial efficacy/safety ratio. These strategies may be utilized in drug development, where many vascular injury-related problems can be tackled locally.

  6. Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

    Science.gov (United States)

    Erxleben, Christian; Klauke, Norbert; Flötenmeyer, Matthias; Blanchard, Marie-Pierre; Braun, Claudia; Plattner, Helmut

    1997-01-01

    In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense. PMID:9024690

  7. Local electrical and dielectric properties of nanocrystalline solid oxide fuel cell electrolytes

    Science.gov (United States)

    Perry, Nicola Helen

    Reducing the operating temperature of solid oxide fuel cells (SOFCs), to improve durability and lower cost, requires an increase in the low temperature oxygen-ion conductivity of the electrolyte. This work investigates whether the electrolyte conductivity could be increased by decreasing the grain size into the nanoscale. Bulk electrolytes - cubic yttria-stabilized zirconia (YSZ, with 8mol% Y2O3), tetragonal zirconia polycrystal (TZP, with 3mol% Y2O3), and Sr- and Mg- co-doped LaGaO3 (LSGM) - were fabricated with grain sizes ranging from 10nm to 3mum, using commercial or sol-gel-derived nanopowders and various sintering techniques. Local grain boundary and grain core conductivities and dielectric constants were analyzed over a range of temperatures and atmospheres using AC-impedance spectroscopy and our novel nano-Grain Composite Model, and interpreted in terms of grain-size dependent defect chemistry (e.g. space charge models, local thermodynamics, and impurity/ acceptor segregation). All three oxides exhibited qualitatively similar electrical/ dielectric behavior. Their single crystal/ grain core dielectric constants exhibited an upturn with temperature, which was attributed to the onset of dipolar relaxation. Grain boundary dielectric constants were consistently higher than grain core dielectric constants at the nanoscale. n-GCM-derived electrical grain boundary half-widths agreed well with measured acceptor dopant segregation widths at grain boundaries. The local grain boundary conductivity was consistently increased in nanocrystalline vs. microcrystalline samples, although the mechanisms responsible for this behavior differed in each material. Grain core conductivity did not change with grain size in each case. Despite the increase in local grain boundary conductivity at the nanoscale, the total conductivity decreased monotonically with decreasing grain size in all three electrolytes; the grain boundaries remain barriers to transport (relative to grain cores

  8. Synthesis and cell localization of self-assembled dinuclear lanthanide bioprobes.

    Science.gov (United States)

    Chauvin, Anne-Sophie; Thomas, Frédéric; Song, Bo; Vandevyver, Caroline D B; Bünzli, Jean-Claude G

    2013-07-28

    Lanthanide bioprobes and bioconjugates are ideal luminescent stains in view of their low propensity to photobleaching, sharp emission lines and long excited state lifetimes permitting time-resolved detection for enhanced sensitivity. In this paper, we expand our previous work which demonstrated that self-assembled dinuclear triple-stranded helicates [Ln2(L(C2X))3] behave as excellent cell and tissue labels in immunocytochemical and immunohistochemical assays. The synthetic strategy of the hexadentate ditopic ligands incorporating dipicolinic acid, benzimidazole units and polyoxyethylene pendants is revisited in order to provide a more straightforward route and to give access to further functionalization of the polyoxyethylene arms by incorporating a terminal function X. Formation of the helicates [Ln2(L(C2X))3] (X=COOH, CH2OH, COEt, NH2, phthalimide) is ascertained by several experimental techniques and their stability tested against diethylenetriaminepentaacetate. Their photophysical properties (quantum yield, lifetime, radiative lifetime and sensitization efficiency) are presented and compared with those of the parent helicates [Ln2(L(C2))3]. Finally, the cellular uptake of five Eu(III) helicates is monitored by time-resolved luminescence microscopy and their localization in HeLa cells established by co-staining experiments.

  9. The localization within plant cells of enzymes involved in arginine biosynthesis.

    Science.gov (United States)

    Shargool, P D; Steeves, T; Weaver, M; Russell, M

    1978-04-01

    Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids. Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.

  10. Characterization of immune cells and cytokine localization in the rat utero-placental unit mid- to late gestation.

    Science.gov (United States)

    Tessier, Daniel R; Raha, Sandeep; Holloway, Alison C; Yockell-Lelièvre, Julien; Tayade, Chandrakant; Gruslin, Andrée

    2015-08-01

    The success of pregnancy is dependent on the precise regulation of the immune response within the utero-placental environment. Rats are beginning to be widely used as a model for human immune-related pregnancy complications. However, our knowledge of immune cells and cytokine localization in the rat utero-placental tissue is limited. The current study aimed to localize the immune cell populations, including uterine natural killer (uNK) cells, neutrophils, and macrophages within the rat utero-placental unit at two crucial gestational ages, gestational days 15.5 and 18.5. In addition, we characterized the distribution of the cytokines TNFα, IFNγ, and IL-10 in the utero-placental regions at both the above-mentioned gestational ages. Our study has demonstrated co-localization TNFα and IFNγ with uNK cells in perivascular regions of the rat mesometrial triangle at both gestational ages. Neutrophils and IL-10-positive cells were localized at the maternal-fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15.5 to GD18.5, which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The current study furthers our knowledge of the localization of uterine immune cells and relevant cytokines, and provides a base from which to research the function of these immune cells and cytokines during rat pregnancy as a model to study human immune-related pregnancy complications.

  11. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury

    Science.gov (United States)

    Ren, Yilong; Ao, Yan; O’Shea, Timothy M.; Burda, Joshua E.; Bernstein, Alexander M.; Brumm, Andrew J.; Muthusamy, Nagendran; Ghashghaei, H. Troy; Carmichael, S. Thomas; Cheng, Liming; Sofroniew, Michael V.

    2017-01-01

    Ependyma have been proposed as adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after spinal cord injury (SCI). This proposal was based on small, midline stab SCI. Here, we tested the generality of this proposal by using a genetic knock-in cell fate mapping strategy in different murine SCI models. After large crush injuries across the entire spinal cord, ependyma-derived progeny remained local, did not migrate and contributed few cells of any kind and less than 2%, if any, of the total newly proliferated and molecularly confirmed scar-forming astrocytes. Stab injuries that were near to but did not directly damage ependyma, contained no ependyma-derived cells. Our findings show that ependymal contribution of progeny after SCI is minimal, local and dependent on direct ependymal injury, indicating that ependyma are not a major source of endogenous neural stem cells or neuroprotective astrocytes after SCI. PMID:28117356

  12. Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells.

    Science.gov (United States)

    Tomlinson, Rebecca L; Abreu, Eladio B; Ziegler, Tania; Ly, Hinh; Counter, Christopher M; Terns, Rebecca M; Terns, Michael P

    2008-09-01

    Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference-mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

  13. Down-regulation of β-catenin Nuclear Localization by Aspirin Correlates with Growth Inhibition of Jurkat Cell Line

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA,and cell cycle of Jurkat cell line (Human T-acute lymphoblastic leukemia). Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin,the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinD1 mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.

  14. Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle

    Institute of Scientific and Technical Information of China (English)

    Aniruddha SENGUPTA; Bhaswati BANERJEE; Rakesh K. TYAGI; Kasturi DATTA

    2005-01-01

    Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitotic nuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process.

  15. Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development.

    Science.gov (United States)

    Zhu, Minghua; Shen, Shudan; Liu, Yan; Granillo, Olivia; Zhang, Weiguo

    2005-01-01

    It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.

  16. Autoradiographic localization of /sup 3/H-digoxin binding by neural cells in the medulla

    Energy Technology Data Exchange (ETDEWEB)

    Traurig, H.H.; Bhagat, A.; Bass, N.H.

    1985-01-01

    The purpose of this investigation was to localize binding sites for the cardiac glycoside digoxin in the medulla of the rat in vivo. Adult male Sprague-Dawley rats were injected (IV) with /sup 3/H-digoxin and killed 30 minutes later. Autoradiographs of medullas showed evidence of /sup 3/H-digoxin binding to small- and medium-sized neural cells in the regions of the nucleus solitarius, dorsal motor nucleus of the vagus, area postrema, and in the zone between the area postrema and the underlying neuropil. However, the parasympathetic preganglionic neurons of the dorsal motor nucleus were not labeled. The /sup 3/H-digoxin-labeled cells in the medulla were located mainly in the commissural and medial portions of nucleus solitarius at the level of the area postrema. Animals injected with unlabeled digoxin followed by /sup 3/H-digoxin showed reduced binding of radioactivity. The small- and medium-sized neurons of the caudal portions of the nucleus solitarius are internuncial in position with respect to cardiovascular afferents of the glossopharyngeal and vagus nerves and sympathetic and parasympathetic cardiovascular efferent neurons of the medulla. The results of this study suggest that these /sup 3/H-digoxin-labeled cells, presumably neurons of nucleus solitarius, may possess high affinity binding sites for digoxin. Further, the area postrema, which lacks a blood-brain barrier, may provide a portal of entry for /sup 3/H-digoxin into regions of the medulla known to contain neurons that play a role in the regulation of cardiac rhythm.

  17. Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

    Science.gov (United States)

    Bártová, Eva; Foltánková, Veronika; Legartová, Soňa; Sehnalová, Petra; Sorokin, Dmitry V; Suchánková, Jana; Kozubek, Stanislav

    2014-01-01

    Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.

  18. 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

    a technique using fluorescent synthetic and protein-based inhibitors. With this approach we have detected a novel serine protease activity with a relative mobility of 37 kDa, localized to the surface of pole cells, the germ-line precursors, in embryos between nuclear cycles 11 and 14 in development. A second...... novel cell-specific protease activity was localized to the tissues of early gastrulating embryos. Microinjection of inhibitors into the perivitelline space of stage 2 embryos perturbed normal embryonic development. Fluorescein-conjugated chymotrypsin inhibitor and Bowman-Birk inhibitor labeled protease...... 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....

  19. 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.

  20. 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

  1. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    Science.gov (United States)

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  2. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Zdenka Drastichova; Jiri Novotny

    2012-01-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors.At least some types of Gprotein o subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists.However,the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol.Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of Gq/1 1α protein and to determine their localization in isolated fractions and stability in naive and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G11α protein.We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the Gq/11 protein,which were found to be membrane-bound.Both of these complexes dissociated after prolonged treatment with TRH.Still other Gq/11α protein complexes of lower molecular weight were determined in the cytosol.These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h)hormone treatment.These results support the notion that a portion of Gq/11α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor.At the same time,these findings indicate that the redistribution of Gq/11α is broughtabout by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the Gq/11α protein.

  3. THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    刘学光; 张志刚; 张秀荣; 朱虹光; 陈琦; 郭慕依

    2002-01-01

    Objective. To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). Methods. A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. Results. A specific monoclonal antibody against AM was successfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. Conclusion. AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

  4. Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib.

    Science.gov (United States)

    Irwin, Mary E; Mueller, Kelly L; Bohin, Natacha; Ge, Yubin; Boerner, Julie L

    2011-09-01

    Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ~30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity.

  5. Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Manuel Modolell

    Full Text Available The balance between T helper (Th 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

  6. Changes in subcellular localization of visfatin in human colorectal HCT-116 carcinoma cell line after cytochalasin B treatment

    Directory of Open Access Journals (Sweden)

    R.J. Bułdak

    2014-09-01

    Full Text Available The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.

  7. 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.

  8. 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.

  9. Local 2D Particle-in-cell simulations of the collisionless MRI

    CERN Document Server

    Riquelme, Mario A; Sharma, Prateek; Spitkovsky, Anatoly

    2012-01-01

    The magnetorotational instability (MRI) is a crucial mechanism of angular momentum transport in a variety of astrophysical accretion disks. In systems accreting at well below the Eddington rate, such as the central black hole in the Milky Way (Sgr A*), the rate of Coulomb collisions between particles is very small, making the disk evolve essentially as a collisionless plasma. We present a nonlinear study of the collisionless MRI using first-principles particle-in-cell (PIC) plasma simulations. In this initial study we focus on local two-dimensional (axisymmetric) simulations, deferring more realistic three-dimensional simulations to future work. For simulations with net vertical magnetic flux, the MRI continuously amplifies the magnetic field until the Alfv\\'en velocity, v_A, is comparable to the speed of light, c (independent of the initial value of v_A/c). This is consistent with the lack of saturation of MRI channel modes in analogous axisymmetric MHD simulations. The amplification of the magnetic field by...

  10. Concerted emission and local potentiometry of light-emitting electrochemical cells.

    Science.gov (United States)

    Rodovsky, Deanna B; Reid, Obadiah G; Pingree, Liam S C; Ginger, David S

    2010-05-25

    We study the operation of polymer light-emitting electrochemical cells (LECs) by combining scanning Kelvin probe microscopy with in situ imaging of the electroluminescence and photoluminescence on planar LECs. By combining these techniques on the same device in the same apparatus we directly map the relationship between the spatial distribution of electroluminescence and the local potential profile across the device. We find that the electroluminescence is always associated with a region of potential drop in LECs made with poly[2-methoxy-5-(3',7'-dimethyl-octyloxy)-p-phenylenevinylene] (MDMO-PPV), poly(ethylene oxide)(PEO), and potassium trifluoromethanesulfonate. Nevertheless, depending on the electrode metal used, we also find significant potential drops at or near the electrode/organic interfaces. We study the effects of using different electrodes and show that both the electroluminescence and potential profiles are strongly dependent on the electrode work function for thin junctions operated at low potentials. These results indicate injection barriers can affect the operation of LECs even in the presence of doping.

