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Sample records for cells reveals intact

  1. Optical manipulation of Saccharomyces cerevisiae cells reveals that green light protection against UV irradiation is favored by low Ca2+ and requires intact UPR pathway.

    Science.gov (United States)

    Farcasanu, Ileana C; Mitrica, Radu; Cristache, Ligia; Nicolau, Ioana; Ruta, Lavinia L; Paslaru, Liliana; Comorosan, Sorin

    2013-11-01

    Optical manipulation of Saccharomyces cerevisiae cells with high density green photons conferred protection against the deleterious effects of UV radiation. Combining chemical screening with UV irradiation of yeast cells, it was noted that the high density green photons relied on the presence of intact unfolded protein response (UPR) pathway to exert their protective effect and that the low Ca(2+) conditions boosted the effect. UPR chemical inducers tunicamycin, dithiotreitol and calcium chelators augmented the green light effect in a synergic action against UV-induced damage. Photo-manipulation of cells was a critical factor since the maximum protection was achieved only when cells were pre-exposed to green light. PMID:24056073

  2. Features of Programmed Cell death in Intact Xenopus Oocytes and Early Embryos Revealed by Near-Infrared Fluorescence and Real-time Monitoring

    OpenAIRE

    Johnson, Carrie E; Freel, Christopher D.; Kornbluth, Sally

    2010-01-01

    Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. Described here is a novel method for monitoring caspase activity in living Xenopus oocytes and early embryos. The approach, utilizing microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enab...

  3. Oligodeoxynucleotide Probes for Detecting Intact Cells

    Science.gov (United States)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  4. Differences in calcium homeostasis between retinal rod and cone photoreceptors revealed by the effects of voltage on the cGMP-gated conductance in intact cells

    OpenAIRE

    1994-01-01

    We measured currents under voltage clamp in intact retinal rod photoreceptors with tight seal electrodes in the perforated patch mode. In the dark, membrane depolarization to voltages > or = +20 mV activates a time- and voltage-dependent outward current in the outer segment. This dark voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course that is voltage dependent. DVAC reaches its maximum enhancement of approximately 30% in 4-6 s at +60 mV. DVAC is entirely supp...

  5. Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo

    OpenAIRE

    Simmons, Alan J; Banerjee, Amrita; McKinley, Eliot T; Scurrah, Cherie' R; Herring, Charles A; Gewin, Leslie S; Masuzaki, Ryota; Karp, Seth J.; Franklin, Jeffrey L.; Gerdes, Michael J.; Irish, Jonathan M.; Coffey, Robert J.; Lau, Ken S.

    2015-01-01

    Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cell...

  6. Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

    Science.gov (United States)

    Simmons, Alan J; Banerjee, Amrita; McKinley, Eliot T; Scurrah, Cherie' R; Herring, Charles A; Gewin, Leslie S; Masuzaki, Ryota; Karp, Seth J; Franklin, Jeffrey L; Gerdes, Michael J; Irish, Jonathan M; Coffey, Robert J; Lau, Ken S

    2015-10-01

    Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues. PMID:26519361

  7. Simultaneous RNA quantification of human and retroviral genomes reveals intact interferon signaling in HTLV-1-infected CD4+ T cell lines

    Directory of Open Access Journals (Sweden)

    Moens Britta

    2012-08-01

    Full Text Available Abstract Background IFN-α contributes extensively to host immune response upon viral infection through antiviral, pro-apoptotic, antiproliferative and immunomodulatory activities. Although extensively documented in various types of human cancers and viral infections, controversy exists in the exact mechanism of action of IFN-α in human immunodeficiency virus type 1 (HIV-1 and human T-lymphotropic virus type 1 (HTLV-1 retroviral infections. Results IFN-α displayed strong anti-HIV-1 effects in HIV-1/HTLV-1 co-infected MT-4 cells in vitro, demonstrated by the dose-dependent inhibition of the HIV-1-induced cytopathic effect (IC50 = 83.5 IU/ml, p 50 = 1.2 IU/ml, p  Conclusions Taken together, our results indicate that both the absence of in vitro antiproliferative and pro-apoptotic activity as well as the modest post-transcriptional antiviral activity of IFN-α against HTLV-1, were not due to a cell-intrinsic defect in IFN-α signalisation, but rather represents a retrovirus-specific phenomenon, considering the strong HIV-1 inhibition in co-infected cells.

  8. Cell-to-cell communication within intact human skin.

    OpenAIRE

    Salomon, D.; Saurat, J. H.; Meda, P.

    1988-01-01

    We have characterized cell-to-cell communication (coupling) within intact human skin by microinjecting single keratinocytes with a gap junction-permeant tracer (Lucifer Yellow). 25-50 keratinocytes from different layers of the epidermis were seen to be coupled after most injections (n = 31). A few noncommunicating cells were also microinjected (n = 3) or observed within large territories of coupled keratinocytes. Microinjections of dermal fibroblasts demonstrated an extensive coupling (greate...

  9. Protocol: optimised electrophyiological analysis of intact guard cells from Arabidopsis

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    Chen Zhong-Hua

    2012-05-01

    Full Text Available Abstract Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells.

  10. Regeneration of transplanted intact cell populations in lethally irradiated hydra

    International Nuclear Information System (INIS)

    A single irradiation of the hydra Pelmatohydra robusta with 0.4 to 10 kR of x rays induced no lethal effects at all, while animals exposed to 40 kR ceased to produce a bud within 24 hr and died within a limited period of time, varying from 10 to 14 days at 250C, and from 28 to 50 days at 100C. This lethal dose of x rays inhibited the proliferation of the cells completely and destroyed interstitial cells. When a small cell mass, which was too small to regenerate alone, was transplanted from the subhypostomal region of an intact animal into the subhypostomal region of the irradiated hydra, the recipient began to produce buds by the proliferation of the graft cells. However, the transplantation of intact peduncle tissue, in which mitotic figures and interstitial cells rarely occurred, failed to produce new buds

  11. SOLITARY CHEMORECEPTOR CELL SURVIVAL IS INDEPENDENT OF INTACT TRIGEMINAL INNERVATION

    OpenAIRE

    Gulbransen, Brian; Silver, Wayne; Finger, Tom

    2008-01-01

    Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al., 2003). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al., 2003) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout m...

  12. Binding of fluorescent and radiolabelled alprenolol to intact cultured brain cells and liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Maderspach, K.; Nemecz, Gy.; Mesude Yigiter (Magyar Tudomanyos Akademia Biologiai Kutato Koezpontja, Szeged)

    1981-01-01

    Beta-adrenergic antagonist L-alprenolol was labelled for radioactivity and UV fluorescence, and the binding to intact cultured chicken brain cells under equilibrium conditions, as the model of the experiments in vivo, was probed. The application of fluorescent label did not explore any cell type or cell particle with enhanced binding. The analysis of the radiolabelled alprenolol-binding revealed a non-specific accumulation on the surface of these cells, which was inhibited by unlabelled alprenolol excess, however, this is the peculiarity of the specific binding. The authors' results emphasize that for the characterization of membrane receptors of intact cells, the accurate separation of specific and non-specific binding is required. The application of a ligand concentration as low as possible and a very effective washing procedure for the removal of the unbound (and the bulk of the nonspecifically bound) label are recommended.

  13. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier

    DEFF Research Database (Denmark)

    Dreier, Jes; Sørensen, Jens A; Brewer, Jonathan R

    2016-01-01

    skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm......In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human...... liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that...

  14. Voltage clamping single cells in intact malpighian tubules of mosquitoes.

    Science.gov (United States)

    Masia, R; Aneshansley, D; Nagel, W; Nachman, R J; Beyenbach, K W

    2000-10-01

    Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways. PMID:10997925

  15. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

    Directory of Open Access Journals (Sweden)

    Jes Dreier

    Full Text Available In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

  16. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

    Science.gov (United States)

    Dreier, Jes; Sørensen, Jens A; Brewer, Jonathan R

    2016-01-01

    In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers. PMID:26751684

  17. The use of fluorescamine as a permeant probe to localize phosphatidylethanolamine in intact friend erythroleukaemic cells

    NARCIS (Netherlands)

    Kamp, J.A.F. op den; Rawyler, A.; Roelofsen, B.

    1984-01-01

    Intact Friend erythroleukaemic cells (Friend cells) were incubated at 0–4°C with increasing amounts of fluorescamine. Phospholipids were extracted and the amounts of phosphatidylethanolamine and of its fluorescamine derivative were determined. (1). The plasma membrane of intact Friend cells appeared

  18. Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

    Science.gov (United States)

    Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

    2016-06-01

    Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted. PMID:27044871

  19. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    Science.gov (United States)

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

  20. Oligodendrocyte plasticity with an intact cell body in vitro.

    Directory of Open Access Journals (Sweden)

    Manabu Makinodan

    Full Text Available Demyelination is generally regarded as a consequence of oligodendrocytic cell death. Oligodendrocyte processes that form myelin sheaths may, however, degenerate and regenerate independently of the cell body, in which case cell death does not necessarily occur. We provide here the first evidence of retraction and regeneration of oligodendrocyte processes with no cell death in vitro, using time-lapse imaging. When processes were severed mechanically in vitro, the cells did not undergo cell death and the processes regenerated in 36 h. In a separate experiment, moderate N-methyl-D-aspartate (NMDA stimuli caused process retraction without apparent cell death, and the processes regained their elaborate morphology after NMDA was removed from the culture medium. These results strongly suggest that demyelination and remyelination can take place without concomitant cell death, at least in vitro. Process regeneration may therefore become a target for future therapy of demyelinating disorders.

  1. Intact individual heart cells isolated from human ventricular tissue.

    OpenAIRE

    Powell, T.; Sturridge, M F; Suvarna, S K; Terrar, D. A.; Twist, V. W.

    1981-01-01

    The intricate architecture of heart muscle, comprising irregularly shaped cells which interdigitate in a complex three-dimensional array, has often compromised clear interpretation of experimental data obtained from the whole organ. One approach to minimise some of the difficulties is to use individual muscle cells in suspension, and data have already been reported using myocytes isolated from mammalian ventricles. It is difficult, however, to extrapolate results obtained from animal tissues ...

  2. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    CERN Document Server

    Kim, Kyoohyun; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mouse were also investigated.

  3. Intact Cell/Spore Mass Spectrometry of Fusarium Macro Conidia for Fast Isolate and Species Differentiation

    Science.gov (United States)

    Dong, Hongjuan; Marchetti-Deschmann, Martina; Winkler, Wolfgang; Lohninger, Hans; Allmaier, Guenter

    The focus of this paper is the development of an approach called intact cell mass spectrometry (ICMS) or intact spore mass spectrometry (ISMS) based on the technique matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the rapid differentiation and identification of Fusarium species. Several parameters, which are known to affect the quality of IC mass spectra, have been investigated in detail by varying the MALDI matrix as well as the solvent system, in which the matrix has been dissolved, the solvent system for sample purification and the type of sample/MALDI matrix deposition technique. In the end characteristic as well as highly reproducible IC or IS mass spectra or peptide/protein fingerprints of three Fusarium species (F. cerealis, F. graminearum and F. poae) including 16 Fusarium isolates derived from different hosts and geographical locations have been obtained. Unscaled hierarchical cluster analysis based on ICMS data of eight selected Fusarium isolates of two species F. graminearum and F. poae revealed significant difference among the peptide/protein pattern of them. The results of the applied cluster analysis proved that, ICMS is a powerful approach for the rapid differentiation of Fusarium species. In addition, an on-target tryptic digestion was applied to Fusarium macro conidia spores to identify proteins using MALDI post source decay (PSD) fragment ion analysis. Two kinds of trypsin, namely bead-immobilized - to favor cleavage of surface-associated proteins - and non-immobilized trypsin were applied and compared. The results showed that the latter is more suitable for generating sequence tags by PSD fragment ion analysis.

  4. Proton nuclear magnetic resonance of intact friend leukemia cells: phosphorylcholine increase during differentiation

    International Nuclear Information System (INIS)

    Proton nuclear magnetic resonance of intact Friend leukemia cells was used to analyze their erythroid-like differentiation. The technique, which requires only 108 to 109 cells and approximately 2 minutes for acquisition of each spectrum, demonstrated the occurrence of many signal changes during differentiation. With cell extracts, 64 signals were assigned to 12 amino acids and 19 other intermediary metabolites, and a dramatic signal change was attributed to a fourfrease in cytoplasmic phosphorylcholines

  5. Fourier transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium Azospirillum brasilense

    Science.gov (United States)

    Kamnev, A. A.; Ristić, M.; Antonyuk, L. P.; Chernyshev, A. V.; Ignatov, V. V.

    1997-06-01

    The data of Fourier transform infrared (FTIR) spectroscopic measurements performed on intact cells of the soil nitrogen-fixing bacterium Azospirillum brasilense grown in a standard medium and under the conditions of an increased metal uptake are compared and discussed. The structural FTIR information obtained is considered together with atomic absorption spectrometry (AAS) data on the content of metal cations in the bacterial cells. Some methodological aspects concerning preparation of bacterial cell samples for FTIR measurements are also discussed.

  6. Intactness of cell wall structure controls the in vitro digestion of starch in legumes.

    Science.gov (United States)

    Dhital, Sushil; Bhattarai, Rewati R; Gorham, John; Gidley, Michael J

    2016-03-16

    Increasing the level of starch that is not digested by the end of the small intestine and therefore enters the colon ('resistant starch') is a major opportunity for improving the nutritional profile of foods. One mechanism that has been shown to be successful is entrapment of starch within an intact plant tissue structure. However, the level of tissue intactness required for resistance to amylase digestion has not been defined. In this study, intact cells were isolated from a range of legumes after thermal treatment at 60 °C (starch not gelatinised) or 95 °C (starch gelatinised) followed by hydrolysis using pancreatic alpha amylase. It was found that intact cells, isolated at either temperature, were impervious to amylase. However, application of mechanical force damaged the cell wall and made starch accessible to digestive enzymes. This shows that the access of enzymes to the entrapped swollen starch is the rate limiting step controlling hydrolysis of starch in cooked legumes. The results suggest that a single cell wall could be sufficient to provide an effective delivery of starch to the large intestine with consequent nutritional benefits, provided that mechanical damage during digestion is avoided. PMID:26786854

  7. Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

    OpenAIRE

    Brown, J.E.; Rothman, S W; Doctor, B P

    1980-01-01

    Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and d...

  8. Embedded Adaptive Optics for Ubiquitous Lab-on-a-Chip Readout on Intact Cell Phones

    OpenAIRE

    Pakorn Preechaburana; Anke Suska; Daniel Filippini

    2012-01-01

    The evaluation of disposable lab-on-a-chip (LOC) devices on cell phones is an attractive alternative to migrate the analytical strength of LOC solutions to decentralized sensing applications. Imaging the micrometric detection areas of LOCs in contact with intact phone cameras is central to provide such capability. This work demonstrates a disposable and morphing liquid lens concept that can be integrated in LOC devices and refocuses micrometric features in the range necessary for LOC evaluati...

  9. Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions

    OpenAIRE

    Kaufmann, Rainer; Schellenberger, Pascale; Seiradake, Elena; Dobbie, Ian M.; Jones, E. Yvonne; Davis, Ilan; Hagen, Christoph; Grünewald, Kay

    2014-01-01

    We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400–500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and...

  10. Accumulation and diffusion of crystallin inside single fiber cells in intact chicken embryo lenses.

    OpenAIRE

    Peetermans, J A; Foy, B.D.; Tanaka, T

    1987-01-01

    The use of microscope laser light-scattering spectroscopy allows for the measurement of dynamic properties of intracellular particles inside single fiber cells at different locations in the intact chicken embryo lens. Profiles of the diffusive properties of the delta-crystallin proteins across the lens are reported for developing chickens from day 5 to day 37. A clear decrease of the diffusion is observed in the lens nucleus relative to the cortex beginning with day 10.

  11. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kim Myung K

    2011-09-01

    Full Text Available Abstract Background Total internal reflection fluorescence microscopy (TIRFM is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein and clathrin light chain (a vesicle coat protein support our theoretical analysis. Conclusions These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

  12. Intact brain cells: a novel model system for studying opioid receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, N.F.; El-Fakahany, E.E.

    1985-07-29

    The use of a novel tissue preparation to study opioid receptor binding in viable, intact cells derived from whole brains of adult rats is presented. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using (/sup 3/H)naloxone as the radioligand, the studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. (/sup 3/H)Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date.