  11. Personalized therapy in locally advanced head and neck squamous-cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sánchez-Escribano R

    2013-11-01

    Full Text Available Introduction: Locally advanced head and neck squamous cell carcinoma patients (LAHNSCC represents a truly heterogeneous population with differences in comorbidities, primary tumor location and etiology. These are key factors in optimal treatment selection. Material and methods: An extensive literature review was made in order to identify the most relevant factor in the therapeutic decision, with special interest in induction chemotherapy as the latest and most debatable option. Results: In the therapeutic decision we have to take into account factors related to the patient, age and performance status are the most important, and others related to the tumor as stage, site of origin and etiology, between this ones l, viral subtypes (EBV and HPV are becoming relevant in the later decades. Chemoradiotherapy is considered the gold standard treatment, supported by several randomized trials and metaanalysis. Induction chemotherapy is one of the later options appeared in the therapeutic arena, improving results in organ preservation and survival. Although a substantial increase in toxicities and lack of prospective comparisons with the standard concurrent chemoradioterapy, warrants a cautious use. Conclusions: Therapeutic choice in the LAHNSCC patient is a complex and multidimensional process, that should be carried in a specialized and multidisciplinary team that can assure the highest efficiency and security for the patient

  12. Neutralizing Interleukin-1β (IL-1β) Induces β-Cell Survival by Maintaining PDX1 Protein Nuclear Localization*

    Science.gov (United States)

    Ardestani, Amin; Sauter, Nadine S.; Paroni, Federico; Dharmadhikari, Gitanjali; Cho, Jae-Hyoung; Lupi, Roberto; Marchetti, Piero; Oberholzer, José; Conte, Julie Kerr; Maedler, Kathrin

    2011-01-01

    The transcription factor PDX1 plays a critical role during β-cell development and in glucose-induced insulin gene transcription in adult β-cells. Acute glucose exposure leads to translocalization of PDX1 to the nucleoplasm, whereas under conditions of oxidative stress, PDX1 shuttles from the nucleus to the cytosol. Here we show that cytosolic PDX1 expression correlated with β-cell failure in diabetes. In isolated islets from patients with type 2 diabetes and from diabetic mice, we found opposite regulation of insulin and PDX1 mRNA; insulin was decreased in diabetes, but PDX1 was increased. This suggests that elevated PDX1 mRNA levels may be insufficient to regulate insulin. In diabetic islets, PDX1 protein was localized in the cytosol, whereas in non-diabetic controls, PDX1 was in the nucleus. In contrast, overexpression of either IL-1 receptor antagonist or shuttling-deficient PDX1 restored β-cell survival and function and PDX1 nuclear localization. Our results show that nuclear localization of PDX1 is essential for a functional β-cell and provides a novel mechanism of the protective effect of IL-1 receptor antagonist on β-cell survival and function. PMID:21393239

  13. 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.

  14. Local transplantation of ex vivo expanded bone marrow-derived CD34-positive cells accelerates fracture healing.

    Science.gov (United States)

    Kawakami, Yohei; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Matsumoto, Tomoyuki; Kuroda, Ryosuke; Shoji, Taro; Fukui, Tomoaki; Masuda, Haruchika; Akimaru, Hiroshi; Mifune, Yutaka; Kuroda, Tomoya; Horii, Miki; Yokoyama, Ayumi; Kurosaka, Masahiro; Asahara, Takayuki

    2012-01-01

    Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

  15. Enhanced local and systemic anti-melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice

    Science.gov (United States)

    Contreras, Amanda; Sen, Siddhartha; Tatar, Andrew J.; Mahvi, David A.; Meyers, Justin V.; Srinand, Prakrithi; Suresh, Marulasiddappa

    2016-01-01

    Adoptive cell transfer (ACT) melanoma immunotherapy typically employs acutely activated effector CD8+ T cells for their ability to rapidly recognize and clear antigen. We have previously observed that effector CD8+ T cells are highly susceptible to melanoma-induced suppression, whereas memory CD8+ T cells are not. Although memory T cells have been presumed to be potentially advantageous for ACT, the kinetics of local and systemic T cell responses after effector and memory ACT have not been compared. B16F10 melanoma cells stably transfected to express very low levels of the lymphocytic choriomeningitis virus (LCMV) peptide GP33 (B16GP33) were inoculated into syngeneic C57BL/6 mice. Equal numbers of bona fide naïve, effector, or memory phenotype GP33-specific CD8+ T cells were adoptively transferred into mice 1 day after B16GP33 inoculation. The efficacy of ACT immunotherapy was kinetically assessed using serial tumor measurements and flow cytometric analyses of local and systemic CD8+ T cell responses. Control of B16GP33 tumor growth, persistence of adoptively transferred CD8+ cells, intratumoral infiltration of CD8+ T cells, and systemic CD8+ T cell responsiveness to GP33 were strongest after ACT of memory CD8+ T cells. Following surgical tumor resection and melanoma tumor challenge, only mice receiving memory T cell-based ACT immunotherapy exhibited durable tumor-specific immunity. These findings demonstrate how the use of non-expanded memory CD8+ T cells may enhance ACT immunotherapeutic efficacy. PMID:27011014

  16. [Ligandin localization in the gonadal cells of rats at various stages of ontogeny].

    Science.gov (United States)

    Bannikov, G A; Chipysheva, T A

    1979-01-01

    Ligandin, a protein binding some carcinogens, steroids and other substances in the rat liver, has been found by means of indirect immunofluorescence in the gonad cells of different types: embryonic and mature Leidig cells in testes, ovarian thecal cells at the maturation stages (theca interna, atretic follicles and interstitial cells) and luteal cells of corpus luteum at pregnancy. Ligandin is found, thus, in cells which belong to various lines of cell differentiation. The functional role of ligandin is discussed.

  17. Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Wang, Li; Xing, Jie; Cheng, Rui; Shao, Ying; Li, Peng; Zhu, Shengtao; Zhang, Shutian

    2015-01-01

    Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC) is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

  18. Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

  19. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

    Science.gov (United States)

    Yang, Yi-Lin; Serrano, Myrna G; Sheoran, Abhineet S; Manque, Patricio A; Buck, Gregory A; Widmer, Giovanni

    2009-11-01

    The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

  20. Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.

    Science.gov (United States)

    Wellner, Robert B; Hong, Sohee; Cotrim, Ana P; Swaim, William D; Baum, Bruce J

    2005-01-01

    To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH

  1. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    Science.gov (United States)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  2. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

    DEFF Research Database (Denmark)

    Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

    1996-01-01

    We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide...... substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types...... organelle....

  3. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  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. Comparison of survival of chronic obstructive pulmonary disease patients with or without a localized non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jeppesen, Stefan Starup; Gerner Hansen, Niels-Christian; Schytte, Tine

    2016-01-01

    OBJECTIVES: Chronic obstructive pulmonary disease (COPD) and non-small cell lung cancer (NSCLC) are often co-existing diseases with poor prognosis. The aim of this study was to compare survival in COPD patients with localized NSCLC treated with stereotactic body radiotherapy (NSCLC group) with COPD...

  6. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, G H; Hösli, E; Belhage, B;

    1991-01-01

    GABAA-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA/benzodiazepine/chloride channel complex. At the...

  7. Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells.

    NARCIS (Netherlands)

    Kurps, J.; Broeke, J.H.; Cijsouw, T.; Kompatscher, A.; Weering, J.R. van; Wit, H. de

    2014-01-01

    BACKGROUND: Adrenal chromaffin cells are a widely used model system to study regulated exocytosis and other membrane-associated processes. Alterations in the amount and localization of the proteins involved in these processes can be visualized with fluorescent probes that report the effect of differ

  8. Differential localization and high expression of SURVIVIN splice variants in human embryonic stem cells but not in differentiated cells implicate a role for SURVIVIN in pluripotency

    Directory of Open Access Journals (Sweden)

    Amber N. Mull

    2014-03-01

    Full Text Available The BIRC5 gene encodes the oncofetal protein SURVIVIN, as well as four additional splice variants (ΔEx3, 2B, 3B and 2α. SURVIVIN, an inhibitor of apoptosis, is also a chromosomal passenger protein (CPP. Previous results have demonstrated that SURVIVIN is expressed at high levels in embryonic stem cells and inhibition of SURVIVIN function results in apoptosis, however these studies have not investigated the other four splice variants. In this study, we demonstrate that all variants are expressed at significantly higher levels in human embryonic stem (hES cells than in differentiated cells. We examined the subcellular localization of the three most highly expressed variants. SURVIVIN displayed canonical CPP localization in mitotic cells and cytoplasmic localization in interphase cells. In contrast, SURVIVIN–ΔEx3 and SURVIVIN–2B did not localize as a CPP; SURVIVIN–ΔEx3 was found constitutively in the nucleus while SURVIVIN–2B was distributed along the chromosomes during mitosis and also to the mitotic spindle poles. We used inducible shRNA against SURVIVIN to inhibit expression in a titratable fashion. Using this system, we reduced the mRNA levels of these three variants to approx. 40%, resulting in a concomitant reduction of OCT4 and NANOG mRNA, suggesting a role for the SURVIVIN variants in pluripotency.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tordeur, M. [Pitie-Salpetriere Teaching Hospital, Radiology Dept., Paris (France); Wybier, M. [Lariboisiere Teaching Hospital, Paris (France); Laporte, J.L. [Pitie-Salpetriere Teaching Hospital, Pathology Dept., Paris (France); Grenier, P. [Pitie-Salpetriere Teaching Hospital, Radiology Dept., Paris (France); Laredo, J.D. [Lariboisiere Teaching Hospital, Paris (France)

    2000-04-15

    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)

  10. Immunohistochemical study on localization of serotonin immunoreactive cells in the gastrointestinal tract of the European catfish (Silurus glanis, L.).

    Science.gov (United States)

    Köprücü, S; Yaman, M

    2015-02-01

    In this study, it was aimed to identify the distribution of serotonin immunoreactive cells within the gastrointestinal tract (GIT) of European catfish (Silurus glanis). For this purpose, the tissue samples were taken from the stomach (cardia, fundus and pylorus region) and intestine (anterior, middle and posterior region). They were examined by applying the avidin-biotin-immunoperoxidase method. The serotonin containing immunoreactive cells are presented in all regions of the GIT. It was determined to be localized generally in different distribution within the stomachs and intestines of S. glanis. It was found that the most intensive regions of immunoreactive cells were the cardia stomach and posterior of intestine.

  11. Localized Langerhans cell histiocytosis masquerading as Brodie's abscess in a 2-year-old child: a case report

    Science.gov (United States)

    Chang, Wei-Fang; Hsu, Yi-Chih; Wu, Yi-Der; Kuo, Chun-Lang; Huang, Guo-Shu

    2016-01-01

    Langerhans cell histiocytosis (LCH), formerly known as histiocytosis X, refers to a spectrum of diseases characterized by idiopathic proliferation of histiocytes that produce either focal (localized LCH) or systemic manifestations (Hand-Schüller-Christian disease and Letterer-Siwe disease). Localized LCH accounts for approximately 60-70 % of all LCH cases. Osseous involvement is the most common manifestation and typically involves the flat bones, along with lesions of the skull, pelvis, and ribs. Localized LCH in bone shows a wide spectrum of clinical manifestations and radiologic features that may mimic those of infections as well as benign and malignant tumors. The diagnostic imaging findings of localized LCH are also diverse and challenging. The penumbra sign is a common and characteristic magnetic resonance imaging (MRI) feature of Brodie's abscess, but is rarely seen in localized LCH. In this report, we describe a case of localized LCH misdiagnosed as Brodie's abscess in a 2-year-old child based on clinical symptoms, laboratory findings, and pre-diagnostic MRI findings (penumbra sign). Therefore, the penumbra sign is not sufficient to clearly establish the diagnosis of Brodie's abscess, and the differential diagnosis of localized LCH should be considered when a child with an osteolytic lesion presents with a penumbra sign. PMID:27065773

  12. Localized Langerhans cell histiocytosis masquerading as Brodie's abscess in a 2-year-old child: a case report.

    Science.gov (United States)

    Chang, Wei-Fang; Hsu, Yi-Chih; Wu, Yi-Der; Kuo, Chun-Lang; Huang, Guo-Shu

    2016-01-01

    Langerhans cell histiocytosis (LCH), formerly known as histiocytosis X, refers to a spectrum of diseases characterized by idiopathic proliferation of histiocytes that produce either focal (localized LCH) or systemic manifestations (Hand-Schüller-Christian disease and Letterer-Siwe disease). Localized LCH accounts for approximately 60-70 % of all LCH cases. Osseous involvement is the most common manifestation and typically involves the flat bones, along with lesions of the skull, pelvis, and ribs. Localized LCH in bone shows a wide spectrum of clinical manifestations and radiologic features that may mimic those of infections as well as benign and malignant tumors. The diagnostic imaging findings of localized LCH are also diverse and challenging. The penumbra sign is a common and characteristic magnetic resonance imaging (MRI) feature of Brodie's abscess, but is rarely seen in localized LCH. In this report, we describe a case of localized LCH misdiagnosed as Brodie's abscess in a 2-year-old child based on clinical symptoms, laboratory findings, and pre-diagnostic MRI findings (penumbra sign). Therefore, the penumbra sign is not sufficient to clearly establish the diagnosis of Brodie's abscess, and the differential diagnosis of localized LCH should be considered when a child with an osteolytic lesion presents with a penumbra sign.

  13. Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines

    Directory of Open Access Journals (Sweden)

    Duncan Alice

    2012-10-01

    Full Text Available Abstract Background Fas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL during luteal regression. In certain cell-types, sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically, keratin 8/18 (K8/K18 filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface. Methods Here, Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5, postovulation and late stages (Days 16–18, postovulation of CL function, and the relationship between Fas expression, K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated. Results Both total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P0.05, n=4 CL/stage, despite evidence these conditions increased Fas expression on HepG2 cells (P0.05 or stage of CL (P>0.05, n= 4 CL/stage on this outcome. Conclusion In conclusion, we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling, however, remain a possibility. Importantly, the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s of Fas in the corpus luteum, particularly during early luteal development.

  14. 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.

  15. Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation.

    Science.gov (United States)

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2015-04-28

    To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.

  16. 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)

  17. Dose escalation for unresectable locally advanced non-small cell lung cancer: end of the line?

    Science.gov (United States)

    Hong, Julian C; Salama, Joseph K

    2016-02-01

    Radiation Therapy Oncology Group (RTOG) 0617 was a randomized trial that investigated both the impact of radiation dose-escalation and the addition of cetuximab on the treatment of non-small cell lung cancer (NSCLC). The results of RTOG 0617 were surprising, with the dose escalation randomization being closed prematurely due to futility stopping rules, and cetuximab ultimately showing no overall survival benefit. Locally advanced unresectable NSCLC has conventionally been treated with concurrent chemoradiation. Though advances in treatment technology have improved the ability to deliver adequate treatment dose, the foundation for radiotherapy (RT) has remained the same since the 1980s. Since then, progressive studies have sought to establish the safety and efficacy of escalating radiation dose to loco-regional disease. Though RTOG 0617 did not produce the anticipated result, much interest remains in dose escalation and establishing an explanation for the findings of this study. Cetuximab was also not found to provide a survival benefit when applied to an unselected population. However, planned retrospective analysis suggests that those patients with high epidermal growth factor receptor (EGFR) expression may benefit, suggesting that cetuximab should be applied in a targeted fashion. We discuss the results of RTOG 0617 and additional findings from post-hoc analysis that suggest that dose escalation may be limited by normal tissue toxicity. We also present ongoing studies that aim to address potential causes for mortality in the dose escalation arm through adaptive or proton therapy, and are also leveraging additional concurrent systemic agents such as tyrosine kinase inhibitors (TKIs) for EGFR-activating mutations or EML4-ALK rearrangements, and poly (ADP-ribose) polymerase (PARP) inhibitors.

  18. Outpatient and Home Chemotherapy with Novel Local Control Strategies in Desmoplastic Small Round Cell Tumor

    Directory of Open Access Journals (Sweden)

    Holly Green

    2008-06-01

    Full Text Available Desmoplastic Small Round Cell Tumor (DSRCT has a very poor prognosis. This report illustrates novel chemotherapy and local control interventions in a 5-year old patient. The patient was treated in the outpatient setting, achieved remission, with excellent quality of life. The patient presented with massive ascites and >1000 abdominal tumors. Neoadjuvant chemotherapy included vincristine (1.5 mg/m2, ifosfamide (3 g/m2/day×3, dexrazoxane/doxorubicin (750/75 mg/m2, and etoposide (150 mg/m2. Continuous hyperthermic peritoneal perfusion (CHPP with cisplatin (100 mg/m2 was given after extensive cytoreductive surgery. This was followed by irinotecan (10 mg/m2/day×5×2  weeks + temozolomide monthly × 2, then abdominal radiation 30 Gy with simultaneous temozolomide (100 mg/m2/day×5. A total of 12 cycles of irinotecan and temozolamide were given. Except for initial chemotherapy, subsequent courses were in the outpatient setting. Focal retroperitoneal relapse at 18 months was treated with IMRT with bevacizumab (5 mg/kg and 2 perihepatic metastases with radio frequency ablation/cryoablation followed by chronic outpatient maintenance chemotherapy (valproic acid, cyclophosphamide, and rapamycin. Almost 2 years from diagnosis, the patient maintained an excellent quality of life. This is a novel approach to the treatment of children with massive abdomino-pelvic DSRCT.

  19. Extrinsic and local glutamatergic inputs of the rat hippocampal CA1 area differentially innervate pyramidal cells and interneurons.

    Science.gov (United States)

    Takács, Virág T; Klausberger, Thomas; Somogyi, Peter; Freund, Tamás F; Gulyás, Attila I

    2012-06-01

    The two main glutamatergic pathways to the CA1 area, the Schaffer collateral/commissural input and the entorhinal fibers, as well as the local axons of CA1 pyramidal cells innervate both pyramidal cells and interneurons. To determine whether these inputs differ in their weights of activating GABAergic circuits, we have studied the relative proportion of pyramidal cells and interneurons among their postsynaptic targets in serial electron microscopic sections. Local axons of CA1 pyramidal cells, intracellularly labeled in vitro or in vivo, innervated a relatively high proportion of interneuronal postsynaptic targets (65.9 and 53.8%, in vitro and in vivo, respectively) in stratum (str.) oriens and alveus. In contrast, axons of in vitro labeled CA3 pyramidal cells in str. oriens and str. radiatum of the CA1 area made synaptic junctions predominantly with pyramidal cell spines (92.9%). The postsynaptic targets of anterogradely labeled medial entorhinal cortical boutons in CA1 str. lacunosum-moleculare were primarily pyramidal neuron dendritic spines and shafts (90.8%). The alvear group of the entorhinal afferents, traversing str. oriens, str. pyramidale, and str. radiatum showed a higher preference for innervating GABAergic cells (21.3%), particularly in str. oriens/alveus. These data demonstrate that different glutamatergic pathways innervate CA1 GABAergic cells to different extents. The results suggest that the numerically smaller CA1 local axonal inputs together with the alvear part of the entorhinal input preferentially act on GABAergic interneurons in contrast to the CA3, or the entorhinal input in str. lacunosum-moleculare. The results highlight differences in the postsynaptic target selection of the feed-forward versus recurrent glutamatergic inputs to the CA1 and CA3 areas.

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

    Directory of Open Access Journals (Sweden)

    Agnieszka Dyrda

    Full Text Available BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. METHODOLOGY/PRINCIPAL FINDINGS: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+ and Cl(- currents were strictly dependent on the presence of Ca(2+. The Ca(2+-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+ permeability (PCa. These results indicate that local membrane deformations can transiently activate a Ca(2+ permeability pathway leading to increased [Ca(2+](i, secondary activation of Ca(2+-sensitive K(+ channels (Gardos channel, IK1, KCa3.1, and hyperpolarization-induced anion currents. CONCLUSIONS/SIGNIFICANCE: The stretch-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

  1. Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

    Science.gov (United States)

    Takaoka, Yousuke; Shigenaga, Miyuki; Imai, Masaki; Nukadzuka, Yuuki; Ishimaru, Yasuhiro; Saito, Kei; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ueda, Minoru

    2016-01-01

    In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

  2. Intracellular ZnO Nanorods Conjugated with Protoporphyrin for Local Mediated Photochemistry and Efficient Treatment of Single Cancer Cell

    Directory of Open Access Journals (Sweden)

    Larsson Per-Olof

    2010-01-01

    Full Text Available Abstract ZnO nanorods (NRs with high surface area to volume ratio and biocompatibility is used as an efficient photosensitizer carrier system and at the same time providing intrinsic white light needed to achieve cancer cell necrosis. In this letter, ZnO nanorods used for the treatment of breast cancer cell (T47D are presented. To adjust the sample for intracellular experiments, we have grown the ZnO nanorods on the tip of borosilicate glass capillaries (0.5 μm diameter by aqueous chemical growth technique. The grown ZnO nanorods were conjugated using protoporphyrin dimethyl ester (PPDME, which absorbs the light emitted by the ZnO nanorods. Mechanism of cytotoxicity appears to involve the generation of singlet oxygen inside the cell. The novel findings of cell-localized toxicity indicate a potential application of PPDME-conjugated ZnO NRs in the necrosis of breast cancer cell within few minutes.

  3. Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells

    Science.gov (United States)

    Nedukha, E. M.

    The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2 rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive pecularity of the cells cultivated on clinostat. It testifies to the fact that increasing of freee calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weighlessness.

  4. 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…

  5. Dynamic localization of tripartite motif-containing 22 in nuclear and nucleolar bodies

    Energy Technology Data Exchange (ETDEWEB)

    Sivaramakrishnan, Gayathri; Sun, Yang; Tan, Si Kee [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Lin, Valerie C.L., E-mail: cllin@ntu.edu.sg [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2009-05-01

    Tripartite motif-containing 22 (TRIM22) exhibits antiviral and growth inhibitory properties, but there has been no study on the localization and dynamics of the endogenous TRIM22 protein. We report here that TRIM22 is dramatically induced by progesterone in MDA-MB-231-derived ABC28 cells and T47D cells. This induction was associated with an increase in TRIM22 nuclear bodies (NB), and an even more prominent increase in nucleolar TRIM22 bodies. Distinct endogenous TRIM22 NB were also demonstrated in several other cell lines including MCF7 and HeLa cells. These TRIM22 NB resemble Cajal bodies, co-localized with these structures and co-immunoprecipitated with p80-coilin. However, IFN{gamma}-induced TRIM22 in HeLa and MCF7 cells did not form NB, implying the forms and distribution of TRIM22 are regulated by specific cellular signals. This notion is also supported by the observation that TRIM22 NB undergoes dynamic cell-cycle dependent changes in distribution such that TRIM22 NB started to form in early G0/G1 but became dispersed in the S-phase. In light of its potential antiviral and antitumor properties, the findings here provide an interesting gateway to study the relationship between the different forms and functions of TRIM22.

  6. 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

  7. A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem.

    Science.gov (United States)

    Burian, Agata; Ludynia, Michal; Uyttewaal, Magalie; Traas, Jan; Boudaoud, Arezki; Hamant, Olivier; Kwiatkowska, Dorota

    2013-12-01

    Cortical microtubules (CMTs) are often aligned in a particular direction in individual cells or even in groups of cells and play a central role in the definition of growth anisotropy. How the CMTs themselves are aligned is not well known, but two hypotheses have been proposed. According to the first hypothesis, CMTs align perpendicular to the maximal growth direction, and, according to the second, CMTs align parallel to the maximal stress direction. Since both hypotheses were formulated on the basis of mainly qualitative assessments, the link between CMT organization, organ geometry, and cell growth is revisited using a quantitative approach. For this purpose, CMT orientation, local curvature, and growth parameters for each cell were measured in the growing shoot apical meristem (SAM) of Arabidopsis thaliana. Using this approach, it has been shown that stable CMTs tend to be perpendicular to the direction of maximal growth in cells at the SAM periphery, but parallel in the cells at the boundary domain. When examining the local curvature of the SAM surface, no strict correlation between curvature and CMT arrangement was found, which implies that SAM geometry, and presumed geometry-derived stress distribution, is not sufficient to prescribe the CMT orientation. However, a better match between stress and CMTs was found when mechanical stress derived from differential growth was also considered.

  8. Phosphorylation switch modulates the interdigitated pattern of PIN1 localization and cell expansion in Arabidopsis leaf epidermis

    Institute of Scientific and Technical Information of China (English)

    Hongjiang Li; Deshu Lin; Pankaj Dhonukshe; Shingo Nagawa; Dandan Chen; Ji(r)í Friml; Ben Scheres; Hongwei Guo; Zhenbiao Yang

    2011-01-01

    Within a multicellular tissue cells may coordinately form a singular or multiple polar axes,but it is unclear whether a common mechanism governs different types of polar axis formation. The phosphorylation status of PIN proteins,which is directly affected by the PINOID (PID) protein kinase and the PP2A protein phosphatase,is known to regulate the apical-basal polarity of PIN localization in bipolar cells of roots and shoot apices. Here,we provide evidence that the phosphorylation status-mediated PIN polarity switch is widely used to modulate cellular processes in Arabidopsis including multipolar pavement cells (PC) with interdigitated lobes and indentations. The degree of PC interdigitation was greatly reduced either when the FYPP1 gene,which encodes a PP2A called phytochromeassociated serine/threonine protein phosphatase,was knocked out or when the PID gene was overexpressed (35S::PID).These genetic modifications caused PIN1 localization to switch from lobe to indentation regions. The PP2A and PID mediated switching of PIN1 localization is strikingly similar to their regulation of the apical-basal polarity switch of PIN proteins in other cells. Our findings suggest a common mechanism for the regulation of PIN1 polarity formation,a fundamental cellular process that is crucial for pattern formation both at the tissue/organ and cellular levels.

  9. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu

    2006-01-01

    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  10. Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals.

    Science.gov (United States)

    Montcouquiol, Mireille; Sans, Nathalie; Huss, David; Kach, Jacob; Dickman, J David; Forge, Andrew; Rachel, Rivka A; Copeland, Neal G; Jenkins, Nancy A; Bogani, Debora; Murdoch, Jennifer; Warchol, Mark E; Wenthold, Robert J; Kelley, Matthew W

    2006-05-10

    Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

  11. Radiation-induced effects on murine kidney tumor cells: role in the interaction of local irradiation and immunotherapy.

    Science.gov (United States)

    Younes, E; Haas, G P; Dezso, B; Ali, E; Maughan, R L; Montecillo, E; Pontes, J E; Hillman, G G

    1995-06-01

    Local tumor irradiation enhances the effect of interleukin-2 (IL-2) therapy in the Renca murine renal adenocarcinoma model. To investigate the mechanism(s) of this interaction, we studied the in vitro and in vivo effects of irradiation on the tumor cells. Tumor cells from in situ irradiated renal tumors had diminished proliferation in vitro. A similar growth inhibition was noted following injection of irradiated Renca cells into naive mice, but this effect could be overcome by injecting more cells. Histologic evaluation of tumors derived from irradiated cells revealed a decrease in mitosis and an increase in multinucleated giant cells, apoptosis and micronecrosis. The presence of irradiated tumor reduced the growth of nonirradiated tumor cells when both were injected into separate flanks of the same animal, suggesting that irradiated tumor cells may trigger a systemic antitumor response. Interleukin-2 therapy given after injection of irradiated tumor cells caused a significant increase in leukocytic infiltrates and micronecrosis. Our findings indicate that radiation directly affects tumor growth and induces a systemic mechanism which could be enhanced by IL-2.

  12. Correlation of Local FOXP3-Expressing T Cells and Th1-Th2 Balance in Perennial Allergic Nasal Mucosa

    Directory of Open Access Journals (Sweden)

    Hideaki Shirasaki

    2011-01-01

    Full Text Available Regulatory T cells (Treg play some important roles in allergic rhinitis. The most specific marker for Treg is FOXP3, a recently identified transcription factor that is essential for Treg development. In order to clarify the levels of Treg in allergic nasal mucosa, we studied the relationship between FOXP3-expressing cells and Th1-Th2 balance in nasal mucosa by means of immunohistochemistry. Human turbinates were obtained after turbinectomy from 26 patients (14 patients with perennial allergic rhinitis and 12 patients with nonallergic rhinitis. To identify the cells expressing the FOXP3 protein, double immunostaining was performed by using anti-FOXP3 antibody and anti-CD3 antibody. There was no significant difference in the percentage of FOXP3+CD3+ cells among CD3+ cells in the nasal mucosa of two groups. The proportion of FOXP3+CD3+ cells tend to be correlated positively with GATA3+CD3+ cells/T-bet+CD3+ cells ratio (=0.56, =0.04. A positive correlation with GATA3+CD3+/T-bet+CD3+ ratio and FOXP3+CD3+/CD3+ ratio suggests the role of local regulatory T cells as a minimal control of the chronic allergen exposure in nasal mucosa.

  13. Localization of the Arabidopsis Senescence- and Cell Death-Associated BFN1 Nuclease: From the ER to Fragmented Nuclei

    Institute of Scientific and Technical Information of China (English)

    Sarit Farage-Barhom; Shaul Burd; Lilian Sonego; Ana Mett; Eduard Belausov; David Gidoni; Amnon Lers

    2011-01-01

    Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms.However,knowledge of their function is limited.Intracellular localization of the Arabidopsis senescenceand PCD-associated nuclease BFN1 was investigated.Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm,which then clustered around the nuclei as the protoplasts senesced.These filamentous structures were identified as being of ER origin.In BFN1GFP-transgenic Arabidopsis plants,similar localization of BFN1-GFP was observed in young leaves,that is,in filamentous structures that reorganized around the nuclei only in senescing cells.In late senescence,BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles.BFN1's postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern.Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.

  14. Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda, Rhabditida).

    Science.gov (United States)

    Yushin, Vladimir V; Claeys, Myriam; Bert, Wim

    2016-10-01

    The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as "major sperm protein", or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of

  15. Morphological study on permeating efficiency and localization of FCLA and HpD through membrane of lung cancer cell

    Science.gov (United States)

    Wu, Yunxia; Xing, Da; Tang, Yonghong

    2004-07-01

    It is reported that apoptosis of cancer cells in photodynamic therapy (PDT) is caused by 1O2 generated in photosensitization. In order to study the mechanism of this kind of 1O2-induced apoptosis, it is necessary to establish a special technique to dynamically detect intracellular production and localization of 1O2. FCLA, as a chemiluminescence probe to detect singlet oxygen (1O2) and superoxide (O2-.), has been used successfully in photodynamic and sonodynamic diagnosis in tissue level, recently. This paper reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. Human lung cancer cell line (ASTC-a-1) was used in the experiment. The result of this research showed that both HpD and FCLA could permeate through cellular membrane and localize to prinuclear area, when HpD or FCLA was incubated with cells. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane was different from that of FCLA. Intracellular FCLA concentration reached a peak after incubation for only 30 - 45 minutes, but amount of HpD in cells approached the equilibrium after incubation for near 22 h. In the experiment, we did not observe the evidence of FCLA or HpD penetrating into nucleolus. This study suggests that it is possibly to use a specific chemiluminescence probe to dynamcially detect the production and localization of 1O2 or 02-. in cell.