  13. Intact brain cells: a novel model system for studying opioid receptor binding

    International Nuclear Information System (INIS)

    The use of a novel tissue preparation to study opioid receptor binding in viable, intact cells derived from whole brains of adult rats is presented. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using [3H]naloxone as the radioligand, the studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. [3H]Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date

  14. Identification of periplasmic nitrate reductase Mo(V) EPR signals in intact cells of Paracoccus denitrificans.

    Science.gov (United States)

    Sears, H J; Bennett, B; Spiro, S; Thomson, A J; Richardson, D J

    1995-08-15

    EPR spectroscopy has been successfully used to detect signals due to molybdenum (V) and ferric iron in intact cells of aerobically grown Paracoccus denitrificans. The signals are ascribed to the catalytic molybdenum centre and to the haem iron of the periplasmic nitrate reductase. These signals are absent from a mutant strain deficient in this enzyme. The Mo(V) signal is due to the High-g Split species which has been well characterized in the purified enzyme. This confirms that the High-g Split is the physiologically relevant signal of a number observed in the previous work on the purified enzyme. PMID:7646461

  15. Hydrogen ions directly regulating the oligomerization state of Photosystem I in intact Spirulina platensis cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    H+ concentration induced-monomerization or trimerization of photosystem I (PSI) in cyanobacteria has never been directly observed. In this work, taking characteristic spectra for the trimers and monomers as the indicators, it was experimentally demonstrated that H+ could induce the oligomeric changes of PSI reaction centers in the intact Spirulina ,platensis cells and also in the isolated thylakoid membrane complexes. Especially, the higher concentration of H+ would induce the monomerization while the lower the trimerization, suggesting the electrostatic interaction should be mainly responsible forchanges in the oligomeric state of PSI in Spirulina platensis.

  16. Multiple opioid receptor binding in dissociated intact guinea pig brain cells

    Energy Technology Data Exchange (ETDEWEB)

    Tam, S.W.; James, D.W.

    1986-03-05

    Dissociated intact guinea pig brain cells were prepared by the method of Rogers and El-Fakahany. Over 95% of these cells are viable as demonstrated by their exclusion of the dye trypan blue. Opioid receptor binding assays were performed in a modified Kreb-Ringers physiological buffer. The following radiolabeled ligands and conditions were used to selectively labeled multiple opioid receptors: mu binding, 1 nM (/sup 3/H)naloxone + 20 nM DADLE + 300 nM U50,488H; kappa binding, 4 nM (-)-(/sup 3/H)-EKC + 100 nM DAGO + 500 nM DADLE; delta binding, 2 nM (/sup 3/H)-DADLE + 100 nM DAGO + 300 nM U50,488H; sigma binding, 4 nM (+)-(/sup 3/H)SKF 10,047. The intact brain cells in physiological buffer demonstrated specific binding for mu, kappa, delta, and sigma receptors. The relative binding potency of naloxone for each of the receptor types is arbitrarily set at 1.

  17. Multiple opioid receptor binding in dissociated intact guinea pig brain cells

    International Nuclear Information System (INIS)

    Dissociated intact guinea pig brain cells were prepared by the method of Rogers and El-Fakahany. Over 95% of these cells are viable as demonstrated by their exclusion of the dye trypan blue. Opioid receptor binding assays were performed in a modified Kreb-Ringers physiological buffer. The following radiolabeled ligands and conditions were used to selectively labeled multiple opioid receptors: mu binding, 1 nM [3H]naloxone + 20 nM DADLE + 300 nM U50,488H; kappa binding, 4 nM (-)-[3H]-EKC + 100 nM DAGO + 500 nM DADLE; delta binding, 2 nM [3H]-DADLE + 100 nM DAGO + 300 nM U50,488H; sigma binding, 4 nM (+)-[3H]SKF 10,047. The intact brain cells in physiological buffer demonstrated specific binding for mu, kappa, delta, and sigma receptors. The relative binding potency of naloxone for each of the receptor types is arbitrarily set at 1

  18. Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform.

    Directory of Open Access Journals (Sweden)

    Claire A Mitchell

    Full Text Available A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant ("diffraction-free" Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.

  19. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

    Directory of Open Access Journals (Sweden)

    FrançoisMarceau

    2013-09-01

    Full Text Available The C-C chemokine receptor-7 (CCR7 is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503 labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination. CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry. The immune complexes were apparent in endosomal structures, colocalized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  20. Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

    DEFF Research Database (Denmark)

    Olsen, M; Krog, L; Edvardsen, K;

    1993-01-01

    density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding to......-s1 and NCAM-s2 and the function of soluble NCAM forms were investigated. It was shown that all three soluble forms could be released from brain membranes with M(r) values identical to the three major membrane-associated forms: the large transmembrane 190,000-M(r) form (NCAM-A), the smaller...... intact soluble form from membranes of cells transfected with this isoform. Thus, NCAM-s1 and NCAM-s2 probably represent intact released transmembrane NCAM-A and NCAM-B. The soluble transmembrane forms are likely to exist in vivo, as NCAM-s1 and NCAM-s2 were readily demonstrated in cerebrospinal fluid. By...

  1. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.

    Science.gov (United States)

    Kaufmann, Rainer; Schellenberger, Pascale; Seiradake, Elena; Dobbie, Ian M; Jones, E Yvonne; Davis, Ilan; Hagen, Christoph; Grünewald, Kay

    2014-07-01

    We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement. PMID:24884378

  2. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

  3. Imaging individual proteins and nanodomains on intact cell membranes with a probe-based optical antenna.

    Science.gov (United States)

    van Zanten, Thomas S; Lopez-Bosque, Maria J; Garcia-Parajo, Maria F

    2010-01-01

    Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 +/- 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 +/- 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range. PMID:19943247

  4. Activation of resting T cells: distinct roles of intact accessory cells, phorbol myristate acetate and interleukin 1

    Energy Technology Data Exchange (ETDEWEB)

    Davis, L.; Lipsky, P.E.

    1986-03-05

    The accessory cell (AC) signals involved in the activation of resting guinea pig T lymphocytes stimulated with mitogen (PHA), or the calcium ionophore, ionomycin (Ion) were examined. Activation of T cells was assessed by cell cycle analysis after acridine orange staining and /sup 3/H-thymidine incorporation. PHA-stimulated T cells depleted of all AC were unable to respond in the presence of phorbol myristate acetate (PMA), and/or interleukin 1 (IL 1). With suboptimal numbers of AC, PMA greatly augmented the number of T cells activated by PHA to enter and progress through the cell cycle, but only when present during the first few hours of culture. By contrast, IL 1 had little effect on the number of cells entering the cell cycle, although it enhanced S phase entry of the activated cells. IL 1 augmented DNA synthesis when added initially or later in culture. In contrast to the effects noted with PHA, PMA promoted activation and DNA synthesis of the majority of Ion stimulated cells in the complete absence of AC. IL 1 alone could not support Ion induced T cell activation although it enhanced T cell DNA synthesis in cultures stimulated by PMA and Ion. These studies indicate that intact AC, IL 1 and PMA-like signals play distinct roles in the progression of T cells through the initial cell cycle. Stimulation by Ion requires only PMA whereas PHA responses require intact AC and can be amplified by PMA. The major effect of IL 1 is to promote S phase entry of activated T cells.

  5. Uptake of Retrograde Tracers by Intact Optic Nerve Axons: A New Way to Label Retinal Ganglion Cells

    OpenAIRE

    Liang, Yu-Xiang; Yang, Jian; Yuan, Ti-Fei; So, Kwok-Fai

    2015-01-01

    Retrograde labelling of retinal ganglion cells with optic nerve transection often leads to degeneration of ganglion cells in prolonged experiments. Here we report that an intact optic nerve could uptake retrograde tracers applied onto the surface of the nerve, leading to high efficiency labelling of ganglion cells in the retina with long-term survival of cells. This method labelled a similar number of ganglion cells (2289±174 at 2 days) as the retrograde labeling technique from the superior c...

  6. Metabolism of fluoranthene in different plant cell cultures and intact plants

    Energy Technology Data Exchange (ETDEWEB)

    Kolb, M.; Harms, H.

    2000-05-01

    The metabolism of fluoranthene was investigated in 11 cell cultures of different plant species using a [{sup 14}C]-labeled standard. Most species metabolized less than 5% of fluoranthene to soluble metabolites and formed less than 5% nonextractable residues during the standardized 48-h test procedure. Higher metabolic rates were observed in lettuce (Lactuca sativa, 6%), wheat (Tricitum aestivum, 9%), and tomato (Lycopersicon esculentum, 15%). A special high metabolic rate of nearly 50% was determined for the rose species Paul's Scarlet. Chromatographic analysis of metabolites extracted from aseptically grown tomato plants proved that the metabolites detected in the cell cultures were also formed in the intact plants. Metabolites produced in tomato and rose cells from [{sup 14}C]-fluoranthene were conjugated with glucose, glucuronic acid, and other cell components. After acid hydrolyses, the main metabolite of both species was 1-hydroxyfluoranthene as identified by gas chromatography-mass spectrometry and high-performance liquid chromatography with diode array detection. The second metabolite formed by both species was 8-hydroxyfluoranthene. A third metabolite in tomatoes was 3-hydroxyfluoranthene.

  7. Layered Double Hydroxide Nanotransporter for Molecule Delivery to Intact Plant Cells

    Science.gov (United States)

    Bao, Wenlong; Wang, Junya; Wang, Qiang; O’Hare, Dermot; Wan, Yinglang

    2016-01-01

    Here we report a powerful method that facilitates the transport of biologically active materials across the cell wall barrier in plant cells. Positively charged delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) with a 0.5‒2 nm thickness and 30‒60 nm diameter exhibit a high adsorptive capacity for negatively charged biomolecules, including fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate isomer I(FITC) and DNA molecules, forming neutral LDH-nanosheet conjugates. These neutral conjugates can shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Furthermore, typical inhibitors of endocytosis and low temperature incubation did not prevent LDH-lactate-NS internalization, suggesting that LDH-lactate-NS penetrated the plasma membrane via non-endocytic pathways, which will widen the applicability to a variety of plant cells. Moreover, the absence of unwanted side effects in our cytological studies, and the nuclear localization of ssDNA-FITC suggest that nano-LDHs have potential application as a novel gene carrier to plants. PMID:27221055

  8. Fast response methods in the radiation chemistry of lethal damage in intact cells

    International Nuclear Information System (INIS)

    The radiation-induced lethal damage in bacteria and mammalian cells involves some important chemical processes which extend up to about 10-2s after the initiating energy deposition. The gas explosion technique, analogous to the use of flash photolysis in photochemistry and pulse radiolysis in radiation chemistry, was applied to examine the kinetics of some of the fast processes within intact cells. The time resolution of this technique is about 100 μs. Five nanosecond electron pulses and the rapid transition from virtually anoxic (2) to well oxygenated (>105 ppm O2) conditions can be used as the fast probes to determine the time scale of the radiosensitization by oxygen. The apparent differences between the observed intracellular kinetics of the reactions of O2 and of TAN (triacetoneamine-N-oxyl) with target damage and the reactions of these agents with DNA radicals support the view that O2 is not sensitized by reacting directly with DNA damage. In general, the processes described in this review appear to conform to simple bimolecular reaction kinetics. The intracellular reaction rates of the various agents tested are all slower than those expected from the rates of their reactions with radicals in dilute aqueous solution probably due to the complex structure of the cells. (Yamashita, S.)

  9. Shifts in oxidation states of cerium oxide nanoparticles detected inside intact hydrated cells and organelles

    Energy Technology Data Exchange (ETDEWEB)

    Szymanski, Craig J.; Munusamy, Prabhakaran; Mihai, Cosmin; Xie, Yumei; Hu, Dehong; Gilles, Marry K.; Tyliszczak, T.; Thevuthasan, Suntharampillai; Baer, Donald R.; Orr, Galya

    2015-09-01

    Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce3+/Ce4+ ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce3+/Ce4+ ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of cells.

  10. Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Davis, L.; Lipsky, P.E.

    1986-05-15

    The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4..beta..-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Results of cell cycle analysis support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen stimulated T cells through the first round of the cell cycle.

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

    Directory of Open Access Journals (Sweden)

    Parr Rebecca D

    2011-06-01

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

  12. In Vivo Real-Time Imaging of Exogenous HGF-Triggered Cell Migration in Rat Intact Soleus Muscles

    International Nuclear Information System (INIS)

    The transplantation of myogenic cells is a potentially effective therapy for muscular dystrophy. However, this therapy has achieved little success because the diffusion of transplanted myogenic cells is limited. Hepatocyte growth factor (HGF) is one of the primary triggers to induce myogenic cell migration in vitro. However, to our knowledge, whether exogenous HGF can trigger the migration of myogenic cells (i.e. satellite cells) in intact skeletal muscles in vivo has not been reported. We previously reported a novel in vivo real-time imaging method in rat skeletal muscles. Therefore, the present study examined the relationship between exogenous HGF treatment and cell migration in rat intact soleus muscles using this imaging method. As a result, it was indicated that the cell migration velocity was enhanced in response to increasing exogenous HGF concentration in skeletal muscles. Furthermore, the expression of MyoD was induced in satellite cells in response to HGF treatment. We first demonstrated in vivo real-time imaging of cell migration triggered by exogenous HGF in intact soleus muscles. The experimental method used in the present study will be a useful tool to understand further the regulatory mechanism of HGF-induced satellite cell migration in skeletal muscles in vivo

  13. Transport of alpha- and beta-D-glucose by the intact human red cell

    International Nuclear Information System (INIS)

    The kinetics of alpha- and beta-D-glucose mutarotation and the transport of these anomers by intact human red cells were determined at 0.6 and 36.6 degrees C. The mutarotation coefficients for alpha- and beta-D-glucose in cell-free tris(hydroxymethyl)aminomethane medium (pH 7.4) at 0.6 degrees C are (2.25 +/- 0.2) and (1.73 +/- 0.42) X 10(-3) min-1, respectively, and at 36.6 degrees C are (69 +/- 12) and (75 +/- 5) X 10(-3) min-1, respectively. These values are in good agreement with previous estimates. At 0.6 degrees C, the red cell contains no detectable mutarotase activity. Initial rates of sugar uptake were measured by using radiolabeled D-glucose and time courses of uptake by turbidimetry. The time courses of alpha- and beta-D-glucose and an equilibrium mixture of alpha- and beta-D-glucose infinite-cis entry are identical at 0.66 degrees C (n = 41) where negligible mutarotation is observed. The apparent Ki values for inhibition of radiolabeled D-glucose initial uptake by unlabeled alpha- or beta-D-glucose at 0.6 degrees C are identical (1.6 mM). The calculated Vmax parameters for uptake of the radiolabeled anomers at this temperature are also indistinguishable. The time courses of infinite-cis alpha- and beta-D-glucose uptake at 36.66 degrees C are identical (n = 40). While D-glucose mutarotation is more rapid at this temperature, the anomers of D-glucose are not transported differently by the red cell hexose transfer system

  14. Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex- periments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi- mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de- tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.

  15. Surface Modification of Intact Poly(dimethylsiloxane) for Cell Culture and Cell Migration

    Institute of Scientific and Technical Information of China (English)

    LI Li; LIU WenMing; WANG JinYi

    2009-01-01

    @@ Cell migration plays a crucial role in various biological processes including embryogenests,wound healing,immune response,and tissue development~([1]).Exploring and understanding the mechanisms and related factors underlying cell migration arc also very important for emerging areas of biotedmology which focus on cellular transplantation and the manufacture of artificial tissues,as well as for the development of new therapeutic strategies for controlling invasive tumor cells~([2]).

  16. Export or recombination of charges in reaction centers in intact cells of photosynthetic bacteria.

    Science.gov (United States)

    Asztalos, Emese; Maróti, Péter

    2009-12-01

    The kinetics and thermodynamics of forward and reverse electron transfer around the reaction center of purple bacterium Rhodobacter sphaeroides were studied in vivo by flash-excited delayed fluorescence, prompt fluorescence (induction) and kinetic difference absorption. By protection of the photomultiplier from intense bacteriochlorophyll prompt fluorescence evoked by laser excitation, the time resolution of the fluorometer was reduced typically 10 micros. Two precursor states of the delayed fluorescence were identified: P(+)Q(A)(-) and cyt c(2)(3+)Q(A)(-) whose enthalpy levels were 340 meV and 1020 meV below A, respectively. The free energy of the P(+)Q(A)(-) state relative to A* was -870 meV in whole cells. Similar values were obtained earlier for isolated reaction center and chromatophore. The free energies of cyt c(2)(3+)Q(A)(-) and P(+)Q(A)(-) states showed no or very weak (-6 meV/pH unit) pH-dependence, respectively, supporting the concept of pH-independent redox midpoint potential of Q(A)/Q(A)(-) in intact cells. In accordance with the multiphasic kinetics of delayed fluorescence, the kinetics of re-opening of the closed reaction center is also complex (it extends up to 1 s) as a consequence of acceptor and donor-side reactions. The control of charge export from the reaction center by light regime, redox agents and inhibitors is investigated. The complex kinetics may arise from the distribution of quinones in different redox states on the acceptor side (Q(B) binding site and pool) and from organization of electron transfer components in supercomplexes. PMID:19555655

  17. Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes.