  16. Knockdown of dystrophin Dp71 impairs PC12 cells cycle: localization in the spindle and cytokinesis structures implies a role for Dp71 in cell division.

    Directory of Open Access Journals (Sweden)

    Marcela Villarreal-Silva

    Full Text Available The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels.

  17. Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Jiang Lu; Dongsheng Li; Kehuan Lu

    2012-01-01

    The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain

  18. 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

    2014-01-01

    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.

  19. 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.

  20. 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.

  1. Differential localization of LGR5 and Nanog in clusters of colon cancer stem cells.

    Science.gov (United States)

    Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Freyhan, Ora; Fabrikant, Yakov; Melzer, Ehud; Givol, David

    2013-05-01

    One paradigm of cancer development claims that cancer emerges at the niche of tissue stem cells and these cells continue to proliferate in the tumor as cancer stem cells. LGR5, a membrane receptor, was recently found to be a marker of normal colon stem cells in colon polyps and is also expressed in colon cancer stem cells. Nanog, an embryonic stem cell nuclear factor, is expressed in several embryonic tissues, but Nanog expression is not well documented in cancerous stem cells. Our aim was to examine whether both LGR5 and Nanog are expressed in the same clusters of colon stem cells or cancer stem cells, using immunocytochemistry with specific antibodies to each antigen. We analyzed this aspect using paraffin embedded tumor tissue sections obtained from 18 polyps and 36 colon cancer specimens at stages I-IV. Antibodies to LGR5 revealed membrane and cytoplasm immunostaining of scattered labeled cells in normal crypts, with no labeling of Nanog. However, in close proximity to the tumors, staining to LGR5 was much more intensive in the crypts, including that of the epithelial cells. In cancer tissue, positive LGR5 clusters of stem cells were observed mainly in poorly differentiated tumors and in only a few scattered cells in the highly differentiated tumors. In contrast, antibodies to Nanog mainly stained the growing edges of carcinoma cells, leaving the poorly differentiated tumor cells unlabeled, including the clustered stem cells that could be detected even by direct morphological examination. In polyp tissues, scattered labeled cells were immunostained with antibodies to Nanog and to a much lesser extent with antibodies to LGR5. We conclude that expression of LGR5 is probably specific to stem cells of poorly differentiated tumors, whereas Nanog is mainly expressed at the edges of highly differentiated tumors. However, some of the cell layers adjacent to the carcinoma cell layers that still remained undifferentiated, expressed mainly Nanog with only a few cells

  2. 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

    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......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...... 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...

  3. Identification and localization of label-retaining cells in hamster epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Bickenbach, J.R.; Mackenzie, I.C.

    1984-06-01

    A subpopulation of basal epithelial cells which retains tritiated thymidine label for extended periods was previously demonstrated in skin and oral mucosae of mice. The present study examined the presence of similar cells in hamsters. Five-day-old hamsters were labeled with tritiated thymidine and the rate at which label was diluted from the basal cells observed. A small percentage of basal cells was found to retain label for up to 69 days. The location of such label-retaining cells (LRCs) in the palatal epithelium and in tongue papillae was examined. Thirty and 69 days after labeling, approximately 80% of LRCs in palate were located in the proximal halves of papillae and 80% of LRCs in tongue were positioned basally with approximately 30% of such LRCs occupying positions previously suggested to be stem cell locations. The finding that slowly cycling keratinocytes are related to patterns of tissue architecture is compatible with a function of these cells as epithelial stem cells.

  4. Regulation of Human p53 Activity and Cell Localization by Alternative Splicing

    OpenAIRE

    2004-01-01

    The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic...

  5. Cell Based Autologous Immune Enhancement Therapy (AIET after Radiotherapy in a Locally Advanced Carcinoma of the Cervix

    Directory of Open Access Journals (Sweden)

    Sumana Premkumar

    2013-01-01

    Full Text Available Radiotherapy is the primary form of treatment in patients with locally advanced cervical carcinoma. However for residual disease in the form of the persistent lymph nodes, surgery or chemotherapy is recommended. As surgery is not acceptable by every patient and chemotherapy has associated side effects, we hereby report the positive outcome of in vitro expanded natural killer cell and activated T lymphocyte based autologous immune enhancement therapy (AIET for the residual lymphadenopathy in a patient with locally advanced cervical cancer after radiation. After six transfusions of AIET, there was complete resolution of residual lymph nodes and there was no evidence of local lesion. The patient also reported improvement in quality of life. As AIET has been reported as the least toxic among the available therapies for cancer, combining AIET with conventional forms of therapy in similar patients might not only improve the outcome but may also help the patients achieve a good quality of life.

  6. An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Damkier, Helle Hasager; Nielsen, Søren; Prætorius, Jeppe

    2006-01-01

    plexus. The anti-NH2-terminal antibody localized NBCn1 to the plasma membrane domains of endothelia and smooth muscle cells in small mesenteric and renal arteries, as well as the capillaries of the heart ventricles, spleen, and salivary glands. NBCn1 was also detected in neuromuscular junctions...... the development of the NH2-terminal antibody allowed the localization of NBCn1 protein to major cardiovascular tissues where NBCn1 mRNA was previously detected. The NBCn1 is a likely candidate for mediating the reported electroneutral Na+-HCO3(-) cotransport in vascular smooth muscle.......The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues...

  7. Effect of mesenchymal stem cells transplantation on glycaemic profile & their localization in streptozotocin induced diabetic Wistar rats

    Directory of Open Access Journals (Sweden)

    Shobhit Bhansali

    2015-01-01

    Full Text Available Background & objectives: Bone marrow is a rich source of adult stem cells that can differentiate into various cell types. Administration of mesenchymal stem cells (MSCs in irradiated diabetic rat model has transiently shown to decrease blood glucose level. This study examines the effect of high dose and multiple injections of MSCs on glycemic profile, their localization and regeneration of islet in diabetic Wistar rat. Methods: The study was carried out in male Wistar rats categorized into three groups (n=6, in each group: Group 1 as control, group 2 streptozotocin (STZ (50 mg/kg induced diabetic group and group 3 experimental group; 5-bromo-2-deoxyuridine (BrdU labelled allogenic MSCs were injected in the non-irradiated diabetic rat of the experimental group through tail vein. The blood glucose profile was subsequently monitored at regular intervals. Rats were sacrificed on day 45 and pancreas was examined for localization of BrdU labelled stem cells by immunofluorescence and islet-neogenesis by immunohistochemistry . Results: There was a significant reduction in blood glucose level after administration of MSCs in the experimental group (P<0.001. The presence of BrdU labelled MSCs in islet suggested their localization in the pancreas. Co-expression of anti-BrdU and anti-insulin antibody indicated trans-differentiation / fusion into insulin producing cells evidenced by significant increase in total number of islet (P=0.004 and insulin positive cells ( P<0.0001 in experimental group. Interpretation & conclusions: Our results showed that the MSCs administration in non-irradiated diabetic Wistar rat reduced hyperglycaemia and was accompanied by increased islet-neogengesis, possibly through trans- differentiation/fusion.

  8. 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.

  9. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Science.gov (United States)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  10. Localized committed differentiation of neural stem cells based on the topographical regulation effects of TiO2 nanostructured ceramics

    Science.gov (United States)

    Mou, Xiaoning; Wang, Shu; Guo, Weibo; Ji, Shaozheng; Qiu, Jichuan; Li, Deshuai; Zhang, Xiaodi; Zhou, Jin; Tang, Wei; Wang, Changyong; Liu, Hong

    2016-07-01

    In this study, a porous-flat TiO2 micropattern was fabricated with flat and nanoporous TiO2 ceramics for investigating the effect of topography on neural stem cell (NSC) differentiation. This finding demonstrates that localized committed differentiation could be achieved in one system by integrating materials with different topographies.In this study, a porous-flat TiO2 micropattern was fabricated with flat and nanoporous TiO2 ceramics for investigating the effect of topography on neural stem cell (NSC) differentiation. This finding demonstrates that localized committed differentiation could be achieved in one system by integrating materials with different topographies. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01874b

  11. EicosaCell - an immunofluorescent-based assay to localize newly synthesized eicosanoid lipid mediators at intracellular sites.

    Science.gov (United States)

    Bandeira-Melo, Christianne; Weller, Peter F; Bozza, Patricia T

    2011-01-01

    Eicosanoids (prostaglandins, leukotrienes and lipoxins) are a family of signaling lipids derived from arachidonic acid that have important roles in physiological and pathological processes. Over the past years, it has been established that successful eicosanoid production is not merely determined by arachidonic acid and eicosanoid-forming enzymes availability, but requires sequential interactions between specific biosynthetic proteins acting in cascade and may involve very unique spatial interactions. Direct assessment of specific subcellular locales of eicosanoid synthesis has been elusive, as those lipid mediators are newly formed, not stored and often rapidly released upon cell stimulation. In this chapter, we discuss the EicosaCell protocol for intracellular detection of eicosanoid-synthesizing compartments by means of a strategy to covalently cross-link and immobilize the lipid mediators at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid.

  12. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

    Science.gov (United States)

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-07-14

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

  13. Heterogeneity in polymer solar cells: local morphology and performance in organic photovoltaics studied with scanning probe microscopy.

    Science.gov (United States)

    Groves, Chris; Reid, Obadiah G; Ginger, David S

    2010-05-18

    The use of organic photovoltaics (OPVs) could reduce production costs for solar cells because these materials are solution processable and can be manufactured by roll-to-roll printing. The nanoscale texture, or film morphology, of the donor/acceptor blends used in most OPVs is a critical variable that can dominate both the performance of new materials being optimized in the lab and efforts to move from laboratory-scale to factory-scale production. Although efficiencies of organic solar cells have improved significantly in recent years, progress in morphology optimization still occurs largely by trial and error, in part because much of our basic understanding of how nanoscale morphology affects the optoelectronic properties of these heterogeneous organic semiconductor films has to be inferred indirectly from macroscopic measurements. In this Account, we review the importance of nanoscale morphology in organic semiconductors and the use of electrical scanning probe microscopy techniques to directly probe the local optoelectronic properties of OPV devices. We have observed local heterogeneity of electronic properties and performance in a wide range of systems, including model polymer-fullerene blends such as poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM), newer polyfluorene copolymer-PCBM blends, and even all polymer donor-acceptor blends. The observed heterogeneity in local photocurrent poses important questions, chiefly what information is contained and what is lost when using average values obtained from conventional measurements on macroscopic devices and bulk samples? We show that in many cases OPVs are best thought of as a collection of nanoscopic photodiodes connected in parallel, each with their own morphological and therefore electronic and optical properties. This local heterogeneity forces us to carefully consider the adequacy of describing OPVs solely by "average" properties such as the bulk carrier mobility

  14. Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid (LPA) and/or 8-chlorophenylthio cAMP (CPT-cAMP). METHODS: Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments (membrane, cytosol, nucleus) and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP+LPA. RESULTS: Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments (membrane, cytosol, nucleus, nucleolus). After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol. CONCLUSION: In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.

  15. 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.

  16. 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...... proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication...

  17. Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells.

    Science.gov (United States)

    Gerelsaikhan, Tudevdagva; Chen, Xiao-Liang; Chander, Avinash

    2011-12-01

    Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.

  18. 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

    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.

  19. TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia

    Directory of Open Access Journals (Sweden)

    Kiyoto Kurima

    2015-09-01

    Full Text Available Mechanosensitive ion channels at stereocilia tips mediate mechanoelectrical transduction (MET in inner ear sensory hair cells. Transmembrane channel-like 1 and 2 (TMC1 and TMC2 are essential for MET and are hypothesized to be components of the MET complex, but evidence for their predicted spatiotemporal localization in stereocilia is lacking. Here, we determine the stereocilia localization of the TMC proteins in mice expressing TMC1-mCherry and TMC2-AcGFP. Functionality of the tagged proteins was verified by transgenic rescue of MET currents and hearing in Tmc1Δ/Δ;Tmc2Δ/Δ mice. TMC1-mCherry and TMC2-AcGFP localize along the length of immature stereocilia. However, as hair cells develop, the two proteins localize predominantly to stereocilia tips. Both TMCs are absent from the tips of the tallest stereocilia, where MET activity is not detectable. This distribution was confirmed for the endogenous proteins by immunofluorescence. These data are consistent with TMC1 and TMC2 being components of the stereocilia MET channel complex.

  20. Localization of motilin-immunopositive cells in the rat intestine by light microscopic immunocytochemistry.

    Science.gov (United States)

    Sakai, T; Satoh, M; Koyama, H; Iesaki, K; Umahara, M; Fujikura, K; Itoh, Z

    1994-01-01

    Motilin-immunopositive cells (Mo cells) are known to exist in the upper small intestine of many species including man. However, the possible presence of Mo cells in the rat gastrointestine has remained obscure because antiserum against it raised in rabbit was found not to cross-react with motilin in the rat gastrointestine. The present study was designed to investigate the distribution of Mo cells in the rat gastrointestine by the peroxidase-conjugated second antibody method using newly raised chicken anti-motilin serum (CPV3). This antiserum was suggested to recognize the N-terminal region of the motilin molecule by enzyme-linked immunosorbent assays and immunocytochemical absorption test. Mo cells detected in the rat gastrointestine by immunocytochemistry were found to be distributed in the duodenum (1.5 cells/mm2), jejunum (2.2 cells/mm2), and ileum (0.028 cells/mm2), and no positive cells were found in the gastric body, gastric antrum, cecum, colon, or pancreas. The immunopositive cells in the rat intestine were spindle shaped or polygonal, scattered throughout the epithelium of the villi and crypts, and similar to those commonly observed in the upper small intestine of other species. These results indicate for the first time that motilin-immunopositive cells do exist in the rat intestine.

  1. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  2. Study on development and localization of CTGF-immunoreactive cells in central nervous system of rats

    Institute of Scientific and Technical Information of China (English)

    SU Bing-yin; CAI Wen-qin; ZHANNG Cheng-gang; B.Perbal

    2001-01-01

    Objective: To study the development of connective tissue growth factor(CTGF) immunoreactive cells in the central nervous system (CNS) of E8-P300 rats. Methods: Immunocytochemistry was employed in our study. Results: No CTGF-immunoreactive cells were detected in the CNS of rats during prenatal stages. A few of CTGF-positive cells were detected in the early postnatal stage. However, the positive cells increased gradually in later stages. CTGF-immunoreactive cells widely distributed in the CNS of rats in the first 30 to 60 days postnatally, and the density of immunoreactive products was the highest in these days. The number and staining intensity of CTGF-positive cells decreased and their area of distribution diminished gradually with age. The positive cells included neurons mainly located in the cingulate cortex,striatum, hippocampus, hypothalamus and cerebellum, and astrocytes in white matter of the spinal cord and ependymal cells of the brain. Most of CTGF-immunoreactive cells were quite big in size with a long process. Conclusion: CTGF-immunoreactive cells were found in the CNS of rats, and their numbers and positive signal decreased with the age.

  3. Target organ localization of memory CD4(+) T cells in patients with chronic beryllium disease.

    Science.gov (United States)

    Fontenot, Andrew P; Canavera, Scott J; Gharavi, Laia; Newman, Lee S; Kotzin, Brian L

    2002-11-01

    Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace, and it remains an important public health concern. Evidence suggests that CD4(+) T cells play a critical role in the development of this disease. Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4(+) T cells in the lungs (bronchoalveolar lavage) of 12 CBD patients ranged from 1.4% to 29% (mean 17.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO(4)). Few, if any, beryllium-specific CD8(+) T cells were identified. In contrast, the frequency of beryllium-responsive CD4(+) T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells as detected by intracellular staining and lymphocyte proliferation in culture after BeSO(4) exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4(+) cells in the lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.

  4. Enhancement of radiation cytotoxicity in breast-cancer cells by localized attachment of gold nanoparticles.

    Science.gov (United States)

    Kong, Tao; Zeng, Jie; Wang, Xiaoping; Yang, Xiaoyan; Yang, Jing; McQuarrie, Steve; McEwan, Alexander; Roa, Wilson; Chen, Jie; Xing, James Z

    2008-09-01

    Gold nanoparticles (GNPs) and modified GNPs having two kinds of functional molecules, cysteamine (AET) and thioglucose (Glu), are synthesized. Cell uptake and radiation cytotoxicity enhancement in a breast-cancer cell line (MCF-7) versus a nonmalignant breast-cell line (MCF-10A) are studied. Transmission electron microscopy (TEM) results show that cancer cells take up functional Glu-GNPs significantly more than naked GNPs. The TEM results also indicate that AET-capped GNPs are mostly bound to the MCF-7 cell membrane, while Glu-GNPs enter the cells and are distributed in the cytoplasm. After MCF-7 cell uptake of Glu-GNPs, or binding of AET-GNPs, the in vitro cytotoxicity effects are observed at 24, 48, and 72 hours. The results show that these functional GNPs have little or no toxicity to these cells. To validate the enhanced killing effect on cancer cells, various forms of radiation are applied such as 200 kVp X-rays and gamma-rays, to the cells, both with and without functional GNPs. By comparison with irradiation alone, the results show that GNPs significantly enhance cancer killing.

  5. Local Microenvironment Controls the Compartmentalization of NK Cell Responses during Systemic Inflammation in Mice.

    Science.gov (United States)

    Rasid, Orhan; Ciulean, Ioana Sonya; Fitting, Catherine; Doyen, Noelle; Cavaillon, Jean-Marc

    2016-09-15

    Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell-intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo-transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell-intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner.

  6. 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

  7. Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells.

    Science.gov (United States)

    Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E; Bringmann, Martin; Lindeboom, Jelmer; Emons, Anne-Mie; Samuels, Lacey; Ketelaar, Tijs; Ehrhardt, David W; Persson, Staffan

    2013-06-01

    The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

  8. Local Imaging of Optoelectronic Properties and Film Degradation in Polymer/Fullerene Solar Cells with Electrostatic Force Microscopy

    Science.gov (United States)

    Cox, Phillip Alexander

    With power conversion efficiencies on the rise, organic photovoltaics (OPVs) hold promise as a next-generation thin-film solar technology. However, both device performance and stability are inextricably linked to local film structure. Methods capable of probing nanoscale electronic properties as a function of film structure are thus a crucial component of the rational design of efficient and robust devices. This dissertation describes the use of three scanning probe methods for studying local charge generation and photodegradation in polymer/fullerene solar cells. First, we show that time-resolved electrostatic force microscopy (trEFM) is capable of resolving local photocurrent from sub-bandgap excitation down to attoampere level currents, a result unattainable by traditional contact-mode methods. We find that the local charging rates measured with trEFM are proportional to external quantum efficiency (EQE) measurements made on completed devices, making trEFM images equivalent to local EQE maps across the entire solar spectrum. For both phase-segregated and well-mixed MDMO-PPV:PCBM film morphologies, we show that the local distribution of photocurrent is invariant to excitation wavelength, providing local evidence for the controversial result that the probability of generating separated charge carriers does not depend on whether excitons are formed at the singlet state or charge transfer state. Next, we describe how local dissipation imaging can be performed with commercially-available frequency-modulated electrostatic force microscopy (FM-EFM) and show that dissipation maps are highly sensitive to photo-oxidative effects in organic semiconductors. We show that photo-oxidation induced changes in cantilever energy dissipation are proportional to device performance losses. We further develop dissipation imaging by implementing ringdown imaging, which directly measures the quality factor of the cantilever, enabling quantitative dissipation mapping. Using organic

  9. Gold Nanorod-Mediated Photothermal Modulation for Localized Ablation of Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yoochan Hong

    2012-01-01

    Full Text Available We estimated the photothermal transduction efficiency of gold nanorod (GNR solutions for different GNR concentrations and irradiation laser power. In particular, we verified that the degree of cell death area could be modulated by GNR concentration and irradiation laser power. The efficacy of GNR-produced photothermal ablation of cancer cells was evaluated by irradiating GNRs in the presence of MDA-MB-231 breast cancer cells with a near-infrared (NIR laser at different laser power densities and irradiation times. GNR-induced photothermal ablation was applied successfully to cancer cells at various NIR laser power densities and irradiation times and was characterized with live-dead cell staining. Through these techniques, we established the system for not only verification of induced photothermal effect using NIR laser and thermocouple, but also identification of uptake efficiency for GNRs and cell viability using dark field and fluorescence imaging, respectively.

  10. Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.

    OpenAIRE

    Patton, S E; Gupta, R. P.; Nishio, S.; Eddy, E M; Jetten, A. M.; Plopper, C. G.; Nettesheim, P; Hook, G E

    1991-01-01

    Highly purified Clara cells (93 +/- 3%) isolated from the lungs of rabbits were used to produce an antiserum against Clara cell secretory proteins. This antiserum was used to identify and study the biosynthesis and secretion of [35S]methionine-labeled proteins from isolated Clara cells. The antiserum recognized one major secretory protein with apparent molecular weight of 6 kDa and reacted weakly with a higher molecular weight protein of about 180 kDa. Biosynthesis and secretion of these prot...

  11. Low-power, Confocal Imaging of Protein Localization in Living Cells (7214-150) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed technology genetically labels intracellular structures and visualizes protein interactions in living cells using a compact, confocal microscope with...

  12. Locally-Delivered T-Cell-Derived Cellular Vehicles Efficiently Track and Deliver Adenovirus Delta24-RGD to Infiltrating Glioma

    Directory of Open Access Journals (Sweden)

    Rutger K. Balvers

    2014-08-01

    Full Text Available Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs, which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007. Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.

  13. Immunohistochemical localization of inflammatory cells and cell cycle proteins in the gills of Loma salmonae infected rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Powell, M. D.; Yousaf, M. N.; Rasmussen, Karina Juhl

    2014-01-01

    Microsporidial gill diseases particularly those caused by Loma salmonae incur significant economic losses to the salmonid aquaculture industry. The gill responses to infection include the formation of xenomas and the acute hyperplastic inflammatory responses once the xenomas rupture releasing...... infective spores. The aim of this work was to characterize the inflammatory responses of the gill to both the presence of the xenomas as well as the hyperplasia associated with L salmonae infection in the rainbow trout gill following an experimental infection using immunohistochemistry. Hyperplastic lesions...... demonstrated numerous cells expressing PCNA as well as an apparent increased expression of caspase-3 and number of apoptotic cells (TUNEL positive cells). There was an expression of TNF alpha in individual cells within the gill and increased expression of a myeloid cell line antigen indicating the presence...

  14. VINDESINE WITH CYCLOPHOSPHAMIDE-EPIRUBICIN-CISPLATIN IN THE TREATMENT LOCALLY ADVANCED NON-SMALL CELL LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    HU Yan-ping; KE Yu-hua; FU Xiao-yu

    1999-01-01

    Objective: To evaluate the addition of vindesine to a cyclophosphamide-epirubicin-cisplatin (CAP) regimen for treating the patients with locally advanced non-small cell lung cancer (NSCLC). Methods: From May 1994to August 1998, 59 previously untreated patients with stage Ⅲa and Ⅲb non-small cell lung cancer were enrolled into this trial. Patients characteristics were the following: the median age was 52 years; the median performance status was 1; there were 19 stage Ⅲa and 40 stage Ⅲb; there were 47 adenocarcinoma, 10squamous cell carcinoma and 2 large cell carcinoma. All patients were treated with vindesine (2 mg/m2, on day 1and day 8), cyclophosphamide (0.6/m2, on day 1),epirubicin (40 mg/m2, on day 1) and cisplatin (60 mg/m2,on day 1) every 3 or 4 weeks. Results: Four achieved a complete response (6.8%), 29 achieved a partial response (49.2%), 15 had stable disease, and 10 had progressive disease. A clinical improvement was in 45 of 59 patients (76.3%). The most frequent major toxic effects were myelosuppression, nausea and vomiting.Conclusion: The vindesine with CAP regimen was active combination chemotherapy in patients with locally advanced NSCLC accompanied by the limited side effects.

  15. AtPGL3 is an Arabidopsis BURP domain protein that is localized to the cell wall and promotes cell enlargement

    Directory of Open Access Journals (Sweden)

    Jiyoung ePark

    2015-06-01

    Full Text Available The BURP domain is a plant-specific protein domain that has been identified in secretory proteins, and some of these are involved in cell wall remodeling. Among Arabidopsis BURP domain proteins, three proteins exhibit strong amino acid similarities with the tomato polygalacturonase 1 beta (PG1β protein that interacts with a pectin-digesting enzyme. To investigate biological roles of the Arabidopsis PG1β-like proteins (AtPGLs, we generated Arabidopsis lines in which expression of AtPGLs is altered. Among the three AtPGLs, AtPGL3 exhibited highest transcriptional activity throughout all developmental stages. When tissue-specific expression pattern of AtPGL3 was examined, the gene was observed to be active in epidermal cell layers of rosette leaves and in the trichomes. AtPGL triple mutant plants were smaller than wild type plants because cells were smaller in the mutant plants. Interestingly, when we overexpressed AtPGL3 using a 35S promoter, cells in transgenic plants grew larger than those of the wild type, suggesting that AtPGL3 plays a role in cell expansion. A C-terminal GFP fusion protein of AtPGL3 complemented phenotypes of the triple mutant plants and localized to the cell wall. A truncated AtPGL3-GFP fusion protein that lacks the BURP domain failed to rescue the mutant phenotypes even though the GFP protein was targeted to the cell wall, indicating that the BURP domain is required for its effect on cell expansion. Quantitative RT-PCR and immunoblot analyses indicated that 2 α-expansin genes are down-regulated and up-regulated in the triple mutant and overexpressor lines, respectively. Taken together, AtPGL3 is a cell wall protein required for normal cell expansion and the coexpression results suggest that AtPGLs regulate cell wall loosening, in conjunction with α-expansins, to promote cell growth.

  16. 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

  17. Truncated SSX protein suppresses synovial sarcoma cell proliferation by inhibiting the localization of SS18-SSX fusion protein.

    Directory of Open Access Journals (Sweden)

    Yasushi Yoneda

    Full Text Available Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18 (p11.2;q11.2 is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18 and the truncated SSX moiety (tSSX of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.

  18. A novel human gene spindlin1,encoding a protein localized in the cell nucleus and inducing NIH3T3 cell's transformation

    Institute of Scientific and Technical Information of China (English)

    GAO Yanhong; QIN Lipeng; ZHANG Peng; CHEN Lin; YUAN Hongfeng; BAI Cixian; YAN Fang; YUE Wen; PEI Xuetao

    2004-01-01

    A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.

  19. 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...

  20. Autonomous Bacterial Localization and Gene Expression Based on Nearby Cell Receptor Density

    Science.gov (United States)

    2013-01-22

    Upon detection of B1–5 mM AI-2, these cells express T7 polymerase that amplifies the native lsr operon response by overexpressing DsRed (see...2 for initiating gene expression (lsr operon ). (B) Indicated densities of PCI-15B or HEK293 cells were seeded to wells followed by mouse anti-EGFR

  1. Treatment Advances in Locally Advanced and Metastatic Non-Small Cell Lung Cancer

    NARCIS (Netherlands)

    V.M.F. Surmont (Veerle)

    2010-01-01

    textabstractLung cancer is the leading cause of cancer mortality in the United States and Europe. Approximately 85% of the patients with lung cancer have non–small cell lung cancer (NSCLC), which can be classified into squamous, adeno, large cell and not otherwise specified (NOS) histologies. The mo

  2. Co-localization of neural cell adhesion molecule and fibroblast growth factor receptor 2 in early embryo development.

    Science.gov (United States)

    Vesterlund, Liselotte; Töhönen, Virpi; Hovatta, Outi; Kere, Juha

    2011-01-01

    During development there is a multitude of signaling events governing the assembly of the developing organism. Receptors for signaling molecules such as fibroblast growth factor receptor 2 (FGFR2) enable the embryo to communicate with the surrounding environment and activate downstream pathways. The neural cell adhesion molecule (NCAM) was first characterized as a cell adhesion molecule highly expressed in the nervous system, but recent studies have shown that it is also a signaling receptor. Using a novel single oocyte adaptation of the proximity ligation assay, we here show a close association between NCAM and FGFR2 in mouse oocytes and 2-cell embryos. Real-time PCR analyses revealed the presence of messenger RNA encoding key proteins in downstream signaling pathways in oocytes and early mouse embryos. In summary these findings show a co-localization of NCAM and FGFR2 in early vertebrate development with intracellular signaling pathways present to enable a cellular response.

  3. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Hösli, E; Belhage, B;

    1991-01-01

    . At the light microscope level specific staining of GABAA-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling...... of GABAA-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations...... to be performed. Quantification of the labeling intensity revealed a higher concentration of GABAA-receptors per microns plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABAA-receptors was observed. No specific labeling of GABAA-receptors was observed...

  4. Estrous cycle-dependent neural plasticity in the caudal brainstem in the female golden hamster: ultrastructural and immunocytochemical studies of axo-dendritic relationships and dynamic remodeling.