    Science.gov (United States)

    Moore, D J; Gioioso, S; Sills, R H; Mendelsohn, R

    1999-01-01

    A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes. PMID:9889394

  18. A system and methodology for high-content visual screening of individual intact living cells in suspension

    Science.gov (United States)

    Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

    2007-02-01

    Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

  19. Low temporal variation in the intact polar lipid composition of North Sea coastal marine water reveals limited chemotaxonomic value

    Science.gov (United States)

    Brandsma, J.; Hopmans, E. C.; Philippart, C. J. M.; Veldhuis, M. J. W.; Schouten, S.; Sinninghe Damsté, J. S.

    2012-03-01

    Temporal variations in the abundance and composition of intact polar lipids (IPLs) in North Sea coastal marine water were assessed over a one-year seasonal cycle, and compared with environmental parameters and the microbial community composition. Sulfoquinovosyldiacylglycerol (SQDG) was the most abundant IPL class, followed by phosphatidylcholine (PC), phosphatidylglycerol (PG) and diacylglyceryl-(N,N,N)-trimethylhomoserine (DGTS) in roughly equal concentrations, and smaller amounts of phosphatidylethanolamine (PE). Although the total concentrations of these IPL classes varied substantially throughout the year, the composition of the IPL pool remained remarkably constant. Statistical analysis yielded negative correlations between IPL concentrations and dissolved inorganic nutrient concentrations, but no changes in the overall planktonic IPL composition due to nutrient limitation were observed. Significant correlations between SQDG, PC, PG and DGTS concentrations and chlorophyll a concentrations and algal abundances indicated that eukaryotic primary producers, in particular Phaeocystis globosa, were the predominant source of IPLs at this site. However, while IPL concentrations in the water were closely tied to total algal abundances, the rapid succession of different algal groups blooming throughout the year resulted in only minor shifts in the IPL composition. Principal component analysis showed that the IPLs were derived from multiple sources, and that no IPL species could be exclusively assigned to a particular algal taxa or (cyano)bacteria. Thus, the most commonly occurring IPLs appear to have limited chemotaxonomic potential, highlighting the need to use targeted assays of more specific biomarker IPLs.

  20. In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

    Directory of Open Access Journals (Sweden)

    Robert C Blake

    2012-04-01

    Full Text Available Electron transfer reactions among colored biomolecules in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579 nanometers in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the oxidized bacterium was always regenerated from that of the iron-reduced bacterium. The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis-Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentraton of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.

  1. Uptake and clearance of plutonium-238 from intact liver and liver cells transplanted into fat pads of F344/N rats

    International Nuclear Information System (INIS)

    An understanding of the role of liver cells and the intact liver in plutonium biokinetics is needed. Liver cells were isolated from rats, injected into fat pads of recipient rats, and allowed 21 days to form cell colonies. Rats then received a single intraperitoneal injection of 1 μCi 238Pu-citrate and were serially sacrificed. Uptake, retention, and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. Intact liver cells took up about twice as much 238Pu as liver cells transplanted into fat pads. However, the retention kinetics of Pu were similar for both the liver cells in the fat pads and the intact liver cells when the retention was expressed as activity per cell. 4 references, 1 figure, 1 table

  2. Transplanting intact donor tissue enhances dopamine cell survival and the predictability of motor improvements in a rat model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Rosemary A Fricker

    Full Text Available Primary cell transplantation is currently the gold standard for cell replacement in Parkinson's disease. However, the number of donors needed to treat a single patient is high, and the functional outcome is sometimes variable. The present work explores the possibility of enhancing the viability and/or functionality of small amounts of ventral mesencephalic (VM donor tissue by reducing its perturbation during preparation and implantation. Briefly, unilaterally lesioned rats received either: (1 an intact piece of half an embryonic day 13 (E13 rat VM; (2 dissociated cells from half an E13 rat VM; or (3 no transplant. D-amphetamine- induced rotations revealed that animals receiving pieces of VM tissue or dissociated cells showed significant improvement in ipsilateral rotation 4 weeks post transplantation. By 6 weeks post transplantation, animals receiving pieces of VM tissue showed a trend for further improvement, while those receiving dissociated cells remained at their 4 week scores. Postmortem cell counts showed that the number of dopaminergic neurons in dissociated cell transplants was significantly lower than that surviving in transplants of intact tissue. When assessing the correlation between the number of dopamine cells in each transplant, and the improvement in rotation bias in experimental animals, it was shown that transplants of whole pieces of VM tissue offered greater predictability of graft function based on their dopamine cell content. Such results suggest that maintaining the integrity of VM tissue during implantation improves dopamine cell content, and that the dopamine cell content of whole tissue grafts offers a more predictable outcome of graft function in an animal model of Parkinson's disease.

  3. Microtubule distribution in cultured cells and intact tissues: improved immunolabeling resolution through the use of reversible embedment cytochemistry.

    OpenAIRE

    Gorbsky, G; Borisy, G G

    1985-01-01

    To investigate the detailed distributions of microtubules in cultured cells and intact tissues we developed a reversible embedment method for antibody labeling of sectioned material. Fixed tissues were infiltrated with fully polymerized polymethylmethacrylate dissolved in an organic solvent. Evaporation of the solvent left the tissue embedded in hard plastic. After sectioning by conventional methods, the plastic was extracted and sections were processed for indirect immunofluorescence to labe...

  4. Intact proinsulin and beta-cell function in lean and obese subjects with and without type 2 diabetes

    DEFF Research Database (Denmark)

    Røder, M E; Dinesen, B; Hartling, S G;

    1999-01-01

    OBJECTIVE: Type 2 diabetes is a heterogeneous disease in which both beta-cell dysfunction and insulin resistance are pathogenetic factors. Disproportionate hyperproinsulinemia (elevated proinsulin/insulin) is another abnormality in type 2 diabetes whose mechanism is unknown. Increased demand due to...... obesity and/or insulin resistance may result in secretion of immature beta-cell granules with a higher content of intact proinsulin. RESEARCH DESIGN AND METHODS: We investigated the impact of obesity on beta-cell secretion in normal subjects and in type 2 diabetic patients by measuring intact proinsulin...... diabetic patients matched for fasting glucose (10.2 +/- 0.6 vs. 10.3 +/- 0.4 mmol/l) were compared with age- and BMI-matched lean (22.4 +/- 0.6 kg/m2) (LC) and obese (30.8 +/- 0.9 kg/m2) (OC) normal control subjects. RESULTS: Diabetic patients (LD vs. LC and OD vs. OC) had elevated fasting levels of intact...

  5. Purification and functional characterization of protoplasts and intact vacuoles from grape cells

    OpenAIRE

    Gerós Hernâni; Lecourieux Fatma; Vignault Céline; Silva Rui; Fontes Natacha; Delrot Serge

    2010-01-01

    Abstract Background During grape berry ripening, the vacuoles accumulate water, sugars and secondary metabolites, causing great impact in plant productivity and wine quality. However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. The present paper describes an isolation method of protoplasts and intact vacuoles from grape b...

  6. Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts

    OpenAIRE

    Levchenko, V.; Guinot, D. R.; Klein, M.; Roelfsema, M. R. G.; Hedrich, R; Dietrich, P

    2008-01-01

    Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca2+ signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+ signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca2+-s...

  7. A surviving intact branch stabilizes remaining axon architecture after injury as revealed by in vivo imaging in the mouse spinal cord.

    Science.gov (United States)

    Lorenzana, Ariana O; Lee, Jae K; Mui, Matthew; Chang, Amy; Zheng, Binhai

    2015-05-20

    The complex morphology of axons presents a challenge in understanding axonal responses to injury and disease. By in vivo two-photon imaging of spinal dorsal column sensory axons, we systematically examined the effect of injury location relative to the main bifurcation point on axon degeneration and regeneration following highly localized laser injuries. Retrograde but not anterograde degeneration was strongly blocked at the bifurcation point at both the acute and subacute phases. Eliminating either the ascending or descending branch led to a poor regenerative response, while eliminating both led to a strong regenerative response. Thus, a surviving intact branch suppresses both retrograde degeneration and regeneration of the injured branch, thereby preserving the remaining axon architecture. Regenerating axons exhibited a dynamic pattern with alternating phases of regeneration and pruning over a chronic period. In vivo imaging continues to reveal new insights on axonal responses to injury in the mammalian spinal cord. PMID:25937174

  8. Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells.

    Science.gov (United States)

    Falconer, R J; O'Neill, B K; Middelberg, A P

    1998-02-20

    A method is presented for the direct extraction of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (Long-R3-IGF-I release and caused an observable increase in viscosity. Advantages of the direct extraction method include its speed, simplicity, and efficiency at releasing product. PMID:10099214

  9. Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals

    International Nuclear Information System (INIS)

    Highlights: • We developed a stable cell line with intact PGC-1α/ERRα axis. • The ERRα repressor, XCT790, down regulates this pathway. • Phytoestrogen, genisten stimulates this pathway. - Abstract: The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway

  10. Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Christina T., E-mail: teng1@niehs.nih.gov [DNTP, BioMolecular Screening Branch, Division, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Beames, Burton; Alex Merrick, B. [DNTP, BioMolecular Screening Branch, Division, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Martin, Negin; Romeo, Charles [DIR, Viral Core Lab, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Jetten, Anton M. [DIR Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States)

    2014-02-07

    Highlights: • We developed a stable cell line with intact PGC-1α/ERRα axis. • The ERRα repressor, XCT790, down regulates this pathway. • Phytoestrogen, genisten stimulates this pathway. - Abstract: The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.

  11. Label-free morphology-based prediction of multiple differentiation potentials of human mesenchymal stem cells for early evaluation of intact cells.

    Directory of Open Access Journals (Sweden)

    Hiroto Sasaki

    Full Text Available Precise quantification of cellular potential of stem cells, such as human bone marrow-derived mesenchymal stem cells (hBMSCs, is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1 the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2 predictions of potentials are generated before differentiation cultures are initiated; (3 prediction of multiple potentials can be provided simultaneously for each sample; and (4 predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21 and cytoskeleton-related genes (PTK2, CD146, and CD49 already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature

  12. Label-free morphology-based prediction of multiple differentiation potentials of human mesenchymal stem cells for early evaluation of intact cells.

    Science.gov (United States)

    Sasaki, Hiroto; Takeuchi, Ichiro; Okada, Mai; Sawada, Rumi; Kanie, Kei; Kiyota, Yasujiro; Honda, Hiroyuki; Kato, Ryuji

    2014-01-01

    Precise quantification of cellular potential of stem cells, such as human bone marrow-derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion

  13. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    International Nuclear Information System (INIS)

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor β-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the β-subunit of the insulin receptor correlated with the occupancy of the β-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the β-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the β-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells

  14. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Beguinot, F.; Smith, R.J.; Kahn, C.R.; Maron, R.; Moses, A.C.; White, M.F.

    1988-05-03

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of (/sup 32/P)-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor ..beta..-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the ..beta..-subunit of the insulin receptor correlated with the occupancy of the ..beta..-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the ..beta..-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the ..beta..-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

  15. Use of fish gill cells in culture to evaluate the cytotoxicity and photocytotoxicity of intact and photomodified creosote

    Energy Technology Data Exchange (ETDEWEB)

    Schirmer, K.; Herbrick, J.S.; Greenberg, B.M.; Dixon, D.G.; Bols, N.C. (Univ. of Waterloo, Ontario (Canada). Dept. of Biology)

    1999-06-01

    The influence of ultraviolet (UV) irradiation on creosote toxicity was investigated with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, and two indicator dyes, alamar Blue[trademark] and 5-carboxyfluorescein diacetate acetoxymethyl ester. These monitor redox potential and membrane integrity, respectively. After solubilization and chemical analysis, creosote was presented to cells in the dark to measure cytotoxicity or concurrently with UV irradiation to evaluate photocytotoxicity. Additionally, creosote was photomodified by 2 h of UV irradiation before presentation to cells in the dark or together with UV. Cytotoxicity was detected only at high nominal creosote concentrations, but photocytotoxicity occurred at creosote concentrations 35-fold lower. All the aromatic hydrocarbons in creosote appeared to contribute to cytotoxicity, but photocytotoxicity was due only to the fluoranthene, pyrene, anthracene, and benzo[a]anthracene in the mixture. Photomodified creosote was much more cytotoxic than intact creosote and this difference was most pronounced in the alamar Blue assay. Likely, this was due to photomodification products that impaired the mitochondrial electron transport chain. Photomodified creosote was slightly less photocytotoxic than intact creosote. Overall these results indicate that UV irradiation potentially enhances the toxicity of creosote to fish in several different but significant ways.

  16. Comparison of DNA double—strand breaks induced by 16O8+ in deproteinized DNA and intact cells

    Institute of Scientific and Technical Information of China (English)

    ZhouGuang-Ming; GaoQing-Xiang; 等

    1998-01-01

    The yield of DNA double-strand breaks(DSBs) is sure to be influenced by the environment around DNA molecule.Inverse pulsed-field gel electrophoresis(PIGE)has been applied to compared the sensitivity of B16 cells and their DNA in DSBs induced by 75MeV/u 16O8+ beam.Results show that the percentages of DNA released from the plug(PR)in both kinds of the samples increase with the dose and approach a similar quasi-threshold of about 81%.A simple new equation was presented to calculated the break level of DNA molecules.Within a certain dose,the relationship between the break level and the dose is linear.THe yield of DSBs in deproteinized DNA was 1.11DSBs/100Mbp/Gy,while that in intact cells was 0.60DSBs/100Mbp/Gy.it is testified that deproteinized DNA is more sensitive to oxygen ions irradiation than intact cells.

  17. Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells.

    Science.gov (United States)

    Falconer, R J; O'Neill, B K; Middelberg, A P

    1999-02-20

    In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized beta-mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. PMID:9921154

  18. Some characteristics of 3H-dihydroalprenolol binding to intact normal granulocytes, lymphocytes and membrane preparations of these cells

    International Nuclear Information System (INIS)

    3H-Dihydroalprenolol (3H-DHA) is a well-known ligand which is used to characterize β-adrenergic receptors on different cell types. In asthma research, circulating blood cells, i.e. granulocytes or lymphocytes, are often used as in-vitro models to study βreceptor function. Most of the studies to date have used rather crude membrane preparations. We therefore compared some 3H-DHA binding characteristics of normal intact granulocytes and lymphocytes and of crude and partly purified membrane preparations of these cells. In a separate series of experiments, we investigated whether 3H-DHA binding to subcellular fragments besides plasma membranes could occur. (author)

  19. Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

    Science.gov (United States)

    Buyel, Johannes F; Hubbuch, Jürgen; Fischer, Rainer

    2016-01-01

    Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy. PMID:27584939

  20. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    OpenAIRE

    Kim Myung K; Hao Huaiqin; Fan Lusheng; Ash William M; Wan Yinglang; Lin Jinxing

    2011-01-01

    Abstract Background Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM) was developed to circumvent this problem. However, the ...

  1. Porcine reproductive and respiratory syndrome virus (PRRSV infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

    Directory of Open Access Journals (Sweden)

    Rowland Raymond RR

    2006-11-01

    Full Text Available Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145 cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs. Results Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i., but the logarithmic infection phase (days 2–3 p.i., was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ, as well as the more-potent experimental antiviral agent AK-2. Conclusion The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small

  2. 2,6-Dichlorobenzamide (BAM) herbicide mineralisation by Aminobacter sp. MSH1 during starvation depends on a subpopulation of intact cells maintaining vital membrane functions

    Energy Technology Data Exchange (ETDEWEB)

    Sjoholm, Ole R.; Nybroe, Ole [Department of Agriculture and Ecology, Section of Genetics and Microbiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Aamand, Jens [Department of Agriculture and Ecology, Section of Genetics and Microbiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Department of Geochemistry, Geological Survey of Denmark and Greenland, Oster Voldgade 10, 1350 Copenhagen K (Denmark); Sorensen, Jan, E-mail: jan@life.ku.d [Department of Agriculture and Ecology, Section of Genetics and Microbiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark)

    2010-12-15

    Mineralisation capability was studied in the 2,6-dichlorobenzamide (BAM)-degrading Aminobacter sp. MSH1 under growth-arrested conditions. Cells were starved in mineral salts (MS) solution or groundwater before {sup 14}C-labelled BAM (0.1 mM) was added. Cell physiology was monitored with a panel of vitality stains combined with flow cytometry to differentiate intact, depolarised and dead cells. Cells starved for up to 3 weeks in MS solution showed immediate growth-linked mineralisation after BAM amendment while a lag-phase was seen after 8 weeks of starvation. In contrast, cells amended with BAM in natural groundwater showed BAM mineralisation but no growth. The cell-specific mineralisation rate was always comparable (10{sup -16} mol C intact cell{sup -1} day{sup -1}) independent of media, growth, or starvation period after BAM amendment; lower rates were only observed as BAM concentration decreased. MSH1 seems useful for bioremediation and should be optimised to maintain an intact cell subpopulation as this seems to be the key parameter for successful mineralisation. - The intact cell population of Aminobacter MSH1 mineralises BAM at a constant rate independent of growth or extended starvation in mineral solution and natural groundwater.