    NARCIS (Netherlands)

    Gerrits, P.O.; Kortekaas, R.; Veening, J.G.; Weerd, H. de; Algra, A.; Mouton, L.J.; Want, J.J. van der

    2008-01-01

    During the short four-day estrous cycle of the female hamster various behavioral (lordosis, vocalization and aggression) and autonomic adaptations occur. Presumably, these changes are under ovarian control. Recently, we described a distinct estrogen receptor-alpha immunoreactive (ER-alpha-IR) cell g

  5. Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

    DEFF Research Database (Denmark)

    Jakobsen, Kristine Raaby; Sørensen, Emilie; Brøndum, Karin Kathrine;

    2013-01-01

    Background Protrusions of cancer cells conferrers a vital function for cell migration and metastasis. Protein and RNA localization mechanisms have been extensively examined and shown to play pivotal roles for the functional presence of specific protein components in cancer cell protrusions. Methods...... To describe genome wide RNA localized in protrusions of the metastatic human breast cancer cell line MDA-MB-231 we used Boyden chamber based methodology followed by direct mRNA sequencing. Results In the hereby identified group of protrusion localized mRNA some previously were described to be localized...... exemplified by mRNA for Ras-Related protein 13 (RAB13) and p0071 (Plakophilin-4/PKP4). For other transcripts, exemplified by mRNA for SH3PXD2A/TKS5 and PPFIA1/Liprin-α1, only the corresponding proteins previously were described to have protrusion localization. Finally, a cohort of MDA-MB-231 protrusion...

  6. 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...

  7. Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 1. Overview and models for probes of structure, properties and function in single cells.

    Science.gov (United States)

    Horobin, R W; Rashid-Doubell, F; Pediani, J D; Milligan, G

    2013-11-01

    Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization. The mechanisms of many such selective accumulations, however, now are known. Moreover, the influence of physicochemical properties of probes on their uptake and localization often can be defined numerically, hence predicted, using quantitative structure activity relations (QSAR) models with its required numerical structure parameters (or "descriptors"). The state of the art of this approach is described. Available QSAR models are summarized for uptake into cells and localization in the cytosol, endoplasmic reticulum, generic biomembranes, Golgi apparatus, lipid droplets, lysosomes/endosomes, mitochondria, eukaryotic nuclei (histones and DNA), plasma membrane, and ribosomal RNA (cytoplasmic and nucleolar). Integration of such core models to both aid understanding and troubleshooting of current fluorescent probes and to assist the design of novel probes is outlined and illustrated using case examples. Limitations and generic problems arising with this approach and comments on application of such approaches to xenobiotics other than probes, e.g., drugs and herbicides, together with a brief note about an alternative approach to prediction, are given.

  8. 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.

  9. Comparative two- and three-dimensional analysis of nanoparticle localization in different cell types by Raman spectroscopic imaging

    Science.gov (United States)

    Bräutigam, Katharina; Bocklitz, Thomas; Silge, Anja; Dierker, Christian; Ossig, Rainer; Schnekenburger, Jürgen; Cialla, Dana; Rösch, Petra; Popp, Jürgen

    2014-09-01

    The increasing production and application of engineered nanomaterials requires a detailed understanding of the potential toxicity of nanoparticles and their uptake in living cells and tissue. For that purpose, a highly sensitive and selective method for detecting single nonlabeled nanoparticles and nanoparticle agglomerations in cells and animal tissue is required. Here, we show that Raman microspectroscopy allows for the specific detection of TiO2 nanoparticles inside cultured NIH/3T3 fibroblasts and RAW 264.7 macrophages. The spatial position of TiO2 nanoparticles and in parallel the relative intracellular concentration and distribution of cellular constituents such as proteins or DNA residues were identified and displayed by construction of two- and three-dimensional Raman maps. The resulting Raman images reflected the significant differences in nanoparticle uptake and intracellular storage of fibroblasts and macrophages. Furthermore, TiO2 nanomaterials could be characterized and the presence of rutile- and anatase-phase TiO2 were determined inside cells. Together, the data shown here prove that Raman spectroscopic imaging is a promising technique for studying the interaction of nanomaterials with living cells and for differentiating intracellular nanoparticles from those localized on the cell membrane. The technology provides a label-free, non-destructive, material-specific analysis of whole cells with high spatial resolution, along with additional information on the current status of the material properties.

  10. Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry

    Indian Academy of Sciences (India)

    Donghua Liu; Ingrid Kottke

    2004-09-01

    The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.

  11. 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.

  12. Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.

    Science.gov (United States)

    Nakajima, W; Hicks, M A; Tanaka, N; Krystal, G W; Harada, H

    2014-02-13

    The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

  13. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  14. Strategies of dose escalation in the treatment of locally advanced non-small cell lung cancer: image guidance and beyond

    Directory of Open Access Journals (Sweden)

    Alexander eChi

    2014-06-01

    Full Text Available Radiation dose in the setting of chemo-radiation for locally advanced non-small cell lung cancer (NSCLC has been historically limited by the risk of normal tissue toxicity and this has been hypothesized to correlate with the poor results in regard to local tumor recurrences. Dose escalation, as a means to improve local control, with concurrent chemotherapy has been shown to be feasible with three-dimensional conformal radiotherapy in early phase studies with good clinical outcome. However, the potential superiority of moderate dose escalation to 74 Gy has not been shown in phase III randomized studies. In this review, the limitations in target volume definition in previous studies; and the factors that may be critical to safe dose escalation in the treatment of locally advanced NSCLC, such as respiratory motion management, image guidance, intensity modulation, FDG-PET incorporation in the treatment planning process, and adaptive radiotherapy, are discussed. These factors, along with novel treatment approaches that have emerged in recent years, are proposed to warrant further investigation in future trials in a more comprehensive and integrated fashion.

  15. 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.

  16. Acute pain induces an instant increase in natural killer cell cytotoxicity in humans and this response is abolished by local anaesthesia

    DEFF Research Database (Denmark)

    Greisen, J.; Hokland, Marianne; Grøfte, Thorbjørn

    1999-01-01

    to abdominal skin for 30 min to an intensity of 8 on a visual analogue scale (0-10). Next, the electric intensity profile was reproduced during local anaesthesia (mepivacaine 10 mg ml-1 s.c. to a total dose of 2.5 mg kg-1). NK cell cytotoxicity was measured using a 4-h 51Cr-release assay against K562 target...... not change either NK cell activity or number. Parallel and significant increases in concentrations of plasma epinephrine and serum cortisol were observed. These changes were abolished by local anaesthesia. We conclude that acute severe pain without tissue injury markedly increased NK cell cytotoxicity. Local...

  17. The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

    Science.gov (United States)

    Takahashi, Noriyuki; Tarumi, Wataru; Itoh, Masanori T; Ishizuka, Bunpei

    2015-12-01

    Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

  18. Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells.

    Science.gov (United States)

    Perry, Jacob L; Ramachandran, Nina K; Utama, Budi; Hyser, Joseph M

    2015-11-15

    Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

  19. Localization of NOS-like protein in guard cells of Vicia faba L. And its possible function

    Institute of Scientific and Technical Information of China (English)

    LIU Xin; WANG YouQun; JIA WenSuo; LOU ChengHou; ZHANG ShuQiu

    2007-01-01

    Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of nitric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS inhibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO generation from NOS pathway is the main pathway in the stress and JA responses. The pharmacological experiment showed that increasing the Ca2+ at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2+-dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.

  20. U.S. Food and Drug Administration approval: vismodegib for recurrent, locally advanced, or metastatic basal cell carcinoma.

    Science.gov (United States)

    Axelson, Michael; Liu, Ke; Jiang, Xiaoping; He, Kun; Wang, Jian; Zhao, Hong; Kufrin, Dubravka; Palmby, Todd; Dong, Zedong; Russell, Anne Marie; Miksinski, Sarah; Keegan, Patricia; Pazdur, Richard

    2013-05-01

    The data and regulatory considerations leading to the U.S. Food and Drug Administration (FDA) January 30, 2012 approval of Erivedge (vismodegib) capsules for the treatment of patients with recurrent, locally advanced, or metastatic basal cell carcinoma (BCC) are described. The FDA's approval decision was based primarily on the results observed in a single-arm, parallel cohort, international trial of vismodegib, administered orally at 150 mg daily until disease progression, in patients with pathologically confirmed, recurrent, locally advanced basal cell carcinoma (laBCC) or metastatic basal cell carcinoma (mBCC). An independent review committee confirmed an overall response rate (ORR) of 30.3% [95% confidence interval (CI): 15.6-48.2] in 33 patients with mBCC and an ORR of 42.9% (95% CI: 30.5-56.0) in 63 patients with laBCC; median response durations were 7.6 months and 7.6 months for patients with mBCC and laBCC, respectively. The most common adverse reactions were muscle spasms, alopecia, dysgeusia, weight loss, fatigue, nausea, diarrhea, decreased appetite, constipation, cough, arthralgias, vomiting, headache, ageusia, insomnia, and upper respiratory tract infection. Animal toxicology studies confirmed that vismodegib is a potent teratogenic agent. Approval was based on durable objective tumor responses supported by knowledge of the pathologic role of Hedgehog signaling in BCC and acceptable toxicity in a population without effective alternative therapies.

  1. 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.

  2. 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.

  3. Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice.

    Science.gov (United States)

    Ward, J. M.; Singh, G.; Katyal, S. L.; Anderson, L. M.; Kovatch, R. M.

    1985-01-01

    The localization of surfactant apoprotein (SAP) and the Clara cell antigen(s) (CCA) was studied in naturally occurring and experimentally induced pulmonary hyperplasias and neoplasms by avidin-biotin peroxidase complex (ABC) immunocytochemistry. Lungs of B6C3F1 and A strain mice with naturally occurring lesions, B6C3F1 mice given injections of N-nitrosodiethylamine (DEN), BALB/c nu/nu or nu/+ mice exposed transplacentally on Day 16 of gestation to ethylnitrosourea (ENU), or BALB/c nu/+ mice exposed to ENU at 8-12 weeks of age were preserved in formalin or Bouin's fixative. After ABC immunocytochemistry, SAP was found in the cytoplasm of normal alveolar Type II cells; in the majority of cells in focal alveolar and solid hyperplasias originating in peribronchiolar or peripheral locations; and in solid, tubular, papillary, and mixed adenomas and carcinomas. The larger mixed-pattern neoplasms and small or large tubular neoplasms usually had the least number of cells with SAP. The majority of large papillary adenomas and carcinomas in BALB/c mice exposed to ENU and in untreated A strain mice contained SAP in the nuclei of many neoplastic cells but only in the cytoplasm of a few neoplastic cells. CCA was found in normal Clara cells of bronchi and bronchioles but not in any hyperplastic or neoplastic lesion of any mouse studied. This study provided immunocytochemical evidence that the vast majority of naturally occurring and experimentally induced pulmonary neoplasms of mice are alveolar Type II cell adenomas and carcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:3883798

  4. Localization of the contacts between Kenyon cells and aminergic neurons in the Drosophila melanogaster brain using SplitGFP reconstitution.

    Science.gov (United States)

    Pech, Ulrike; Pooryasin, Atefeh; Birman, Serge; Fiala, André

    2013-12-01

    The mushroom body of the insect brain represents a neuronal circuit involved in the control of adaptive behavior, e.g., associative learning. Its function relies on the modulation of Kenyon cell activity or synaptic transmitter release by biogenic amines, e.g., octopamine, dopamine, or serotonin. Therefore, for a comprehensive understanding of the mushroom body, it is of interest not only to determine which modulatory neurons interact with Kenyon cells but also to pinpoint where exactly in the mushroom body they do so. To accomplish the latter, we made use of the GRASP technique and created transgenic Drosophila melanogaster that carry one part of a membrane-bound splitGFP in Kenyon cells, along with a cytosolic red fluorescent marker. The second part of the splitGFP is expressed in distinct neuronal populations using cell-specific Gal4 drivers. GFP is reconstituted only if these neurons interact with Kenyon cells in close proximity, which, in combination with two-photon microscopy, provides a very high spatial resolution. We characterize spatially and microstructurally distinct contact regions between Kenyon cells and dopaminergic, serotonergic, and octopaminergic/tyraminergic neurons in all subdivisions of the mushroom body. Subpopulations of dopaminergic neurons contact complementary lobe regions densely. Octopaminergic/tyraminergic neurons contact Kenyon cells sparsely and are restricted mainly to the calyx, the α'-lobes, and the γ-lobes. Contacts of Kenyon cells with serotonergic neurons are heterogeneously distributed over the entire mushroom body. In summary, the technique enables us to localize precisely a segmentation of the mushroom body by differential contacts with aminergic neurons.

  5. Expression and localization of inwardly rectifying potassium channel Kit2.1 in glia cells of native bovine retina

    Institute of Scientific and Technical Information of China (English)

    PAN Ai-hua; LUO Xue-gang

    2005-01-01

    Objective The purpose of this study is to identify the molecular basis of the contacting -neuron membrane K+ conductance in glia cells of native bovine retina. Methods RT-PCR, Northern blot and Western blot analyses were used to detect the expression of the inwardly rectifying K+ (Kir) channel subunits Kir2.1 in native bovine RPE and neural retina. The distribution of Kir2.1 protein was determined in frozen sections of bovine retina-RPEchoroid by indirect immunofluorescence analysis. Results RT-PCR analysis reveals Kir2.1 transcript in both RPE and neural retina. In Northern blots, Kir2.1 probe hybridizes to an appropriately sized-transcript in neural retina but not in RPE. In Western blots, Kir2.1 antibody recognizes a major monomer of about 60 kDa in neural retina but not in RPE. Immunofluorescence reveals that Kir2.1 immunostaining is expressed at many parts of Muller cells, especially in the membrane domains of Muller cells that contact retinal neurons, i. e. , along the two stem processes,over the soma, and in the side branches extending into the synaptic layers. No immunostaining is seen in RPE. Doubling staining shows that Kir2.1 proteins and glutamine synthetase proteins which are a marker of Muller cell co-localized well. Conclusions These results reveal that Kir2.1 is localized in the Muller cells, no Kir2.1 in RPE. These data suggests that Kir2.1 may be involved in the transport of K+ in the bovine neural retina.