  3. Mitochondrial Stress Tests Using Seahorse Respirometry on Intact Dictyostelium discoideum Cells.

    Science.gov (United States)

    Lay, Sui; Sanislav, Oana; Annesley, Sarah J; Fisher, Paul R

    2016-01-01

    Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum. PMID:27271893

  4. Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells.

    Science.gov (United States)

    Chen, Chun-Rong; Salazar, Larry M; McLachlan, Sandra M; Rapoport, Basil

    2015-07-01

    The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody. PMID:25860033

  5. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle. PMID:198130

  6. Transferring Gus gene into intact rice cells by low energy ion beam

    Science.gov (United States)

    Zengliang, Yu; Jianbo, Yang; Yuejin, Wu; Beijiu, Cheng; Jianjun, He; Yuping, Huo

    1993-06-01

    A new technique of transferring genes by low energy ion beam has been reported in this paper. The Gus and CAT (chloramphenicol acetyltransferase) genes, as "foreign" genetic materials, were introduced into the suspension cells and ripe embryos or rice by implantation of 20-30 keV Ar + at doses ranging from 1 × 10 15 to 4 × 10 15 ions/cm 2. The activities of CAT and Gus were detected in the cells and embryos after several weeks. The results indicate that the transfer was a success.

  7. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [32P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  8. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))

    1991-06-01

    The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

  9. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    International Nuclear Information System (INIS)

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa β-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 μM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa β-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases

  10. 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

  11. Ca++- and cyclic AMP-induced changes in intact cell phosphorylation of ileal microvillus membrane proteins

    International Nuclear Information System (INIS)

    Pieces of rabbit distal ileal mucosa, with the muscularis propria and serosa removed, were incubated for 90 minutes in Krebs-Ringer bicarborate buffer (KRB) with 32PO4 to label the intracellular nucleotide pools. After rinsing, the mucosal pieces were transferred to KRB in the absence and presence of 10 μM A23187 or 10 mM theophylline. After a further 10 minutes the cells were scraped off and microvillus membranes prepared. The membranes were solubilized, subjected to two dimensional gel electrophoresis and autoradiography, and analyzed by densitometry. A23187 increased the phosphorylation of four microvillus membrane proteins with M/sub r/ of 32, 52, 110 and 116K. Increased phosphorylation of the 52 and 116K proteins has also been detected in microvillus membranes subjected to Ca++ and calmodulin in the presence of γ-32P-ATP. Theophylline increased the phosphorylation of the same 32 and 52K proteins and, additionally, of a second 32K peptide. While any of these proteins could be involved in the control of electrolyte transport, it is noteworthy that increased Ca++, and increased cyclic AMP levels exert similar effects upon intestinal electrolyte transport. That A23187 and theophylline both increase the phosphorylation of the 32 and 52K proteins increases the possibility that these are involved in ion transport

  12. Direct visualization of vaults within intact cells by electron cryo-tomography.

    Science.gov (United States)

    Woodward, Cora L; Mendonça, Luiza M; Jensen, Grant J

    2015-09-01

    The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex's actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault. PMID:25864047

  13. Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

    International Nuclear Information System (INIS)

    Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an M/sub r/ 95,000 protein (identified as the insulin receptor β subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) anti-sera) and an M/sub r/ 180,000 protein. After purification and tryptic digestion of the M/sub r/ 95,000 protein, tryptic peptides containing Tyr (P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. Approximately 80% of all β subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Try-Glu-Thr-Asp-Try-Try-Arg from the tyrosine kinase domain. A second tryptic peptide is located near the carboxyl terminus; this contains 20-30% of β subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. In a summary, the insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells involves at least 6 of the 13 tyrosine residues located on the β subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin

  14. Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks.

    Science.gov (United States)

    Valletta, Elisa; Kučera, Lukáš; Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

    2016-01-01

    Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general. PMID:26821236

  15. Valine pyrrolidide preserves intact glucose-dependent insulinotropic peptide and improves abnormal glucose tolerance in minipigs with reduced beta-cell mass

    DEFF Research Database (Denmark)

    Larsen, Marianne Olholm; Rolin, Bidda; Ribel, Ulla; Wilken, Michael; Deacon, Carolyn F; Svendsen, Ove; Gotfredsen, Carsten F; Carr, Richard David

    2003-01-01

    for type 2 diabetes was evaluated in a new large animal model of insulin-deficient diabetes and reduced beta-cell mass, the nicotinamide (NIA) (67 mg/kg) and streptozotocin (STZ) (125 mg/kg)-treated minipig, using the DPPIV inhibitor, valine pyrrolidide (VP) (50 mg/kg). VP did not significantly affect...... levels of intact GLP-1 but increased levels of intact GIP (from 4543 +/- 1880 to 9208 +/- 3267 pM x min; P <.01), thus improving glucose tolerance (area under the curve [AUC] for glucose reduced from 1904 +/- 480 to 1582 +/- 353 mM x min; P =.05). VP did not increase insulin levels during the oral......; however, additional mechanisms for the effect of DPPIV inhibition cannot be excluded. The authors conclude that DPPIV inhibitors may be useful to treat type 2 diabetes, even when this is due to reduced beta-cell mass....

  16. Single cell transcriptional analysis reveals novel innate immune cell types

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  17. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by β-chloroalanine

    International Nuclear Information System (INIS)

    The effects of β-chloroalanine (β-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with β-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with β-Cl-alanine and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of β-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although β-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that β-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis

  18. Phosphorylation substrates for protein kinase C in intact pituitary cells: characterization of a receptor-mediated event using novel gonadotropin-releasing hormone analogues

    International Nuclear Information System (INIS)

    The involvement of protein kinase C in the signal transduction of gonadotropin-releasing hormone (GnRH) action was investigated with a GnRH superagonist, partial agonists, and antagonists in intact rat pituitary cells. Exposure of 32P-labeled cells to GnRH or to the superagonist [D-Nal(2)6]GnRH induced the enhanced phosphorylation of 42-, 34-, 11-, and 10-kDa proteins and the dephosphorylation of a 15-kDa protein as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. This effect was blocked in a dose-dependent manner by potent GnRG antagonists. Downregulation of protein kinase C by prolonged incubation of the pituitary cells with high concentrations of active phorbol esters abolished protein kinase C activity and also prevented the phosphorylation induced by GnRN, or [D-Nal(2)6]GnRH. The same effect was obtained by preincubating the cells with the protein kinase C inhibitor H-7. In this study the authors identify for the first time physiological substrates for protein kinase C in intact pituitary cells. They demonstrate a close quantitative correlation between the extent of translocation of protein kinase C, levels of phosphorylation of specific substrates in the intact cells, and the biological activity of the GnRH analogues with varying affinity for the GnRH receptor. These data strengthen the contention that the physiological effects of GnRH are primarily mediated via the phosphatidylinositol/Ca2+ signal transfer system and represent a first step toward defining the physiological substrates of protein kinase C and their role in the cascade of events that starts upon binding of GnRH to its receptor

  19. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -chloroalanine

    Energy Technology Data Exchange (ETDEWEB)

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-09-06

    The effects of ..beta..-chloroalanine (..beta..-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with ..beta..-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with ..beta..-Cl-alanine and this treatment inhibited (/sup 14/C)serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of ..beta..-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, (/sup 14/C)serine uptake was not blocked, total lipid biosynthesis from (/sup 14/C)acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and (/sup 3/H)thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of (/sup 3/H)leucine, which was only decreased by 14%. Although ..beta..-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that ..beta..-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

  20. Quantification of intact and truncated stromal cell-derived factor-1α in circulation by immunoaffinity enrichment and tandem mass spectrometry.

    Science.gov (United States)

    Wang, Weixun; Choi, Bernard K; Li, Wenyu; Lao, Zhege; Lee, Anita Y H; Souza, Sandra C; Yates, Nathan A; Kowalski, Timothy; Pocai, Alessandro; Cohen, Lucinda H

    2014-04-01

    Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α's two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions. PMID:24500701

  1. Quantification of Intact and Truncated Stromal Cell-Derived Factor-1α in Circulation by Immunoaffinity Enrichment and Tandem Mass Spectrometry

    Science.gov (United States)

    Wang, Weixun; Choi, Bernard K.; Li, Wenyu; Lao, Zhege; Lee, Anita Y. H.; Souza, Sandra C.; Yates, Nathan A.; Kowalski, Timothy; Pocai, Alessandro; Cohen, Lucinda H.

    2014-04-01

    Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α's two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions.

  2. Cloning of adeno-associated virus into pBR322: Rescue of intact virus from the recombinant plasmid in human cells

    OpenAIRE

    1982-01-01

    We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescu...

  3. Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

    OpenAIRE

    Belousov, E S; Afonina, I A; Kutyavin, I V; Gall, A A; Reed, M W; Gamper, H B; Wydro, R M; Meyer, R. B.

    1998-01-01

    A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the...

  4. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    International Nuclear Information System (INIS)

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa β-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor β-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission

  5. The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Aksmann, Anna, E-mail: bioaak@ug.edu.pl [Department of Plant Physiology, University of Gdansk, Al. Marszalka Pilsudskiego 46, 81-378 Gdynia (Poland); Shutova, Tatiana; Samuelsson, Goeran [Umea Plant Science Centre, Department of Plant Physiology, Umea University, SE 90187 Umea (Sweden); Tukaj, Zbigniew [Department of Plant Physiology, University of Gdansk, Al. Marszalka Pilsudskiego 46, 81-378 Gdynia (Poland)

    2011-08-15

    Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

  6. The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

  7. Inotropic effect, binding properties, and calcium flux effects of the calcium channel agonist CGP 28392 in intact cultured embryonic chick ventricular cells

    International Nuclear Information System (INIS)

    CGP 28392 is a recently described dihydropyridine derivative with positive inotropic properties. To study the mechanism of action of this putative calcium channel agonist, we have related the effects of CGP 28392 on contraction (measured with an optical video system) and radioactive calcium uptake to ligand-binding studies in cultured, spontaneously beating chick embryo ventricular cells. CGP 28392 produced a concentration-dependent increase in amplitude and velocity of contraction (EC50 = 2 x 10(-7) M; maximum contractile effect = 85% of the calcium 3.6 mM response). Nifedipine produced a shift to the right of the concentration-effect curve for CGP 28392 without decreasing the maximum contractile response, suggesting competitive antagonism (pA2 = 8.3). Computer analysis of displacement of [3H]nitrendipine binding to intact heart cells by unlabeled CGP 28392 indicated a K /sub D/ = 2.2 +/- 0.95 x 10(-7) M, in good agreement with the EC50 for the inotropic effect. CGP 28392 increased the rate of radioactive calcium influx (+39% at 10 seconds) without altering beating rate, while nifedipine decreased radioactive calcium influx and antagonized the CGP 28392-induced increase in calcium influx. Our results indicate that, in intact cultured myocytes, CGP 28392 acts as a calcium channel agonist and competes for the dihydropyridine-binding site of the slow calcium channel. In contrast to calcium channel blockers, CGP 28392 increases calcium influx and enhances the contractile state

  8. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns

    DEFF Research Database (Denmark)

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone;

    2015-01-01

    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties....... Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and coexisting PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human...... hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly...

  9. Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

    Directory of Open Access Journals (Sweden)

    Kanji Oshima

    Full Text Available The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation

  10. 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

    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 by such...

  11. Metabolism of the persistent plasticizer chemical bis(2-ethylhexyl) phthalate in cell suspension cultures of wheat (Triticum aestivum L.). Discrepancy from the intact plant

    International Nuclear Information System (INIS)

    Cell suspension cultures of wheat (Triticum aestivum L.) metabolized the persistent plasticizer chemical bis(2-ethylhexyl) phthalate (DEHP; 1 ppm) predominantly to β-D-glucosyl conjugates. After incubation for 48 h at 270C, 23% of the applied radioactively labeled chemical was recovered in the total polar metabolite fraction. Prior heat treatment of freeze-thawing of the wheat cells abolished conjugate formation and led to mono(2-ethylhexyl) phthalate (MEHP)) as the predominant metabolite (up to 10% conversion). Direct feeding of MEHP to native wheat cells led to 93% conversion to polar metabolites, again consisting largely of β-D-glucosyl conjugates. This suggested that MEHP was a metabolic intermediate and that DEHP esterase activity was rate limiting in DEHP metabolism. The rate of cellular DEHP metabolism in fact agreed with the rate of the DEHP esterase reaction determined in crude cell-free extracts. Therefore, no significant permeation barrier between the intracellular enzyme and external DEHP appeared to exist in cell suspension cultures. In contrast, the DEHP esterase activity of intact leaves has previously been found to be inaccessible to external DEHP

  12. Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells

    OpenAIRE

    Hug, H; Los, Marek Jan; Hirt, W; Debatin, K. M.

    1999-01-01

    Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is...

  13. FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.

    Science.gov (United States)

    Börner, Sebastian; Schwede, Frank; Schlipp, Angela; Berisha, Filip; Calebiro, Davide; Lohse, Martin J; Nikolaev, Viacheslav O

    2011-04-01

    Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d. PMID:21412271

  14. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

    2016-02-15

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  15. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin

  16. Regulation of the Na(+)-K+ pump activity and estimation of the reserve capacity in intact rat peritoneal mast cells

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1990-01-01

    Evidence is provided that regulation of the Na(+)-K+ pump activity in rat peritoneal mast cells occurs mainly through stimulation of the pump from inside the plasma membrane by sodium. It is demonstrated that there is a large reserve capacity for the exchange of intracellular sodium with extracel......Evidence is provided that regulation of the Na(+)-K+ pump activity in rat peritoneal mast cells occurs mainly through stimulation of the pump from inside the plasma membrane by sodium. It is demonstrated that there is a large reserve capacity for the exchange of intracellular sodium with...

  17. Monitoring Intact Viruses Using Aptamers.

    Science.gov (United States)

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  18. Monitoring Intact Viruses Using Aptamers

    Directory of Open Access Journals (Sweden)

    Penmetcha K. R. Kumar

    2016-08-01

    Full Text Available Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review.

  19. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Science.gov (United States)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  20. MRI of intact plants

    NARCIS (Netherlands)

    As, van H.; Scheenen, T.; Vergeldt, F.J.

    2009-01-01

    Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transpor

  1. MRI of intact plants.

    NARCIS (Netherlands)

    As, H. van; Scheenen, T.W.J.; Vergeldt, F.J.

    2009-01-01

    Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transpor

  2. Gold nanoparticles assisted laser desorption/ionization mass spectrometry and applications: from simple molecules to intact cells.

    Science.gov (United States)

    Abdelhamid, Hani Nasser; Wu, Hui-Fen

    2016-07-01

    Gold nanoparticles (AuNPs) assisted laser desorption/ionization mass spectrometry (GALDI-MS) provided new horizons and offered many functions for various applications. This review summarized AuNPs applications for analytical, biotechnology and proteomics. AuNPs efficiently absorbed the laser radiation and transferred the energy to the analyte for the desorption/ionization process. The unique features of AuNPs such as large surface area and high absorption coefficient lead not only to high resolution, low interference and low limit of detection, but also offered selective detection for certain species. AuNPs provided an excellent surface for the analysis of several species such as small molecules, biomarkers, proteins and cells (pathogenic bacteria or cancer cells). AuNPs played many roles such as surface for LDI-MS, probe and stationary phase for separation or preconcentration. AuNPs modified various surface chemistry was applied for a wide range of different wavelength. AuNPs severed as a source of Au(+) ions that were suitable for analyte cationisation. Characterization of Au nanoclusters (AuNCs) by mass spectrometry, pros and cons were also highlighted. Graphical Abstract Schematic representation of the analysis by Gold Nanoparticles Assisted Laser Desorption/Ionization Mass Spectrometry (GALDI-MS). PMID:26973236

  3. CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity.

    Science.gov (United States)

    Daly, Owen M; Gaboriau, David; Karakaya, Kadin; King, Sinéad; Dantas, Tiago J; Lalor, Pierce; Dockery, Peter; Krämer, Alwin; Morrison, Ciaran G

    2016-05-01

    Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR. PMID:26966185

  4. Inhibition of Na(+) -K+ pump activity by divalent cations in intact peritoneal mast cells of the rat

    DEFF Research Database (Denmark)

    Knudsen, T; Berthelsen, Carsten; Johansen, Torben

    1990-01-01

    1. The inhibition by the divalent cations magnesium, barium and strontium and the trivalent ion lanthanum of the Na(+) -K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure mast cell populations by measurement of the ouabain-sensitive uptake of the radioactive potassium......-resistant uptake was not changed. Half maximum decrease in the ouabain-sensitive K+(86Rb+)-uptake was observed with 1.8 mM magnesium, 1.2mM barium and 0.7 mM strontium. 4. The trivalent ion lanthanum blocked almost completely the ouabain-sensitive K+(86Rb+)-uptake at a concentration of 1 microM as does 1 m......M calcium. Combining either of these ions with magnesium had no further inhibitory effect on the ouabain-sensitive uptake. 5. In conclusion, in addition to the previously suggested modulation by calcium of the activity of the Na+ (-)K+ pump, evidence is provided in this investigation that the modulation may...

  5. Intact Cohesion, Anaphase, and Chromosome Segregation in Human Cells Harboring Tumor-Derived Mutations in STAG2.

    Directory of Open Access Journals (Sweden)

    Jung-Sik Kim

    2016-02-01

    Full Text Available Somatic mutations of the cohesin complex subunit STAG2 are present in diverse tumor types. We and others have shown that STAG2 inactivation can lead to loss of sister chromatid cohesion and alterations in chromosome copy number in experimental systems. However, studies of naturally occurring human tumors have demonstrated little, if any, correlation between STAG2 mutational status and aneuploidy, and have further shown that STAG2-deficient tumors are often euploid. In an effort to provide insight into these discrepancies, here we analyze the effect of tumor-derived STAG2 mutations on the protein composition of cohesin and the expected mitotic phenotypes of STAG2 mutation. We find that many mutant STAG2 proteins retain their ability to interact with cohesin; however, the presence of mutant STAG2 resulted in a reduction in the ability of regulatory subunits WAPL, PDS5A, and PDS5B to interact with the core cohesin ring. Using AAV-mediated gene targeting, we then introduced nine tumor-derived mutations into the endogenous allele of STAG2 in cultured human cells. While all nonsense mutations led to defects in sister chromatid cohesion and a subset induced anaphase defects, missense mutations behaved like wild-type in these assays. Furthermore, only one of nine tumor-derived mutations tested induced overt alterations in chromosome counts. These data indicate that not all tumor-derived STAG2 mutations confer defects in cohesion, chromosome segregation, and ploidy, suggesting that there are likely to be other functional effects of STAG2 inactivation in human cancer cells that are relevant to cancer pathogenesis.

  6. Ferromagnetic resonance of intact cells and isolated crystals from cultured and uncultured magnetite-producing magnetotactic bacteria

    International Nuclear Information System (INIS)

    Most magnetotactic bacteria (MB) produce stable, single-domain magnetite nanocrystals with species-specific size, shape and chain arrangement. In addition, most crystals are elongated along the [111] direction, which is the easy axis of magnetization in magnetite, chemically pure and structurally perfect. These special characteristics allow magnetite crystal chains from MB to be recognized in environmental samples including old sedimentary rocks. Ferromagnetic resonance (FMR) has been proposed as a powerful and practical tool for screening large numbers of samples possibly containing magnetofossils. Indeed, several studies were recently published on FMR of cultured MB, mainly Magnetospirillum gryphiswaldense. In this work, we examined both uncultured magnetotactic cocci and the cultured MB M. gryphiswaldense using transmission electron microscopy (TEM) and FMR from 10 K to room temperature (RT). The TEM data supported the FMR spectral characteristics of our samples. The FMR spectra of both bacteria showed the intrinsic characteristics of magnetite produced by MB, such as extended absorption at the low field region of the spectra and a Verwey transition around 100 K. As previously observed, the spectra of M. gryphiswaldense isolated crystals were more symmetrical than the spectra obtained from whole cells, reflecting the loss of chain arrangement due to the small size and symmetrical shape of the crystals. However, the FMR spectra of magnetic crystals isolated from magnetotactic cocci were very similar to the FMR spectra of whole cells, because the chain arrangement was maintained due to the large size and prismatic shape of the crystals. Our data support the use of FMR spectra to detect magnetotactic bacteria and magnetofossils in samples of present and past environments. Furthermore, the spectra suggest the use of the temperature transition of spectral peak-to-peak intensity to obtain the Verwey temperature for these systems. (papers)

  7. Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Traber, M.G.; Thellman, C.A.; Rindler, M.J.; Kayden, H.J.

    1988-09-01

    The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 (PEG 1000)) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. (/sup 14/C)TPGS (synthesized from (/sup 14/C)PEG 1000) was taken up by Caco-2 cells but (/sup 14/C)PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the (/sup 14/C)TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule.

  8. Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

    International Nuclear Information System (INIS)

    The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 [PEG 1000]) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. [14C]TPGS (synthesized from [14C]PEG 1000) was taken up by Caco-2 cells but [14C]PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the [14C]TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule

  9. Effects of Incubation Conditions on Cr(VI) Reduction by c-type Cytochromes in Intact Shewanella oneidensis MR-1 Cells

    Science.gov (United States)

    Han, Rui; Li, Fangbai; Liu, Tongxu; Li, Xiaomin; Wu, Yundang; Wang, Ying; Chen, Dandan

    2016-01-01

    It is widely recognized that the outer membrane c-type cytochromes (OM c-Cyts) of metal-reducing bacteria play a key role in microbial metal reduction processes. However, the in situ redox status of OM c-Cyts during microbial metal reduction processes remain poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy is used to investigate the in situ spectral reaction of Cr(VI) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. The Cr(VI) reduction rates decreased with increasing initial Cr(VI) concentrations, and Cr(III) was identified as a reduced product. The presence of Cr(III) substantially inhibited Cr(VI) reduction and the recovery of reduced c-Cyts, indicating that Cr(III) might inhibit cell growth. Cr(VI) reduction rates increased with increasing cell density. The highest Cr(VI) reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30°C, with sodium lactate serving as an electron donor. The presence of O2 strongly inhibited Cr(VI) reduction, suggesting that O2 might compete with Cr(VI) as an electron acceptor in cells. This study provides a case of directly examining in vivo reaction properties of an outer-membrane enzyme during microbial metal reduction processes under non-invasive physiological conditions. PMID:27242759

  10. Optoperforation of Intact Plant Cells, Spectral Characterization of Alloy Disorder in InAsP Alloys, and Bimetallic Concentric Surfaces for Metal-Enhanced Fluorescence in Upconverting Nanocrystals

    Science.gov (United States)

    Merritt, Travis R.

    The techniques of optoperforation, spectral characterization of alloy disorder, and metal-enhanced uorescence were applied to previously unconsidered or disregarded systems in order to demonstrate that such applications are both feasible and consequential. These applications were the subject of three disparate works and, as such, are independently discussed. Despite being ostensibly restricted to mammalian cells, optoperforation was demonstrated in intact plant cells by means of successful femtosecond-laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into cells of vital Arabidopsis seedling stems. By monitoring the rate of dye uptake, and the reaction of both CFP-expressing vacuoles and nanocellulose substrates, the intensity and exposure time of the perforating laser were adjusted to values that both preserved cell vitality and permitted the laser-assisted uptake of the uorophore. By using these calibrated laser parameters, dye was injected and later observed in targeted cells after 72 hours, all without deleteriously affecting the vital functions of those cells. In the context of alloy disorder, photoluminescence of excitonic transitions in two InAsxP1--x alloys were studied through temperature and magnetic field strength dependencies, as well as compositionally-dependent time-resolved behavior. The spectral shape, behavior of the linewidths at high magnetic fields, and the divergence of the peak positions from band gap behavior at low temperatures indicated that alloy disorder exists in the x=0.40 composition while showing no considerable presence in the x=0.13 composition. The time-resolved photoluminescence spectrum for both compositions feature a fast and slow decay, with the slow decay lifetime in x=0.40 being longer than that of x=0.13, which may be due to carrier migration between localized exciton states in x=0.40. In order to achieve broadband metal-enhanced uorescence in upconverting NaYF4:Yb,Er nanocrystals, two nanocomposite

  11. MRI of intact plants

    OpenAIRE

    As, van, H.; Scheenen, T.; Vergeldt, F.J.

    2009-01-01

    Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transport in the stem, e.g., as a function of environmental (stress) conditions. Non-spatially resolved portable NMR is becoming available to study leaf water content and distribution of water in different...

  12. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  13. Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

    OpenAIRE

    van Lierop, M J; Wagenaar, J P; Van Noort, J M; Hensen, E J

    1995-01-01

    Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.

  14. (Photosynthesis in intact plants)

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  15. The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant, Kalanchoe daigremontiana Hamet et Perr.

    Science.gov (United States)

    Rygol, J; Winter, K; Zimmermann, U

    1987-12-01

    Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 μeq H(+)·g(-1) fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO2-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (ΔH=160 μeq H(+)·g(-1) fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM. PMID:24226067

  16. Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

    Science.gov (United States)

    Mizutani, Tatsuaki; Neugebauer, Nina; Putz, Eva M.; Moritz, Nadine; Simma, Olivia; Zebedin-Brandl, Eva; Gotthardt, Dagmar; Warsch, Wolfgang; Eckelhart, Eva; Kantner, Hans-Peter; Kalinke, Ulrich; Lienenklaus, Stefan; Weiss, Siegfried; Strobl, Birgit; Müller, Mathias; Sexl, Veronika; Stoiber, Dagmar

    2012-01-01

    Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance. PMID:23170251

  17. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  18. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  19. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    Science.gov (United States)

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation. PMID:26000486

  20. Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

    OpenAIRE

    Garz, Andreas; Sandmann, Michael; Rading, Michael; Ramm, Sascha; Menzel, Ralf; Steup, Martin

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cel...

  1. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  2. Biomimetic emulsions reveal the effect of homeostatic pressure on cell-cell adhesion

    CERN Document Server

    Pontani, Lea-Laetitia; Viasnoff, Virgile; Brujic, Jasna

    2012-01-01

    Cell-cell contacts in tissues are continuously subject to mechanical forces due to homeostatic pressure and active cytoskeleton dynamics. While much is known about the molecular pathways of adhesion, the role of mechanics is less well understood. To isolate the role of pressure we present a dense packing of functionalized emulsion droplets in which surface interactions are tuned to mimic those of real cells. By visualizing the microstructure in 3D we find that a threshold compression force is necessary to overcome electrostatic repulsion and surface elasticity and establish protein-mediated adhesion. Varying the droplet interaction potential maps out a phase diagram for adhesion as a function of force and salt concentration. Remarkably, fitting the data with our theoretical model predicts binder concentrations in the adhesion areas that are similar to those found in real cells. Moreover, we quantify the adhesion size dependence on the applied force and thus reveal adhesion strengthening with increasing homeos...

  3. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  4. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

    Directory of Open Access Journals (Sweden)

    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  5. Capillary hydrodynamic chromatography reveals temporal profiles of cell aggregates.

    Science.gov (United States)

    Tang, Ya-Ru; Huang, Hsin-Yi; Hu, Jie-Bi; Rattinam, Rajesh; Li, Chun-Hsien; Chen, Yu-Chie; Urban, Pawel L

    2016-03-01

    Microbial cells are known to form aggregates. Such aggregates can be found in various matrices; for example, functional drinks. Capillary hydrodynamic chromatography (HDC) enables separation of particles by size using nanoliter-scale volumes of samples. Here we propose an approach based on HDC for characterisation of real samples containing aggregated and non-aggregated bacterial and fungal cells. Separation of cells and cell aggregates in HDC arises from the parabolic flow profile under laminar flow conditions. In the presented protocol, hydrodynamic separation is coupled with different on-line and off-line detectors (light absorption/scattering and microscopy). The method has successfully been applied in the monitoring of dynamic changes in the microbiome of probiotic drinks. Chromatographic profiles of yogurt and kefir samples obtained at different times during fermentation are in a good agreement with microscopic images. Moreover, thanks to the implementation of an area imaging detector, capillary HDC could be multiplexed and used to profile spatial gradients in cell suspensions, which arise in the course of sedimentation of cells and cell aggregates. This result shows compatibility of sedimentation analysis and capillary HDC. We believe that the approach may find applications in the profiling of functional foods and other matrices containing aggregated bioparticles. PMID:26873471

  6. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  7. Transcriptome network analysis reveals candidate genes for renal cell carcinoma

    OpenAIRE

    Wei Zhai; Yun-Fei Xu; Min Liu; Jun-Hua Zheng

    2012-01-01

    Context: Renal cell carcinoma (RCC) is a kidney cancer that originates in renal parenchyma and it is the most common type of kidney cancer with approximately 80% lethal cases. Aims: To interpret the mechanism, explore the regulation of TF-target genes and TF-pathway, and identify the potential key genes of renal cell carcinoma. Settings and Design: After constructing a regulation network from differently expressed genes and transcription factors, pathway regulation network and gene onto...

  8. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    OpenAIRE

    Laura-Roxana Stingaciu; Hugh O’Neill; Michelle Liberton; Urban, Volker S.; Himadri B. Pakrasi; Michael Ohl

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membran...

  9. Cellular and biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2 and inactivated influenza A (H3N2 virus at 12x and 30x in the MDCK cells

    Directory of Open Access Journals (Sweden)

    Mariah Marcondes

    2011-09-01

    Full Text Available Biotherapics are homeopathic remedies prepared from organic products that are chemically undefined and can be used for treatment of diseases like influenza. There are several classes of biotherapics and, among these, there are some called "living biotherapics" or "Roberto Costa’s Biotherapics". This study aimed to compare the cellular and biochemical effects of biotherapics prepared from intact influenza virus diluted in water and the one obtained from the same viral sample inactivated by ethanol 70% (v / v, both in the potencies of 12x and 30x. Transmission electron microscopy (TEM analyses were performed on both preparations to assess the integrity of viral particles, which showed that ethanol 70% (v/v induced a complete denaturation of viral particles. In contrast, the integrity of virus particles was preserved when water was used as the biotherapic solvent. Cellular and biochemical alterations induced by the preparations on MDCK cells were analyzed and compared with those induced by respective controls (water 30x-treated and untreated cells. Cellular viability analyzed by MTT method showed statistically significant differences (p <0.05 in MDCK cells treated with intact biotherapic for 5 (3 stimuli and 30 (18 stimuli days in comparison with untreated control. TEM analysis did not show significant cellular changes when the different experimental groups were compared. The enzymatic activity of phosphofructokinase 1 (PFK, an important enzyme in the glycolytic pathway, presented a statistically significant increase (p <0.05 after 30 days of treatment when compared to control groups. The results obtained suggest that inactivation of viral sample with ethanol 70% induces lysis and disruption of viral particles. In addition, preliminary results indicated that treatment with intact biotherapic seems to induce higher variations on MDCK cells responses when compared to inactivated-biotherapic-treated cells. Further analyses are ongoing

  10. Nanoparticle-Based Immunocytochemistry Reveals Microarchitecture of the Cell Nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Kyoto : International Federation of Societies for Histochemistry and Cytochemistry, Japan Society of Histochemistry, 2012. [14th International Congress of Histochemistry and Cytochemistry. Kyoto (JP), 26.08.2012-29.08.2012] R&D Projects: GA ČR GAP305/11/2232; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MPO(CZ) FRTI3588 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : PIP2 * NMI * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  11. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Mérida : CIASEM, AMM, 2011. ---. [Inter-American Congress on Microscopy /11./. 25.09.2011-29.09.2011, Mérida] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  12. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Istanbul: Turkish Society for Electron Microscopy, 2011. ---. [National Electron Microscopy Congress /20./. 25.10.2011-28.10.2011, Kemer] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MŠk 2B06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  13. Single cell activity reveals direct electron transfer in methanotrophic consortia

    Science.gov (United States)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  14. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    Science.gov (United States)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  15. A new mechanism for the regulation of stomatal aperture size in intact leaves: accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba

    International Nuclear Information System (INIS)

    At various times after pulse-labeling broad bean (Vicia faba L.) leaflets with 14CO2, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents, whereas those from rinsed peels contained only symplastic contents. Sucrose (Suc)-specific radioactivity peaked (111 GBq mol-1) in palisade cells at 20 min. In contrast, the 14C content and Suc-specific radioactivity were very low in guard cells for 20 min, implying little CO2 incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum Suc-specific radioactivity (204 GBq mol-1) and a high Suc influx rate (0.05 pmol stoma-1 min-1). These and other comparisons implied the presence of (a) multiple Suc pools in mesophyll cells, (b) a localized mesophyll-apoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived Suc in guard-cell walls sufficient to diminish stomatal opening by approximately 3 micrometers. Factors expected to enhance Suc accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic Suc concentration, which is elevated when mesophyll Suc efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal aperture size by this previously unrecognized mechanism

  16. Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

    DEFF Research Database (Denmark)

    Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retin...