  6. Expression and Subcellular Localization of Recombinant SDF-1 and Its Mutant Intrakine in Transfected COS-7 Cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-yuan; GUO Zhi-gang; TAN-yi; MA Wei-feng; CAI Shao-xi; DU-jun; CAI Shao-hui

    2006-01-01

    Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-1/54 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein. This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface. Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-Dec/PET-30a(+)with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 /SDF-1/KDEL, pcDNA3.1 /SDF-1/54/KDEL, pEGFP/SDF-1/KDEL and pEGFP/SDF-1/54/KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate, they were transferred into COS-7 cells with liposome. The exogenous expressions were observed, fusion protein SDF-1/His and SDF-1/54/His were confirmed by Western blot, and the SDF-1/EGFP and SDF-1/54/EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1/KDEL/His and SDF-1/54 KDEL/His expressed in COS-7 cells. Subcelluar localization analysis showed that SDF-1/KDEL/EGFP and SDF-1/54/KDEL/EGFP were located mainly in endoplasmic reticulum. Conclusion: Four expression vectors pcDNA3. 1/ SDF -1/KDEL, pcDNA3.1/SDF - 1/54/KDEL, pEGFP/SDF - 1/KDEL and pEGFP/SDF-1/54/KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum.

  7. 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.

  8. Th2-Associated Alternative Kupffer Cell Activation Promotes Liver Fibrosis without Inducing Local Inflammation

    Science.gov (United States)

    López-Navarrete, Giuliana; Ramos-Martínez, Espiridión; Suárez-Álvarez, Karina; Aguirre-García, Jesús; Ledezma-Soto, Yadira; León-Cabrera, Sonia; Gudiño-Zayas, Marco; Guzmán, Carolina; Gutiérrez-Reyes, Gabriela; Hernández-Ruíz, Joselín; Camacho-Arroyo, Ignacio; Robles-Díaz, Guillermo; Kershenobich, David; Terrazas, Luis I.; Escobedo, Galileo

    2011-01-01

    Cirrhosis is the final outcome of liver fibrosis. Kupffer cell-mediated hepatic inflammation is considered to aggravate liver injury and fibrosis. Alternatively-activated macrophages are able to control chronic inflammatory events and trigger wound healing processes. Nevertheless, the role of alternative Kupffer cell activation in liver harm is largely unclear. Thus, we evaluated the participation of alternatively-activated Kupffer cells during liver inflammation and fibrosis in the murine model of carbon tetrachloride-induced hepatic damage. To stimulate alternative activation in Kupffer cells, 20 Taenia crassiceps (Tc) larvae were inoculated into BALBc/AnN female mice. Six weeks post-inoculation, carbon tetrachloride or olive oil were orally administered to Tc-inoculated and non-inoculated mice twice per week during other six weeks. The initial exposure of animals to T. crassiceps resulted in high serum concentrations of IL-4 accompanied by a significant increase in the hepatic mRNA levels of Ym-1, with no alteration in iNOS expression. In response to carbon tetrachloride, recruitment of inflammatory cell populations into the hepatic parenchyma was 5-fold higher in non-inoculated animals than Tc-inoculated mice. In contrast, carbon tetrachloride-induced liver fibrosis was significantly less in non-inoculated animals than in the Tc-inoculated group. The latter showed elevated IL-4 serum levels and low IFN-γ concentrations during the whole experiment, associated with hepatic expression of IL-4, TGF-β, desmin and α-sma, as well as increased mRNA levels of Arg-1, Ym-1, FIZZ-1 and MMR in Kupffer cells. These results suggest that alternative Kupffer cell activation is favored in a Th2 microenvironment, whereby such liver resident macrophages could exhibit a dichotomic role during chronic hepatic damage, being involved in attenuation of the inflammatory response but at the same time exacerbation of liver fibrosis. PMID:22110380

  9. Th2-Associated Alternative Kupffer Cell Activation Promotes Liver Fibrosis without Inducing Local Inflammation

    Directory of Open Access Journals (Sweden)

    Giuliana López-Navarrete, Espiridión Ramos-Martínez, Karina Suárez-Álvarez, Jesús Aguirre-García, Yadira Ledezma-Soto, Sonia León-Cabrera, Marco Gudiño-Zayas, Carolina Guzmán, Gabriela Gutiérrez-Reyes

    2011-01-01

    Full Text Available Cirrhosis is the final outcome of liver fibrosis. Kupffer cell-mediated hepatic inflammation is considered to aggravate liver injury and fibrosis. Alternatively-activated macrophages are able to control chronic inflammatory events and trigger wound healing processes. Nevertheless, the role of alternative Kupffer cell activation in liver harm is largely unclear. Thus, we evaluated the participation of alternatively-activated Kupffer cells during liver inflammation and fibrosis in the murine model of carbon tetrachloride-induced hepatic damage. To stimulate alternative activation in Kupffer cells, 20 Taenia crassiceps (Tc larvae were inoculated into BALBc/AnN female mice. Six weeks post-inoculation, carbon tetrachloride or olive oil were orally administered to Tc-inoculated and non-inoculated mice twice per week during other six weeks. The initial exposure of animals to T. crassiceps resulted in high serum concentrations of IL-4 accompanied by a significant increase in the hepatic mRNA levels of Ym-1, with no alteration in iNOS expression. In response to carbon tetrachloride, recruitment of inflammatory cell populations into the hepatic parenchyma was 5-fold higher in non-inoculated animals than Tc-inoculated mice. In contrast, carbon tetrachloride-induced liver fibrosis was significantly less in non-inoculated animals than in the Tc-inoculated group. The latter showed elevated IL-4 serum levels and low IFN-γ concentrations during the whole experiment, associated with hepatic expression of IL-4, TGF-β, desmin and α-sma, as well as increased mRNA levels of Arg-1, Ym-1, FIZZ-1 and MMR in Kupffer cells. These results suggest that alternative Kupffer cell activation is favored in a Th2 microenvironment, whereby such liver resident macrophages could exhibit a dichotomic role during chronic hepatic damage, being involved in attenuation of the inflammatory response but at the same time exacerbation of liver fibrosis.

  10. 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.

  11. Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus.

    Science.gov (United States)

    Kühn, Juliane; Briegel, Ariane; Mörschel, Erhard; Kahnt, Jörg; Leser, Katja; Wick, Stephanie; Jensen, Grant J; Thanbichler, Martin

    2010-01-20

    The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.

  12. 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

  13. Expression of IL-4 and IL-13 predicts recurrence and survival in localized clear-cell renal cell carcinoma.

    Science.gov (United States)

    Chang, Yuan; Xu, Le; An, Huimin; Fu, Qiang; Chen, Lian; Lin, Zongming; Xu, Jiejie

    2015-01-01

    Interleukin-4 (IL-4) and IL-13 are anti-inflammatory and immunoregulatory cytokines that can influence cancer-directed immunosurveillance. However, they are not evaluated as biomarkers for ccRCC outcomes. The aim of this study was to investigate the prognostic value of tumor-derived IL-4 and IL-13 in patients with localized ccRCC after surgery. Our study comprised 194 consecutive patients with localized ccRCC undergoing nephrectomy in a single center. Clinical characteristics, recurrence-free survival (RFS) and overall survival (OS) were recorded. We assessed IL-4 and IL-13 expression as continuous variables and dichotomized as low versus high by immunohistochemistry. For associations with RFS and OS, we used the Kaplan-Meier method and Cox regression models. Concordance index was calculated for predictive accuracy. We found that high expression levels of IL-4 and IL-13 were associated with increased recurrence (P IL-13 expression (IL-4/IL-13 signature) was an independent prognostic factor for RFS and OS (P = 0.009 and P = 0.016, respectively). When applied to UISS score, IL-4/IL-13 signature improved the predictive accuracy. Notably, this improvement in prediction was mainly observed in patients with low-risk disease. To conclude, IL-4/IL-13 signature is an independent predictor of outcomes in patients with localized ccRCC, and the prognostic value is more prominent among patients with low-risk disease. Evaluation of IL-4 and IL-13 expression provides the opportunity to optimize postsurgical management and develop novel targeted therapies for ccRCC patients.

  14. Tissue specific localization of root infection by fungal pathogens: role of root border cells.

    Science.gov (United States)

    Gunawardena, Uvini; Hawes, Martha C

    2002-11-01

    When roots of pea seedlings were inoculated uniformly with spores of Nectria haematocca or other pea pathogenic fungi, more than 90% developed lesions in the region of elongation within 3 days. More mature regions of most roots as well as the tip showed no visible signs of infection. Yet, microscopic observation revealed that 'mantles,' comprised of fungal hyphae intermeshed with populations of border cells, covered the tips of most roots. After physical detachment of the mantle, the underlying tip of most roots was found to be free of infection. Mantle-covered root tips did not respond to invasion of their border cells by activation of known defense genes unless there was invasion of the tip itself, as revealed by the presence of a lesion. Concomitant with the activation of defense genes was the induction of a cell-wall degrading enzyme whose expression is a marker for renewed production of border cells. Mantle formation did not occur in response to nonpathogens. The data are consistent with the hypothesis that border cells serve as a host-specific 'decoy' that protects root meristems by inhibiting fungal infection of the root tip.

  15. CHI3L1 nuclear localization in monocyte derived dendritic cells.

    Science.gov (United States)

    Di Rosa, Michelino; Tibullo, Daniele; Saccone, Salvatore; Distefano, Gisella; Basile, Maria Sofia; Di Raimondo, Francesco; Malaguarnera, Lucia

    2016-02-01

    Chitinase-3-like-1 protein (CHI3L1) is a glycosyl hydrolase (GH) highly expressed in a variety of inflammatory diseases at infectious and non-infectious etiology. CHI3L1 is produced by a wide variety of cells including monocyte-derived macrophages cell lines such as polarized M1 and M2 type macrophages, osteoclasts and Kupffer cells. In this study we have examined the expression of CHI3L1 during the differentiation and maturation of dendritic cells. Magnetically-isolated peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs) through a combination of factors and cytokines. Our result showed, for the first time, that CHI3L1 is expressed during the process of differentiation and maturation of dendritic cells in time dependent manner. Furthermore, the CHI3L1 is evenly distributed in cytoplasm and in the nucleus of both the iDCs and mDCs. These results suggest that CHI3L1 may play crucial role in the DCs immunoresponse.

  16. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

    Science.gov (United States)

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A; Irudayaraj, Joseph M K

    2015-02-01

    Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

  17. New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

    Science.gov (United States)

    Vedyaykin, Alexey D; Vishnyakov, Innokentii E; Polinovskaya, Vasilisa S; Khodorkovskii, Mikhail A; Sabantsev, Anton V

    2016-06-01

    FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction.

  18. Development of the CAS-LIBB single-particle microbeam for localized irradiation of living cells

    Institute of Scientific and Technical Information of China (English)

    WANG Xufei; XU Mingliang; WU Lijun; WANG Shaohu; FENG Huiyun; ZHAN Furu; PENG Shixiang; HU Chundong; ZHANG Shuqing; CHENG Jianjun; SHI Zhongtao; WANG Xiaohua; YUAN Hang; YUAN Haitao; YU Zengliang; CHEN Lianyun; HU Zhiwen; LI Jun; WU Yu; CHEN Bin; HU Suhua; ZHANG Jun

    2004-01-01

    A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering (LIBB), Chinese Academy of Sciences (CAS). The system was designed to deliver a defined numbers of hydrogen ions, produced by a van de Graaff accelerator, in an energy range of 2.0-3.0 MeV, into an area smaller than that of the nucleus of an individual living cell. The beam is collimated by a borosilicate glass capillary that forms the beam-line exit. An integrated computer program recognizes the cells and locates them one by one over the microbeam exit for irradiation. We present technical details of the CAS-LIBB microbeam facility, particularly on the collimator, hardware, control program, as well as cell irradiation protocols available. Various factors contributing to the targeting and positioning precision are discussed along with accuracy measurement results.

  19. Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Ai Leen [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States); Department of Mechanical Engineering, Stanford University, Stanford, CA 94305 (United States)], E-mail: alkoh@stanford.edu; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P. [Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305 (United States); Sinclair, Robert [Department of Materials Science and Engineering, Stanford University, Durand Building Room 139, 496 Lomita Mall, Stanford, CA 94305 (United States); Stanford Nanocharacterization Laboratory, Stanford University, Stanford, CA 94305 (United States)

    2008-12-15

    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.

  20. Acute esophagitis for patients with local-regional advanced non small cell lung cancer treated with concurrent chemoradiotherapy

    DEFF Research Database (Denmark)

    Pan, Yi; Brink, Carsten; Knap, Marianne

    2016-01-01

    PURPOSE: Esophagitis is common in patients treated with definitive radiotherapy for local-regional advanced non small cell lung cancer (NSCLC). The purpose of this study was to estimate the dose-effect relationship using clinical and dosimetric parameters in patients receiving intensity modulated...... radiotherapy (IMRT) and concomitant chemotherapy (CCT). METHODS: Between 2009 and 2013, 117 patients with stages IIB-IIIB NSCLC were treated in a multicenter randomized phase II trial with 2 cycles of induction chemotherapy followed by IMRT and CCT. The esophagitis was prospectively scored using the Common...

  1. 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.

  2. Activated myeloid dendritic cells accumulate and co-localize with CD3+ T cells in coronary artery lesions in patients with Kawasaki disease.

    Science.gov (United States)

    Yilmaz, Atilla; Rowley, Anne; Schulte, Danica J; Doherty, Terence M; Schröder, Nicolas W J; Fishbein, Michael C; Kalelkar, Mitra; Cicha, Iwona; Schubert, Katja; Daniel, Werner G; Garlichs, Christoph D; Arditi, Moshe

    2007-08-01

    Emerging evidence implicating the participation of dendritic cells (DCs) and T cells in various vascular inflammatory diseases such as giant cell arteritis, Takayasu's arteritis, and atherosclerosis led us to hypothesize that they might also participate in the pathogenesis of coronary arteritis in Kawasaki disease (KD). Coronary artery specimens from 4 patients with KD and 6 control patients were obtained. Immunohistochemical and computer-assisted histomorphometric analyses were performed to detect all myeloid DCs (S-100(+), fascin(+)), all plasmacytoid DCs (CD123(+)) as well as specific DC subsets (mature myeloid DCs [CD83(+)], myeloid [BDCA-1(+)] and plasmacytoid DC precursors [BDCA-2(+)]), T cells (CD3(+)), and all antigen-presenting cells (HLA-DR(+)). Co-localization of DCs with T cells was assessed using double immunostaining. Significantly more myeloid DCs at a precursor, immature or mature stage were found in coronary lesions of KD patients than in controls. Myeloid DC precursors were distributed equally in the intima and adventitia. Mature myeloid DCs were particularly abundant in the adventitia. There was a significant correlation between mature DCs and HLA-DR expression. Double immunostaining demonstrated frequent contacts between myeloid DCs and T cells in the outer media and adventitia. Plasmacytoid DC precursors were rarely found in the adventitia. In conclusion, coronary artery lesions of KD patients contain increased numbers of mature myeloid DCs with high HLA-DR expression and frequent T cell contacts detected immunohistochemically. This suggests that mature arterial myeloid DCs might be activating T cells in situ and may be a significant factor in the pathogenesis of coronary arteritis in KD.

  3. Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry.