  17. Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    XuanWang; YanyanLu; HuanqingZhang; KunWang; QihuaHe; YueWang; XianyuLiu; LinsongLi; XiaominWang

    2005-01-01

    Neural progenitor cells have shown tile effectiveness in the treatment of Parkinson's disease, but tile therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity ofpre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50ng/ml fibroblast growth factor 8, 10ng/ml glial cell line-derived neurotrophic factor and 10μM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum ofparkinsonian rats, only pre-differentiated grafts resulted in an elevated production ofdopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intactprogenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance ofpre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.

  18. Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

    OpenAIRE

    Rowland Raymond RR; Ward-Demo Pam; Said Suleman; Wong Grace HW; Duman Richard G; Cafruny William A; Nelson Eric A

    2006-01-01

    Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experime...

  19. Language and Williams syndrome: how intact is "intact"?

    Science.gov (United States)

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  20. HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells

    International Nuclear Information System (INIS)

    HIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293 T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC-Top2A, BAC-DNMT1, or BAC-BACH2, but only BAC-DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-α resulted in virus production from four of five BAC-Top2A and all BAC-DNMT1 cell lines, but not from the BAC-BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency.

  1. Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery

    Science.gov (United States)

    Hu, Xiongwei; Fan, Wufa; Yu, Zhou; Lu, Yi; Qi, Jianping; Zhang, Jian; Dong, Xiaochun; Zhao, Weili; Wu, Wei

    2016-03-01

    Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and simulated mixed micelles (SMMs) to intestinal mucus, but no evidence of penetration of integral nanocarriers. Both SLNs and SMMs exhibit significant cellular uptake, but fail to penetrate cell monolayers. Confocal laser scanning microscopy reveals that nanocarriers mainly concentrate on the surface of the monolayers, and no evidence of penetration of intact vehicles can be obtained. The mucous layer acts as a barrier to the penetration of both SLNs and SMMs. Both bile salt-decoration and SMM formulation help to strengthen the interaction with biomembranes. It is concluded that evidence does not support absorption of intact SLNs via oral delivery.Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and

  2. Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation

    OpenAIRE

    Sugiyama, Takuya; Benitez, Cecil M.; Ghodasara, Amar; Liu, Lucy; McLean, Graeme W.; Lee, Jonghyeob; Blauwkamp, Timothy A; Nusse, Roeland; Wright, Christopher V.E.; Gu, Guoqiang; Kim, Seung K.

    2013-01-01

    Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A...

  3. Dendritic Cell Activation and Cytokine Production Induced by Group B Neisseria meningitidis: Interleukin-12 Production Depends on Lipopolysaccharide Expression in Intact Bacteria

    OpenAIRE

    Dixon, Garth L. J.; Newton, Phillippa J.; Chain, Benjamin M; Katz, David; Andersen, Svein Rune; Wong, Simon; van der Ley, Peter; Klein, Nigel; Callard, Robin E.

    2001-01-01

    Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of...

  4. Live-Cell Imaging of Dual-Labeled Golgi Stacks in Tobacco BY-2 Cells Reveals Similar Behaviors for Different Cisternae during Movement and Brefeldin A Treatment

    Institute of Scientific and Technical Information of China (English)

    Stephanie L. Madison; Andreas Nebenführ

    2011-01-01

    In plant cells,the Golgi apparatus consists of numerous stacks that,in turn,are composed of several flattened cisternae with a clear cis-to-trans polarity.During normal functioning within living cells,this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility,constant membrane flux through the cisternae,and Golgi enzyme recycling through the ER.In order to further investigate various aspects of Golgi stack dynamics and integrity,we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments,movement,and brefeldin A (BFA)-induced disassembly.A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm.The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead,but trans cisternae were also found at the leading edge.During BFA treatments,the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however,no consistent order could be detected.In contrast,the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected.Our results thus demonstrate a remarkable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER.In addition,we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

  5. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2009-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  6. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin.

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G

    2009-02-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  7. A gallbladder tumor revealing metastatic clear cell renal carcinoma: report of case and review of literature

    OpenAIRE

    Ghaouti Merieme; Znati Kaoutar; Jahid Ahmed; Zouaidia Fouad; Bernoussi Zakiya; Fakir Youssef El; Mahassini Najat

    2013-01-01

    Abstract Metastatic renal cell carcinoma in the gallbladder is extremely rare, with reported frequencies of less than 0.6% in large autopsy reviews. Only 40 cases were reported in the literature. We report a first case of gallbladder polypoid tumor revealing metastatic clear cell renal cell carcinoma, which demonstrates the importance of radiological tests, histology and immunohistochemistry when making a definitive diagnosis. These examinations also allow differentiating metastatic clear cel...

  8. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    Science.gov (United States)

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  9. Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node

    Science.gov (United States)

    Miller, Mark J.; Wei, Sindy H.; Parker, Ian; Cahalan, Michael D.

    2002-06-01

    Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to ``swarms'' and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.

  10. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

    Science.gov (United States)

    Gokce, Ozgun; Stanley, Geoffrey M; Treutlein, Barbara; Neff, Norma F; Camp, J Gray; Malenka, Robert C; Rothwell, Patrick E; Fuccillo, Marc V; Südhof, Thomas C; Quake, Stephen R

    2016-07-26

    The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states. PMID:27425622

  11. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

    Full Text Available The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  12. Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

    OpenAIRE

    Fobert, P R; Coen, E S; Murphy, G. J.; Doonan, J H

    1994-01-01

    Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstainin...

  13. Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses

    DEFF Research Database (Denmark)

    Benninger, Richard K P; Vanherberghen, Bruno; Young, Stephen;

    2009-01-01

    We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into...... absent from the center of the mature synapse. Understanding the role of such extensive membrane ruffling in the assembly of cytolytic synapses is an intriguing new goal....

  14. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation

    DEFF Research Database (Denmark)

    Klochendler, Agnes; Weinberg-Corem, Noa; Moran, Maya;

    2012-01-01

    biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to......Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive...... reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of...

  15. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    Science.gov (United States)

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells. PMID:27074779

  16. Single-Cell Transcriptome Analyses Reveal Signals to Activate Dormant Neural Stem Cells

    OpenAIRE

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary

    2015-01-01

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133+/GFAP− ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133+/GFAP− quiescent cells were enriched...

  17. Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.

    OpenAIRE

    Luhmann, Heiko,; Dzhala, V.I.; Ben-Ari, Yehezkel

    2000-01-01

    We examined the generation, propagation and pharmacology of 4-aminopyridine (4-AP)-induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0-7) rat in vitro. Whole-cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4-AP-induced EA as early as P0-1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal co...

  18. Epithelial structure revealed by chemical dissection and unembedded electron microscopy

    OpenAIRE

    Fey, E G; Capco, D G; Krochmalnic, G; Penman, S

    1984-01-01

    Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton w...

  19. Kinetic modeling reveals a common death niche for newly formed and mature B cells.

    Directory of Open Access Journals (Sweden)

    Gitit Shahaf

    Full Text Available BACKGROUND: B lymphocytes are subject to elimination following strong BCR ligation in the absence of appropriate second signals, and this mechanism mediates substantial cell losses during late differentiation steps in the bone marrow and periphery. Mature B cells may also be eliminated through this mechanism as well as through normal turnover, but the population containing mature cells destined for elimination has not been identified. Herein, we asked whether the transitional 3 (T3 subset, which contains most newly formed cells undergoing anergic death, could also include mature B cells destined for elimination. METHODOLOGY/PRINCIPAL FINDINGS: To interrogate this hypothesis and its implications, we applied mathematical models to previously generated in vivo labeling data. Our analyses reveal that the death rate of T3 B cells is far higher than the death rates of all other splenic B cell subpopulations. Further, the model, in which the T3 pool includes both newly formed and mature primary B cells destined for apoptotic death, shows that this cell loss may account for nearly all mature B cell turnover. CONCLUSIONS/SIGNIFICANCE: This finding has implications for the mechanism of normal mature B cell turnover.

  20. Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

    OpenAIRE

    Alvarez-Dominguez, Juan R.; Hu, Wenqian; Yuan, Bingbing; Shi, Jiahai; Park, Staphany S.; Gromatzky, Austin A.; van Oudenaarden, Alexander; Lodish, Harvey F.

    2014-01-01

    Global lncRNA discovery reveals novel erythroid-specific lncRNAs that are dynamically expressed and targeted by GATA1, TAL1, and KLF1.Multiple types of lncRNAs promote red cell maturation by regulating neighboring loci, including DLEU2 and a novel Band 3 enhancer lncRNA.

  1. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    OpenAIRE

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M.

    2012-01-01

    Rubin and colleagues describe the first structures of full-length and phosphorylated Retinoblastoma (Rb) protein. These structures reveal the mechanism of Rb inactivation and provide valuable insight into this critical tumor suppressor protein's allosteric inhibition via multisite Cdk phosphorylation and its E2F and cell cycle regulation.

  2. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  3. Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

    International Nuclear Information System (INIS)

    Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

  4. Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape.

    Science.gov (United States)

    Li, Qin; Wennborg, Anders; Aurell, Erik; Dekel, Erez; Zou, Jie-Zhi; Xu, Yuting; Huang, Sui; Ernberg, Ingemar

    2016-03-01

    The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker-Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer. PMID:26929366

  5. Principles of bacterial cell-size determination revealed by cell wall synthesis perturbations

    OpenAIRE

    Carolina Tropini; Timothy K. Lee; Jen Hsin; Samantha M. Desmarais; Tristan Ursell; Russell D. Monds; Kerwyn Casey Huang

    2014-01-01

    Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cyto...

  6. Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq.

    Science.gov (United States)

    Chan, Sunny Sun-Kin; Chan, Howe H W; Kyba, Michael

    2016-06-01

    Mesp1 is a transcription factor that promotes differentiation of pluripotent cells into different mesoderm lineages including hematopoietic, cardiac and skeletal myogenic. This occurs via at least two transient cell populations: a common hematopoietic/cardiac progenitor population and a common cardiac/skeletal myogenic progenitor population. It is not established whether Mesp1-induced mesoderm cells are intrinsically heterogeneous, or are simply capable of multiple lineage decisions. In the current study, we applied single-cell RNA-seq to analyze Mesp1+ mesoderm. Initial whole transcriptome analysis showed a surprising homogeneity among Mesp1-induced mesoderm cells. However, this apparent global homogeneity masked an intrinsic heterogeneity revealed by interrogating a panel of early mesoderm patterning factors. This approach enabled discovery of subpopulations primed for hematopoietic or cardiac development. These studies demonstrate the heterogeneic nature of Mesp1+ mesoderm. PMID:27131741

  7. Genomic instability of micronucleated cells revealed by single-cell comparative genomic hybridization.

    NARCIS (Netherlands)

    Imle, A.; Polzer, B.; Alexander, S.; Klein, C.A.; Friedl, P.H.A.

    2009-01-01

    Nuclear variation in size and shape and genomic instability are hallmarks of dedifferentiated cancer cells. Although micronuclei are a typical long-term consequence of DNA damage, their contribution to chromosomal instability and clonal diversity in cancer disease is unclear. We isolated cancer cell

  8. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    Science.gov (United States)

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  9. Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

    Directory of Open Access Journals (Sweden)

    Dmitri Papatsenko

    2015-08-01

    Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

  10. An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

    Directory of Open Access Journals (Sweden)

    Cecil M Benitez

    2014-10-01

    Full Text Available The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  11. In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering

    OpenAIRE

    Brune, T.; Borel, A.; Gilbert, T. W.; J P Franceschi; Badylak, S.F.; Sommer, P.

    2007-01-01

    The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of ...

  12. Genetically Induced Cell Death in Bulge Stem Cells Reveals Their Redundancy for Hair and Epidermal Regeneration

    OpenAIRE

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2014-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Set...

  13. In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Chien-Chih Ke

    Full Text Available Mesenchymal stromal cells (MSCs are multipotent adult stem cells which are recruited to the tumor microenvironment (TME and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

  14. Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human β Cell Function.

    Science.gov (United States)

    Arda, H Efsun; Li, Lingyu; Tsai, Jennifer; Torre, Eduardo A; Rosli, Yenny; Peiris, Heshan; Spitale, Robert C; Dai, Chunhua; Gu, Xueying; Qu, Kun; Wang, Pei; Wang, Jing; Grompe, Markus; Scharfmann, Raphael; Snyder, Michael S; Bottino, Rita; Powers, Alvin C; Chang, Howard Y; Kim, Seung K

    2016-05-10

    Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function. PMID:27133132

  15. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    Science.gov (United States)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  16. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    Science.gov (United States)

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. PMID:27523609

  17. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    Science.gov (United States)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  18. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    Science.gov (United States)

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  19. An integrated workflow for characterizing intact phosphoproteins from complex mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Si; Yang, Feng; Zhao, Rui; Tolic, Nikola; Robinson, Errol W.; Camp, David G.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2009-05-08

    The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography-mass spectrometry (LC-MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection.

  20. Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

    OpenAIRE

    Meierhofer, D.; Mayr, J. A.; Fink, K.; Schmeller, N.; Kofler, B; Sperl, W.

    2006-01-01

    Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHP...

  1. Functional Features of Trans-differentiated Hair Cells Mediated by Atoh1 Reveals a Primordial Mechanism

    OpenAIRE

    Yang, Juanmei; Bouvron, Sonia; Lv, Ping; Chi, Fanglu; Yamoah, Ebenezer N.

    2012-01-01

    Evolution has transformed a simple ear with few vestibular maculae into a complex 3-dimensional structure consisting of nine distinct endorgans. It is debatable whether the sensory epithelia underwent progressive segregation or emerged from distinct sensory patches. To address these uncertainties we examined the morphological and functional phenotype of trans-differentiated rat hair cells to reveal their primitive or endorgan-specific origins. Additionally, it is uncertain how Atoh1-mediated ...

  2. Early events leading to erythroid differentiation in mouse Friend cells revealed by cell fusion experiments

    International Nuclear Information System (INIS)

    Cell fusion with two genetically marked Friend (murine erythroleukemia) cells has made it possible to characterize the very early events leading to erythroid differentiation, particularly the nature of reactions initiated by inducers such as dimethyl sulfoxide. We have found that brief exposure of Friend cells to dimethyl sulfoxide (as well as butyric acid or hexamethylene-bisacetamide) induces an early cellular activity required for erythroid differentiation which is detected only by fusion with ultraviolet-irradiated cells. The induction process of this activity consists of at least two distinct stages. In the first stage, the reaction proceeds without supply of metabolites from the medium and exhibits sensitivity to tumor promoters. The second stage is tightly coupled to cellular metabolic activity, notably protein synthesis. Under normal conditions, the induced activity is short-lived, suggesting turnover of the molecules responsible for this activity. There appears to be a signal produced following dimethyl sulfoxide pulse which acts as an inducer for this activity. The signal remains active for as long as 40 hr when protein synthesis is blocked

  3. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy. PMID:25556991

  4. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

    Directory of Open Access Journals (Sweden)

    Kaoru Miyazaki

    Full Text Available BACKGROUND: Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. METHODOLOGY/PRINCIPAL FINDINGS: ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. CONCLUSIONS/SIGNIFICANCE: We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo

  5. Cryo-STEM Tomography of Intact Vitrified Fibroblasts

    OpenAIRE

    David Kirchenbuechler; Yael Mutsafi; Ben Horowitz; SmadarLevin-Zaidman; Deborah Fass; Wolf, Sharon G.; Michael Elbaum

    2015-01-01

    Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). He...

  6. Radially expanding transglial calcium waves in the intact cerebellum

    OpenAIRE

    Hoogland, Tycho M; Kuhn, Bernd; Göbel, Werner; Huang, Wenying; Nakai, Junichi; HELMCHEN, Fritjof; Flint, Jane; Wang, Samuel S.-H.

    2009-01-01

    Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of ...

  7. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  8. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    Directory of Open Access Journals (Sweden)

    Schuren Frank H

    2008-12-01

    Full Text Available Abstract Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.

  9. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    Science.gov (United States)

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  10. Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

    International Nuclear Information System (INIS)

    Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34+ cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML

  11. Proton gradients in intact cyanobacteria

    Science.gov (United States)

    Belkin, S.; Mehlhorn, R. J.; Packer, L.

    1987-01-01

    The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.

  12. Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

    Science.gov (United States)

    Yu, Wenmin; Li, Yiping; Wang, Zhi; Liu, Lei; Liu, Jing; Ding, Fengan; Zhang, Xiaoyi; Cheng, Zhengyuan; Chen, Pingsheng; Dou, Jun

    2016-09-01

    Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK‑2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK‑2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK‑2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma. PMID:27432315

  13. Robustness and adaptation reveal plausible cell cycle controlling subnetwork in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Jiun-Yan; Huang, Chi-Wei; Kao, Kuo-Ching; Lai, Pik-Yin

    2013-04-10

    Biological systems are often organized spatially and temporally by multi-scale functional subsystems (modules). A specific subcellular process often corresponds to a subsystem composed of some of these interconnected modules. Accurate identification of system-level modularity organization from the large scale networks can provide valuable information on subsystem models of subcellular processes or physiological phenomena. Computational identification of functional modules from the large scale network is the key approach to solve the complexity of modularity in the past decade, but the overlapping and multi-scale nature of modules often renders unsatisfactory results in these methods. Most current methods for modularity detection are optimization-based and suffered from the drawback of size resolution limit. It is difficult to trace the origin of the unsatisfactory results, which may be due to poor data, inappropriate objective function selection or simply resulted from natural evolution, and hence no system-level accurate modular models for subcellular processes can be offered. Motivated by the idea of evolution with robustness and adaption as guiding principles, we propose a novel approach that can identify significant multi-scale overlapping modules that are sufficiently accurate at the system and subsystem levels, giving biological insights for subcellular processes. The success of our evolution strategy method is demonstrated by applying to the yeast protein-protein interaction network. Functional subsystems of important physiological phenomena can be revealed. In particular, the cell cycle controlling network is selected for detailed discussion. The cell cycle subcellular processes in yeast can be successfully dissected into functional modules of cell cycle control, cell size check point, spindle assembly checkpoint, and DNA damage check point in G2/M and S phases. The interconnections between check points and cell cycle control modules provide clues on the

  14. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

    Science.gov (United States)

    Ng, Kiaw Kiaw; Motoda, Yoko; Watanabe, Satoru; Sofiman Othman, Ahmad; Kigawa, Takanori; Kodama, Yutaka; Numata, Keiji

    2016-01-01

    In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology. PMID:27100681

  15. [Acute intestinal obstruction revealing enteropathy associated t-cell lymphoma, about a case].

    Science.gov (United States)

    Garba, Abdoul Aziz; Adamou, Harissou; Magagi, Ibrahim Amadou; Brah, Souleymane; Habou, Oumarou

    2016-01-01

    Enteropathy associated T-cell lymphoma (EATL) is a rare complication of celiac disease (CD). We report a case of EATL associated with CD revealed by acute intestinal obstruction. A North African woman of 38 years old with a history of infertility and chronic abdominal pain was admitted in emergency with acute intestinal obstruction. During the surgery, we found a tumor on the small intestine with mesenteric lymphadenopathy. Histology and immunohistochemistry of the specimen objectified a digestive T lymphoma CD3+ and immunological assessment of celiac disease was positive. The diagnosis of EATL was thus retained. Chemotherapy (CHOEP protocol) was established as well as gluten-free diet with a complete response to treatment. The EATL is a rare complication of CD that can be revealed by intestinal obstruction. The prognosis can be improved by early treatment involving surgery and chemotherapy. Its prevention requires early diagnosis of celiac and gluten-free diets. PMID:27217874

  16. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

    OpenAIRE

    Owens, Kelly,; Cunningham, Dale,; Macdonald, Glen; Rubel, Edwin,; Raible, David,; Pujol, Remy

    2007-01-01

    Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycos...

  17. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas

    DEFF Research Database (Denmark)

    Cirilo, Priscila Daniele Ramos; Marchi, Fábio Albuquerque; Barros Filho, Mateus de Camargo;

    2013-01-01

    integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell...... transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs....

  18. Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2011-11-01

    Full Text Available As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium and prototrophic (minimal medium culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens and an important target.

  19. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  20. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

    Full Text Available BACKGROUND: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. METHODOLOGY: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins. PRINCIPAL FINDINGS: We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved. CONCLUSIONS/SIGNIFICANCE: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  1. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  2. Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons.

    Directory of Open Access Journals (Sweden)

    Pascaline Clerc

    Full Text Available Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.

  3. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Liang-Hui Chu

    Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

  4. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    Science.gov (United States)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  5. Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

    Science.gov (United States)

    Kolinko, Sebastian; Jogler, Christian; Katzmann, Emanuel; Wanner, Gerhard; Peplies, Jörg; Schüler, Dirk

    2012-07-01

    Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum. PMID:22003954

  6. Combined in silico and in vivo analyses reveal role of Hes1 in taste cell differentiation.

    Directory of Open Access Journals (Sweden)

    Masato S Ota

    2009-04-01

    Full Text Available The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction, the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP, we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/ (- mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.

  7. CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Yue Hu

    Full Text Available We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC and adenocarcinoma (AC subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.

  8. Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles.

    Science.gov (United States)

    Muranaka, Tomoaki; Oyama, Tokitaka

    2016-07-01

    Recent advances in single-cell analysis have revealed the stochasticity and nongenetic heterogeneity inherent to cellular processes. However, our knowledge of the actual cellular behaviors in a living multicellular organism is still limited. By using a single-cell bioluminescence imaging technique on duckweed, Lemna gibba, we demonstrate that, under constant conditions, cells in the intact plant work as individual circadian clocks that oscillate with their own frequencies and respond independently to external stimuli. Quantitative analysis uncovered the heterogeneity and instability of cellular clocks and partial synchronization between neighboring cells. Furthermore, we found that cellular clocks in the plant body under light-dark cycles showed a centrifugal phase pattern in which the effect of cell-to-cell heterogeneity in period lengths was almost masked. The inherent heterogeneity in the properties of cellular clocks observed under constant conditions is corrected under light-dark cycles to coordinate the daily rhythms of the plant body. These findings provide a novel perspective of spatiotemporal architectures in the plant circadian system. PMID:27453946

  9. Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles

    Science.gov (United States)

    Muranaka, Tomoaki; Oyama, Tokitaka

    2016-01-01

    Recent advances in single-cell analysis have revealed the stochasticity and nongenetic heterogeneity inherent to cellular processes. However, our knowledge of the actual cellular behaviors in a living multicellular organism is still limited. By using a single-cell bioluminescence imaging technique on duckweed, Lemna gibba, we demonstrate that, under constant conditions, cells in the intact plant work as individual circadian clocks that oscillate with their own frequencies and respond independently to external stimuli. Quantitative analysis uncovered the heterogeneity and instability of cellular clocks and partial synchronization between neighboring cells. Furthermore, we found that cellular clocks in the plant body under light-dark cycles showed a centrifugal phase pattern in which the effect of cell-to-cell heterogeneity in period lengths was almost masked. The inherent heterogeneity in the properties of cellular clocks observed under constant conditions is corrected under light-dark cycles to coordinate the daily rhythms of the plant body. These findings provide a novel perspective of spatiotemporal architectures in the plant circadian system. PMID:27453946

  10. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. DNA-based digital tension probes reveal integrin forces during early cell adhesion

    Science.gov (United States)

    Zhang, Yun; Ge, Chenghao; Zhu, Cheng; Salaita, Khalid

    2014-10-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. Here to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20- to 30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA hairpins with tunable force response thresholds, ligands and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized RGD over linear RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

  12. Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types

    Directory of Open Access Journals (Sweden)

    Villegas Rosario

    2012-06-01

    Full Text Available Abstract Background Understanding the cellular mechanisms regulating axon degeneration and regeneration is crucial for developing treatments for nerve injury and neurodegenerative disease. In neurons, axon degeneration is distinct from cell body death and often precedes or is associated with the onset of disease symptoms. In the peripheral nervous system of both vertebrates and invertebrates, after degeneration of detached fragments, axons can often regenerate to restore function. Many studies of axonal degeneration and regeneration have used in vitro approaches, but the influence of extrinsic cell types on these processes can only be fully addressed in live animals. Because of its simplicity and superficial location, the larval zebrafish posterior lateral line (pLL nerve is an ideal model system for live studies of axon degeneration and regeneration. Results We used laser axotomy and time-lapse imaging of pLL axons to characterize the roles of leukocytes, Schwann cells and target sensory hair cells in axon degeneration and regeneration in vivo. Immune cells were essential for efficient removal of axonal debris after axotomy. Schwann cells were required for proper fasciculation and pathfinding of regenerating axons to their target cells. Intact target hair cells were not themselves required for regeneration, but chemical ablation of neuromasts caused axons to transiently deviate from their normal paths. Conclusions Macrophages, Schwann cells, and target sensory organs are required for distinct aspects of pLL axon degeneration or regeneration in the zebrafish larva. Our work introduces a powerful vertebrate model for analyzing axonal degeneration and regeneration in the living animal and elucidating the role of extrinsic cell types in these processes.

  13. Bone marrow‑derived mesenchymal stem cells rescue injured H9c2 cells via transferring intact mitochondria through tunneling nanotubes in an in vitro simulated ischemia/reperfusion model.

    Science.gov (United States)

    Han, Hui; Hu, Jinquan; Yan, Qiang; Zhu, Jinzhou; Zhu, Zhengbin; Chen, Yanjia; Sun, Jiateng; Zhang, Ruiyan

    2016-02-01

    The transplantation of mesenchymal stem cells (MSCs) is considered to be a promising treatment for ischemic heart disease; however, the therapeutic effects and underlying mechanisms of action require further evaluation. Mitochondrial dysfunction is a key event in simulated ischemia/reperfusion (SI/R) injury. The purpose of the present study was to investigate the mechanism of mitochondrial transfer, which may be involved the antiapoptotic action of co-culture with MSCs. An in vitro model of simulated ischemia/reperfusion (SI/R) was used in the present study. The apoptotic indexes were significantly increased when H9c2 cardiomyocytes were induced in the SI/R group. Following co-culture with bone marrow-derived (BM)‑MSCs, H9c2 cells exhibited marked resistance against the SI/R-induced apoptotic process. Besides, mitochondrial transfer via a tunneling nanotube (TNT) like structure was detected by confocal fluorescent microscopy. In addition, following pretreated with latrunculin-A (LatA), an inhibitor of TNT formation, the BM-MSCs were not able to rescue injured H9c2 cells from apoptosis, as previously observed. In conclusion, the anti‑apoptotic ability of BM‑MSCs may be partially attributed to the recovery of mitochondrial dysfunction in SI/R, and the formation of TNTs appears to be involved in this action of mitochondrial transfer between adjacent cells. PMID:26718099

  14. Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD

    Science.gov (United States)

    Cortez, Eliane; Gladh, Hanna; Braun, Sebastian; Bocci, Matteo; Cordero, Eugenia; Björkström, Niklas K.; Miyazaki, Hideki; Michael, Iacovos P.; Eriksson, Ulf; Folestad, Erika; Pietras, Kristian

    2016-01-01

    Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis. PMID:26831065

  15. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    Science.gov (United States)

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  16. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  17. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  18. Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

    Directory of Open Access Journals (Sweden)

    Daniel J Anderson

    Full Text Available Oncogenic mutations in the mitogen activated protein kinase (MAPK pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2. We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

  19. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  20. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

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    Lulu Cao

    Full Text Available The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76 plays a critical scaffolding role in T cell receptor (TCR signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

  1. Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

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    Liu Liwen

    2009-09-01

    Full Text Available Abstract Background Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast. Results By integrating genome-wide data from multiple time course cell cycle microarray experiments we reconstructed a gene regulatory network. Based on the network, we discovered in addition to previously known regulatory hubs in M phase, a new putative regulatory hub in the form of the HMG box transcription factor SPBC19G7.04. Further, we inferred periodic activities of several less known transcription factors over the course of the cell cycle, identified over 500 putative regulatory targets and detected many new phase-specific and conserved cis-regulatory motifs. In particular, we show that SPBC19G7.04 has highly significant periodic activity that peaks in early M phase, which is coordinated with the late G2 activity of the forkhead transcription factor fkh2. Finally, using an enhanced Bayesian algorithm to co-cluster the expression data, we obtained 31 clusters of co-regulated genes 1 which constitute regulatory modules from different phases of the cell cycle, 2 whose phase order is coherent across the 10 time course experiments, and 3 which lead to identification of phase-specific control elements at both the transcriptional and post-transcriptional levels in S. pombe. In particular, the ribosome biogenesis clusters expressed in G2 phase reveal new, highly conserved RNA motifs. Conclusion Using a systems-level analysis of the phase-specific nature of the S. pombe cell cycle gene regulation, we have provided new testable evidence for post-transcriptional regulation in the G2 phase of the fission yeast cell cycle

  2. Quantitative trait loci mapping reveals candidate pathways regulating cell cycle duration in Plasmodium falciparum

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    Siwo Geoffrey

    2010-10-01

    Full Text Available Abstract Background Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of Plasmodium falciparum through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration. Results Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle. Conclusions We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c, a Zinc finger transcription factor (PFL0465c both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c.

  3. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

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    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  4. In situ microscopy reveals reversible cell wall swelling in kelp sieve tubes: one mechanism for turgor generation and flow control?

    Science.gov (United States)

    Knoblauch, Jan; Tepler Drobnitch, Sarah; Peters, Winfried S; Knoblauch, Michael

    2016-08-01

    Kelps, brown algae (Phaeophyceae) of the order Laminariales, possess sieve tubes for the symplasmic long-distance transport of photoassimilates that are evolutionarily unrelated but structurally similar to the tubes in the phloem of vascular plants. We visualized sieve tube structure and wound responses in fully functional, intact Bull Kelp (Nereocystis luetkeana [K. Mertens] Postels & Ruprecht 1840). In injured tubes, apparent slime plugs formed but were unlikely to cause sieve tube occlusion as they assembled at the downstream side of sieve plates. Cell walls expanded massively in the radial direction, reducing the volume of the wounded sieve elements by up to 90%. Ultrastructural examination showed that a layer of the immediate cell wall characterized by circumferential cellulose fibrils was responsible for swelling and suggested that alginates, abundant gelatinous polymers of the cell wall matrix, were involved. Wall swelling was rapid, reversible and depended on intracellular pressure, as demonstrated by pressure-injection of silicon oil. Our results revive the concept of turgor generation and buffering by swelling cell walls, which had fallen into oblivion over the last century. Because sieve tube transport is pressure-driven and controlled physically by tube diameter, a regulatory role of wall swelling in photoassimilate distribution is implied in kelps. PMID:26991892

  5. Revealing nonergodic dynamics in living cells from a single particle trajectory

    Science.gov (United States)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  6. Characterization of KATP channels in intact mammalian skeletal muscle fibres

    OpenAIRE

    Barrett-Jolley, Richard; McPherson, Grant A

    1998-01-01

    The aim of this study was to characterize the KATP channel of intact rat skeletal muscle (rat flexor digitorum brevis muscle). Changes in membrane currents were recorded with two-electrode voltage-clamp of whole fibres.The KATP channel openers, levcromakalim and pinacidil (10–400 μM), caused a concentration-dependent increase in whole-cell chord conductance (up to approximately 1.5 mScm−2). The activated current had a weak inwardly rectifying current-voltage relation, a reversal potential nea...

  7. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  8. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.

    Science.gov (United States)

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E

    2016-03-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions. PMID:26883038

  9. Effects of Triclocarban on Intact Immature Male Rat

    Science.gov (United States)

    Duleba, Antoni J.; Ahmed, Mohamed I.; Sun, Meng; Gao, Allen C.; Villanueva, Jesus; Conley, Alan J.; Turgeon, Judith L.; Benirschke, Kurt; Gee, Nancy A.; Chen, Jiangang; Green, Peter G.; Lasley, Bill L.