    Science.gov (United States)

    Azanza, M J; Pes, N; Pérez-Bruzón, R N; Aisa, J; Raso, M; Junquera, C; Lahoz, J M; Maestú, C; Martínez-Ciriano, C; Pérez-Castejón, C; Vera-Gil, A; Del Moral, A

    2007-05-01

    The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

  4. 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.

  5. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    Science.gov (United States)

    Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao

    2015-01-01

    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.

  6. Tunable Multifunctional Thermal Metamaterials: Manipulation of Local Heat Flux via Assembly of Unit-Cell Thermal Shifters

    Science.gov (United States)

    Park, Gwanwoo; Kang, Sunggu; Lee, Howon; Choi, Wonjoon

    2017-01-01

    Thermal metamaterials, designed by transformation thermodynamics are artificial structures that can actively control heat flux at a continuum scale. However, fabrication of them is very challenging because it requires a continuous change of thermal properties in materials, for one specific function. Herein, we introduce tunable thermal metamaterials that use the assembly of unit-cell thermal shifters for a remarkable enhancement in multifunctionality as well as manufacturability. Similar to the digitization of a two-dimensional image, designed thermal metamaterials by transformation thermodynamics are disassembled as unit-cells thermal shifters in tiny areas, representing discretized heat flux lines in local spots. The programmed-reassembly of thermal shifters inspired by LEGO enable the four significant functions of thermal metamaterials—shield, concentrator, diffuser, and rotator—in both simulation and experimental verification using finite element method and fabricated structures made from copper and PDMS. This work paves the way for overcoming the structural and functional limitations of thermal metamaterials. PMID:28106156

  7. Laser Fired Local Back Contact C-Si Solar Cells Using Phosphoric Acid for Back Surface Field

    Science.gov (United States)

    Balaji, Nagarajan; Park, Cheolmin; Ju, Minkyu; Lee, Seunghwan; Kim, Jungmo; Chung, Sungyoun; Raja, Jayapal; Yi, Junsin

    2015-04-01

    We report on a laser doping process for the formation of a local back surface field (BSF) using phosphoric acid (H3PO4) for n-type passivated emitter rear totally diffused silicon solar cells. The sheet resistance of the BSF layer was varied by changing the H3PO4 concentration. The BSF layer was passivated using SiN x . With the passivated BSF, the LBC solar cell shows an improved open circuit voltage. A laser power of 44 mW with 10 kHz resulted in a 45-Ω/sq BSF layer with effective lifetime of 290 μs and a higher V oc of 623 mV. With the optimized laser parameters, devices with the best electrical results yielded a short circuit current density of 36 mA/cm2 and an efficiency of 18.26%.

  8. Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mps1 and Bub1/BubR1. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubR1 and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mps1. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.

  9. Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.

  10. Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

    NARCIS (Netherlands)

    T. Yokomizo (Tomomasa); T. Yamada-Inagawa (Tomoko); A.D. Yzaguirre (Amanda); M.J. Chen (Michael); N.A. Speck (Nancy); E.A. Dzierzak (Elaine)

    2012-01-01

    textabstractWe describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development

  11. FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines

    Indian Academy of Sciences (India)

    Fan-Lun Liu; Ting-Yi Liu; Fan-Lu Kung

    2014-03-01

    One of the pathological hallmarks of Alzheimer’s disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer’s disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.

  12. STK31 is a cell-cycle regulated protein that contributes to the tumorigenicity of epithelial cancer cells.

    Directory of Open Access Journals (Sweden)

    Pao-Lin Kuo

    Full Text Available Serine/threonine kinase 31 (STK31 is one of the novel cancer/testis antigens for which its biological functions remain largely unclear. Here, we demonstrate that STK31 is overexpressed in many human colorectal cancer cell lines and tissues. STK31 co-localizes with pericentrin in the centrosomal region throughout all phases of the cell cycle. Interestingly, when cells undergo mitosis, STK31 also localizes to the centromeres, central spindle, and midbody. This localization behavior is similar to that of chromosomal passenger proteins, which are known to be the important players of the spindle assembly checkpoint. The expression of STK31 is cell cycle-dependent through the regulation of a putative D-box near its C-terminal region. Ectopically-expressed STK31-GFP increases cell migration and invasive ability without altering the proliferation rate of cancer cells, whereas the knockdown expression of endogenous STK31 by lentivirus-derived shRNA results in microtubule assembly defects that prolong the duration of mitosis and lead to apoptosis. Taken together, our results suggest that the aberrant expression of STK31 contributes to tumorigenicity in somatic cancer cells. STK31 might therefore act as a potential therapeutic target in human somatic cancers.

  13. Palliative Local Radiotherapy in the Treatment of Tumor-stage Cutaneous T-cell Lymphoma/Mycosis Fungoides

    Institute of Scientific and Technical Information of China (English)

    Chen-chen Xu; Tao Zhang; Tao Wang; Jie Liu; Yue-hua Liu

    2014-01-01

    Objective To determine the efficacy of palliative radiotherapy in treating tumor-stage cutaneous T-cell lymphoma/mycosis fungoides (MF). Methods From January 2008 to January 2013, a total of 11 patients with tumor-stage MF were treated with local radiation therapy in Peking Union Medical College Hospital. The median age of these patients was 53.36±14.45 years. Female-male ratio was 1:1.2. The average course of disease was 10.82±3.37 years. All the patients were treated with local electronic beam irradiation with a total median dosage of 48.55±9.51 (40-74) Gy in an average of 24.55±5.57 (20-40) fractions, 5 fractions per week. Results The median follow-up time was 55.27±29.3 (13-103) months. No severe acute or chronic side effects of irradiation were observed. Complete clinical response (CR) rate of the radiated sites was 54.5%(6/11), partial response (PR) rate was 36.4%(4/11), and the overall response rate (CR+PR) was 90.9%. One patient showed no response. Conclusion Local radiotherapy with psolaren plus ultraviolet A and/or interferon maintaining treatment is an effective palliative therapy in the treatment of tumor-stage MF patients.

  14. Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells.

    Directory of Open Access Journals (Sweden)

    Isabel Weinheimer

    Full Text Available Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae encodes a Class 1 RNase III endoribonuclease (RNase3 that suppresses post-transcriptional RNA interference (RNAi and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas. For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3 and RNA-dependent RNA polymerase 6 (RDR6. In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.

  15. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    Science.gov (United States)

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  16. Systemic localization of seven major types of carbohydrates on cell membranes by dSTORM imaging

    OpenAIRE

    Junling Chen; Jing Gao; Min Zhang; Mingjun Cai; Haijiao Xu; Junguang Jiang; Zhiyuan Tian; Hongda Wang

    2016-01-01

    Carbohydrates on the cell surface control intercellular interactions and play a vital role in various physiological processes. However, their systemic distribution patterns are poorly understood. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we systematically revealed that several types of representative carbohydrates are found in clustered states. Interestingly, the results from dual-color dSTORM imaging indicate that these carbohydrate clusters are prone...

  17. Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins.

    Science.gov (United States)

    Schofield, Whitman B; Lim, Hoong Chuin; Jacobs-Wagner, Christine

    2010-09-15

    What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.

  18. Local communication among mucosal immune cells in patients with celiac disease.

    Science.gov (United States)

    van Bergen, Jeroen; Mulder, Chris J; Mearin, M Luisa; Koning, Frits

    2015-05-01

    In patients with celiac disease, gluten consumption causes inflammation of the duodenum, and, to a lesser extent, the proximal jejunum. Immune-dominant gluten peptides are modified by the enzyme TG2, leading to their high-affinity binding to HLA-DQ2 or HLA-DQ8 molecules, present in people with a predisposition to celiac disease. Gluten peptide-loaded HLA-DQ2 or HLA-DQ8 molecules are recognized by highly conserved receptors on CD4(+) T cells in the lamina propria. B cells specific for TG2 and modified gluten peptides are also abundant in the lamina propria of patients with celiac disease. In the epithelium, interleukin-15 activates intraepithelial lymphocytes that promote destruction of epithelial cells. However, it is not clear how the immune responses in the lamina propria and the epithelium, separated by a basement membrane, are linked. We review the immune processes that occur in the lamina propria and their potential effects on epithelial pathology in celiac disease.

  19. Power electronics for local fuel cell/-battery plants; Leistungselektronik fuer dezentrale Brennstoffzellen/-Batterieanlagen

    Energy Technology Data Exchange (ETDEWEB)

    Krykunov, Oleksandr

    2009-10-13

    With their high efficiency and modular structure, fuel cells are an attractive option for decentral power supply. An important component of decentral power supply systems is the power-electronic control element for supply of electric power from the fuel cell to the three-phase electricity grid. Control elements can be constructed of a unidirectional DC/DC converter with a current inverter connnected in series. The investigation focused on the development of the DC/DC converter with minimum constructional and control requirements and optimum adaption of the DC/DC converter to the characteristics of the fuel cell. (orig.) [German] Die Brennstoffzelle stellt mit ihrem hohen Wirkungsgrad und ihrem modularen Aufbau eine attraktive Option fuer die Verwendung in einem dezentralen Energieversorgungssystem dar. Eine wichtige Komponente des dezentralen Energieversorgungssystems sind die leistungselektronischen Stellglieder fuer die Einspeisung der elektrischen Energie aus der Brennstoffzelle in das dreiphasige Netz. Die leistungselektronischen Stellglieder koennen aus einem undirektionalen DC/DC-Wandler und einem nachgeschalteten Wechselrichter realisiert werden. Die Entwicklung des DC/DC-Wandlers mit einem moeglichst geringeren Bauelemente- und Steuerungsaufwand fuer diese leistungselektronischen Stellglieder und die Anpassung des DC/DC-Wandlers an die Eigenschaften der Brennstoffzelle war das Ziel dieser Arbeit. (orig.)

  20. Calmodulin Involvement in Stress-Activated Nuclear Localization of Albumin in JB6 Epithelial Cells.

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Thomas J.; Negash, Sewite; Smallwood, Heather S.; Ramos, Kenneth S.; Thrall, Brian D.; Squier, Thomas C.

    2004-06-15

    We report that in response to oxidative stress, albumin is translocated to the nucleus where it binds in concert with known transcription factors to an antioxidant response element (ARE), which controls the expression of glutathione-S-transferase and other antioxidant enzymes, functioning to mediate adaptive cellular responses. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein, calmodulin (CaM), co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients, and associated increases in reactive oxygen species, contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin to mediate the transcriptional regulation of antioxidant responsive elements.

  1. Regulation of ErbB2 localization and function in breast cancer cells by ERM proteins

    Science.gov (United States)

    Asp, Nagham; Kvalvaag, Audun; Sandvig, Kirsten; Pust, Sascha

    2016-01-01

    The ERM protein family is implicated in processes such as signal transduction, protein trafficking, cell proliferation and migration. Consequently, dysregulation of ERM proteins has been described to correlate with carcinogenesis of different cancer types. However, the underlying mechanisms are poorly understood. Here, we demonstrate a novel functional interaction between ERM proteins and the ErbB2 receptor tyrosine kinase in breast cancer cells. We show that the ERM proteins ezrin and radixin are associated with ErbB2 receptors at the plasma membrane, and depletion or functional inhibition of ERM proteins destabilizes the interaction of ErbB2 with ErbB3, Hsp90 and Ebp50. Accompanied by the dissociation of this protein complex, binding of ErbB2 to the ubiquitin-ligase c-Cbl is increased, and ErbB2 becomes dephosphorylated, ubiquitinated and internalized. Furthermore, signaling via Akt- and Erk-mediated pathways is impaired upon ERM inhibition. Finally, interference with ERM functionality leads to receptor degradation and reduced cellular levels of ErbB2 and ErbB3 receptors in breast cancer cells. PMID:27029001

  2. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

    Directory of Open Access Journals (Sweden)

    Jesse E. Jun

    2013-09-01

    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  3. Injection of sup 32 P colloid into squamous cell carcinoma of the esophagus for local disease control

    Energy Technology Data Exchange (ETDEWEB)

    Perakos, P.G.; Scheer, T.F. (Memorial Hospital of Laramie County and Wyoming College of Human Medicine, Wyoming (USA))

    1989-10-01

    Local treatment of squamous cell carcinoma of the esophagus is only modestly successful. To increase local control, we have developed a procedure to inject a boost dose of radiation into the tumor bed after completion of external beam radiotherapy. The boost dose is given with {sup 32}P, a readily available radiocolloid. {sup 32}P is a pure emitter and poses no significant radiation hazards. It can penetrate 10approx15 mm into the tumor mass and has a half-life of 14.3 days. After determination of the volume to be treated, the colloid is injected with endoscopic guidance using the same technique as used in injection scierotherapy of esophageal varices. We use the Pentax FG 34 JA operating gastroscope and a Bard disposable 0.5 cm 25 Ga retractable injection sclerotherapy needle. We deliver 150approx200 microCurie of {sup 32}P colloid diluted to 20 ml with normal saline at 10 to 20 injection sites. This boosts the radiotherapy dose of 5,500approx6,000 cGy to the range of 7,500approx8,000 cGy. We have treated five patients so far, with length of follow-up ranging from 8approx28 months. Local control and survival results have been excellent and no complications have been associated with the procedure. A combination of external beam radiotherapy and interstitial boost treatment with colloidal {sup 32}P appears to be a safe and effective method of managing squamous cell carcinoma of the esophagus. (author).

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

    Science.gov (United States)

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

    2014-07-01

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

  5. Neoadjuvant therapy and surgical resection for locally advanced non-small cell lung cancer.

    Science.gov (United States)

    Meko, J; Rusch, V W

    2000-10-01

    During the past 15 years, treatment of stage IIIA (N2) non-small cell lung cancer has evolved considerably because of improvements in patients selection, staging, and combined modality therapy. Results of several clinical trials suggest that induction chemotherapy or chemoradiation and surgical resection is superior to surgery alone. However, the optimal induction regimen has not been defined. An intergroup trial is also underway to determine whether chemoradiation and surgical resection leads to better survival than chemotherapy and radiation alone. Future studies will assess ways to combine radiation and novel chemotherapeutic agents, and will identify molecular abnormalities that predict response to induction therapy.

  6. Preparation of Cells for Assessing Ultrastructural Localization of Nanoparticles with Transmission Electron Microscopy

    Science.gov (United States)

    2010-01-01

    300 mesh (EM Sciences, cat. no. FCF300-Cu)  crItIcal It is important to have a supporting film on grids for thin cell sections that are delicate...support film such as formvar/carbon), being careful to avoid the cutting edge of the knife. 39| Transfer sections by capillary action with a loop by... dopamine depletion. J. Tox. Sci. 92, 456–463 (2006). 8. Skebo, J.E., Grabinski, C.M., Schrand, A.M., Schlager, J.J. & Hussain, S.M. Assessment of metal

  7. The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Gian Carlo Alghisi

    Full Text Available Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC cultured on the beta1 ligands fibronectin and collagen I. We show tha