    2011-01-01

    Triclocarban (TCC; 3,4,4′-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age-matched male rats and on human prostate LNCaP and C4–2B cells. Seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for 10 days. Triclocarban induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum luteinizing hormone (LH) and testosterone were not significantly altered by TCC treatment. In prostate cancer-derived LNCaP and C4–2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells. PMID:20889956

  10. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    Science.gov (United States)

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  11. Measurement of diffusive properties of intact rock

    International Nuclear Information System (INIS)

    In the Postclosure Assessment of a Reference System for the Disposal of Canada's Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the 'exclusion zone.' A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (Do) for 1291 and 14C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (Di) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs

  12. Silica Aerogel Captures Cosmic Dust Intact

    Science.gov (United States)

    Tsou, P.

    1994-01-01

    The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

  13. Age ultrastructural features of sclera in intact rats

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    Ulyanova N.A.

    2013-12-01

    Full Text Available Background. The absence of adequate experimental myopia model is the actual problem in pathophysiological studying of the myopia progression. Studies of the chick eye have formed the basis for several hypotheses of myopic development. The most pathogenesis reasonable myopia animal model is a form-deprivation myopia in chicks. The introduction of form-deprivation myopia animal model in mammals in particular rats, is necessary to study the dynamics of age morphological changes in sclera. Objective. To examine age-related features of the sclera in intact rats on the ultra-structural level. Methods. It was investigated the sclera ultrastructure by using electron microscopy method in intact rats at 14, 21, 30, 90 days. Results. It was set that in rats’ eyes at the age of 14-21 days the sclera presence of fibroblasts, characterizing the active generation of plastic materials, mainly on collagen. At the age of 30-90 days, the number of fibroblasts decreases and normal cells become mature look. However, practically, in all study periods, but with advantage aged 14-21 days, some of the cells are eliminated by apoptosis. At the age of 14-21 days is not formed clear scleral tissue architecture, part of the collagen fibrils stacked in bundles, part loosely arranged. At the age of 90 days, collagen scleral stroma is ordered. Conclusion. We identified a significant amount of fibroblasts decreasing, fibroblasts functional activity reduction, and collagen fibers ordering in sclera of the intact rats during the period from 14 to 90 days of postnatal ontogenesis. The highest intensity of the restructuring in the sclera is observed between 14-30 days of postnatal ontogenesis. In our opinion, this period is optimal for experimental modeling myopia in rats.

  14. Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Cheng, Caixia; Zhou, Yong; Li, Hongyi; Xiong, Teng; Li, Shuaicheng; Bi, Yanghui; Kong, Pengzhou; Wang, Fang; Cui, Heyang; Li, Yaoping; Fang, Xiaodong; Yan, Ting; Li, Yike; Wang, Juan; Yang, Bin; Zhang, Ling; Jia, Zhiwu; Song, Bin; Hu, Xiaoling; Yang, Jie; Qiu, Haile; Zhang, Gehong; Liu, Jing; Xu, Enwei; Shi, Ruyi; Zhang, Yanyan; Liu, Haiyan; He, Chanting; Zhao, Zhenxiang; Qian, Yu; Rong, Ruizhou; Han, Zhiwei; Zhang, Yanlin; Luo, Wen; Wang, Jiaqian; Peng, Shaoliang; Yang, Xukui; Li, Xiangchun; Li, Lin; Fang, Hu; Liu, Xingmin; Ma, Li; Chen, Yunqing; Guo, Shiping; Chen, Xing; Xi, Yanfeng; Li, Guodong; Liang, Jianfang; Yang, Xiaofeng; Guo, Jiansheng; Jia, JunMei; Li, Qingshan; Cheng, Xiaolong; Zhan, Qimin; Cui, Yongping

    2016-02-01

    Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs. PMID:26833333

  15. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  16. The metabolic enzyme ManA reveals a link between cell wall integrity and chromosome morphology.

    OpenAIRE

    Maya Elbaz; Sigal Ben-Yehuda

    2010-01-01

    Author Summary The bacterial cell is resistant to extremes of osmotic pressure and protected against mechanical damages by the existence of a rigid outer shell defined as the cell wall. The strength of the cell wall is achieved by the presence of long glycan strands cross-linked by peptide side bridges. The cell wall is a dynamic structure continuously being synthesized and modified to allow for cell growth and division. Damaging the cell wall leads to abnormal cellular morphologies and cell ...

  17. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

    Science.gov (United States)

    Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

  18. Single nanoparticle tracking of [Formula: see text]-methyl-d-aspartate receptors in cultured and intact brain tissue.

    Science.gov (United States)

    Varela, Juan A; Ferreira, Joana S; Dupuis, Julien P; Durand, Pauline; Bouchet, Delphine; Groc, Laurent

    2016-10-01

    Recent developments in single-molecule imaging have revealed many biological mechanisms, providing high spatial and temporal resolution maps of molecular events. In neurobiology, these techniques unveiled that plasma membrane neurotransmitter receptors and transporters laterally diffuse at the surface of cultured brain cells. The photostability of bright nanoprobes, such as quantum dots (QDs), has given access to neurotransmitter receptor tracking over long periods of time with a high spatial resolution. However, our knowledge has been restricted to cultured systems, i.e., neurons and organotypic slices, therefore lacking several aspects of the intact brain rheology and connectivity. Here, we used QDs to track single glutamatergic [Formula: see text]-methyl-d-aspartate receptors (NMDAR) in acute brain slices. By delivering functionalized nanoparticles in vivo through intraventricular injections to rats expressing genetically engineered-tagged NMDAR, we successfully tracked the receptors in native brain tissue. Comparing NMDAR tracking to different classical brain preparations (acute brain slices, cultured organotypic brain slices, and cultured neurons) revealed that the surface diffusion properties shared several features and are also influenced by the nature of the extracellular environment. Together, we describe the experimental procedures to track plasma membrane NMDAR in dissociated and native brain tissue, paving the way for investigations aiming at characterizing receptor diffusion biophysics in intact tissue and exploring the physiopathological roles of receptor surface dynamics. PMID:27429996

  19. Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

    Science.gov (United States)

    Libantová, Jana; Kämäräinen, Terttu; Moravcíková, Jana; Matusíková, Ildikó; Salaj, Jan

    2009-05-01

    The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated. PMID:18437530

  20. Study on sickle cell disease haplotypes reveals the African origin of Amapá's population

    Directory of Open Access Journals (Sweden)

    Natália de Morais Castelo

    2014-04-01

    Full Text Available Introduction:Sickle cell disease (SCD is a hereditary, hematologic, multifactorial disease, with high prevalence worldwide; its cause is a mutation in the sixth codon of the beta globin gene (βs.Objective:To identify the haplotypes present in people with SCD in Amapá, and relate them to African descent.Methods:We analyzed, by molecular techniques, 46 blood samples from people with SCD in Macapá, the capital of Amapá, with the purpose of obtaining information about haplotype frequency distribution, which helps understand the ethnic background of Amapá's population.Results:Our study revealed that the most frequent haplotype is Bantu (61.2%, followed by Benin (26.6% and Senegal (12.2%. Results showed statistical differences from studies conducted in other regions. A high frequency of the Senegal haplotype stands out, in comparison with some Brazilian studies.Conclusion:Amapá's results exhibit unique characteristics when compared to haplotypes in other regions, with high frequency of Senegal and Benin haplotypes, absence of atypical, Cameroon and Saudi, confirming that Brazil shows ethnic background diversity, as well as different haplotype frequencies.

  1. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. PMID:25525749

  2. Single-cell genomics reveal metabolic strategies for microbial growth and survival in an oligotrophic aquifer

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Michael J.; Kennedy, David W.; Castelle, Cindy; Field, Erin; Stepanauskas, Ramunas; Fredrickson, Jim K.; Konopka, Allan

    2014-02-09

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3­-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra-and extra­-cellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.

  3. Pushing structural limits to reveal fundamental mechanisms of organic solar cell operation

    Science.gov (United States)

    Rand, Barry

    2015-03-01

    Organic-based solar cells are beginning to emerge with the potential to compete with other thin film photovoltaic technologies, with efficiencies of 12% recently demonstrated. Unique to the function of organic photovoltaics are the creation of tightly bound excitons that can only be efficiently separated at a donor/acceptor (D/A) interface capable of providing the necessary energetic driving force for dissociation. The consequences of this are the need for long exciton diffusion lengths and the presence of charge transfer (CT) states, ground state complexes that exist at the D/A interface. We have found that charge transfer states are more easily separated into free charge if they are delocalized; an aspect that becomes most feasible for highly ordered systems. I will discuss our recent efforts to template and control film morphology and molecular orientation. These studies allow us to understand the importance of molecular orientation, crystallite size, and crystal phase. We will show templated devices utilizing neat films as well as bulk heterojunctions, with crystallite dimensions spanning from the more standard nano-sized grains to those with highly ordered micron-sized crystalline domains revealing unprecedented thin film exciton diffusion lengths of 100s of nm. In these highly ordered films, owing to significant delocalization, we are able to directly measure photocurrent from multiple CT states, an aspect which has important consequences for the design of more efficient photocurrent generation. We acknowledge support from DOE BES Grant #11493344.

  4. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells

    Energy Technology Data Exchange (ETDEWEB)

    Abou-El-Ardat, Khalil, E-mail: kabouela@sckcen.be [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Monsieurs, Pieter [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Anastasov, Natasa; Atkinson, Mike [Department of Radiation Sciences, Helmholtz Zentrum Muenchen, Munich (Germany); Derradji, Hanane [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); De Meyer, Tim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Bekaert, Sofie [Clinical Research Center, Faculty for Medicine and Health Sciences, Universiteit Gent, 185 De Pintelaan, 9000 Ghent (Belgium); Van Criekinge, Wim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); and others

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4 Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4 Gy in both RET/PTC-positive systems but no common genes at 62.5 mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

  5. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells.

    Science.gov (United States)

    Abou-El-Ardat, Khalil; Monsieurs, Pieter; Anastasov, Nataša; Atkinson, Mike; Derradji, Hanane; De Meyer, Tim; Bekaert, Sofie; Van Criekinge, Wim; Baatout, Sarah

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent. PMID:22027090

  6. Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance

    OpenAIRE

    Luo, Yuchun; Ellis, Lixia Z; Dallaglio, Katiuscia; Takeda, Moe; Robinson, William A.; Robinson, Steven; Liu, Weimin; Lewis, Karl D.; McCarter, Martin D; Gonzalez, Rene; David A Norris; Roop, Dennis R.; Spritz, Richard A.; Ahn, Natalie G.; Fujita, Mayumi

    2012-01-01

    Side population (SP) is identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a sub...

  7. Sorption of cesium in intact rock

    Energy Technology Data Exchange (ETDEWEB)

    Puukko, E. [Univ. of Helsinki, Dept. of Chemistry (Finland)

    2014-04-15

    The mass distribution coefficient K{sub d} is used in performance assessment (PA) to describe sorption of a radionuclide on rock. The R{sub d} is determined using crushed rock which causes uncertainty in converting the R{sub d} values to K{sub d} values for intact rock. This work describes a method to determine the equilibrium of sorption on intact rock. The rock types of the planned Olkiluoto waste disposal site were T-series mica gneiss (T-MGN), T-series tonalite granodiorite granite gneiss (T-TGG), P-series tonalite granodiorite granite gneiss (P-TGG) and pegmatitic granite (PGR). These rocks contain different amount of biotite which is the main sorbing mineral. The sorption of cesium on intact rock slices was studied by applying an electrical field to speed up migration of cesium into the rock. Cesium is in the solution as a noncomplex cation Cs{sup +} and it is sorbed by ion exchange. The tracer used in the experiments was {sup 134}Cs. The experimental sorption on the intact rock is compared with values calculated using the in house cation exchange sorption model (HYRL model) in PHREEQC program. The observed sorption on T-MGN and T-TGG rocks was close to the calculated values. Two PGR samples were from a depth of 70 m and three samples were from a depth of 150 m. Cesium sorbed more than predicted on the two 70 m PGR samples. The sorption of Cs on the three 150 m PGR samples was small which was consistent with the calculations. The pegmatitic granite PGR has the smallest content of biotite of the four rock types. In the case of P-TGG rock the observed values of sorption were only half of the calculated values. Two kind of slices were cut from P-TGG drill core. The slices were against and to the direction of the foliation of the biotite rims. The sorption of cesium on P-TGG rock was same in both cases. The results indicated that there was no effect of the directions of the electric field and the foliation of biotite in the P-TGG rock. (orig.)

  8. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  9. A Nanoprinted Model of Interstitial Cancer Migration Reveals a Link between Cell Deformability and Proliferation.

    Science.gov (United States)

    Panagiotakopoulou, Magdalini; Bergert, Martin; Taubenberger, Anna; Guck, Jochen; Poulikakos, Dimos; Ferrari, Aldo

    2016-07-26

    Metastatic progression of tumors requires the coordinated dissemination of cancerous cells through interstitial tissues and their replication in distant body locations. Despite their importance in cancer treatment decisions, key factors, such as cell shape adaptation and the role it plays in dense tissue invasion by cancerous cells, are not well understood. Here, we employ a 3D electrohydrodynamic nanoprinting technology to generate vertical arrays of topographical pores that mimic interstitial tissue resistance to the mesenchymal migration of cancerous cells, in order to determine the effect of nuclear size, cell deformability, and cell-to-substrate adhesion on tissue invasion efficiency. The high spatial and temporal resolution of our analysis demonstrates that the ability of cells to deform depends on the cell cycle phase, peaks immediately after mitosis, and is key to the invasion process. Increased pore penetration efficiency by cells in early G1 phase also coincided with their lower nuclear volume and higher cell deformability, compared with the later cell cycle stages. Furthermore, artificial decondensation of chromatin induced an increase in cell and nuclear deformability and improved pore penetration efficiency of cells in G1. Together, these results underline that along the cell cycle cells have different abilities to dynamically remodel their actin cytoskeleton and induce nuclear shape changes, which determines their pore penetration efficiency. Thus, our results support a mechanism in which cell proliferation and pore penetration are functionally linked to favor the interstitial dissemination of metastatic cells. PMID:27268411

  10. 7 CFR 160.29 - Containers to remain intact.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Containers to remain intact. 160.29 Section 160.29... STANDARDS FOR NAVAL STORES Analysis, Inspection, and Grading on Request § 160.29 Containers to remain intact... the containers holding such naval stores remain intact as sampled until the analysis,...

  11. A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division

    OpenAIRE

    Scott A Hoose; Jeremy A Rawlings; Kelly, Michelle M.; M Camille Leitch; Ababneh, Qotaiba O; Robles, Juan P.; David Taylor; Hoover, Evelyn M.; Bethel Hailu; McEnery, Kayla A.; S Sabina Downing; Deepika Kaushal; Yi Chen; Alex Rife; Kirtan A Brahmbhatt

    2012-01-01

    Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene dele...

  12. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    OpenAIRE

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2008-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an inc...

  13. Intestinal crypt homeostasis revealed at single stem cell level by in vivo live-imaging

    Science.gov (United States)

    Zomer, Anoek; Snippert, Hugo J.; de Sauvage, Frederic J.; Simons, Benjamin D.; Clevers, Hans; van Rheenen, Jacco

    2014-01-01

    Summary The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. Alongside Lgr5, intestinal stem cells have been associated with various markers, which are expressed heterogeneously within the crypt base region1-6. Previous quantitative clonal fate analyses have proposed that homeostasis occurs as the consequence of neutral competition between dividing stem cells7-9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here, we established the short-term dynamics of intestinal stem cells using a novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem cell maintenance in which a dynamically heterogeneous cell population is able to function long-term as a single stem cell pool. PMID:24531760

  14. Bacterial clearance in the intact and regenerating liver

    International Nuclear Information System (INIS)

    The Kupffer cells in the liver play an important role in reticuloendothelial system (RES) function by clearing particulate matter and bacteria from the blood stream. While hepatocyte regeneration and function have been extensively studied following partial hepatectomy, little information is available concerning RES function in the regenerating liver. This study investigates hepatic RES function by evaluating bacterial clearance (live E. coli) in the intact and regenerating liver. Thirty-four young male Sprague Dawley rats were studied. Twenty-two animals underwent a standard 70% partial hepatectomy using ligature technique and 12 had a sham operation. Both groups of rats received 10(9) organism of 35S labeled E coli, intravenously at 24 hours, 72 hours, 2 1/2 weeks, and 6 weeks postoperatively. Rats were killed 10 minutes following injection and liver, lung, spleen, and kidney harvested, fixed, and radioactivity was determined using a scintillation spectrometer interfaced with a micro-computer counting the 35S radiolabel. The total organ count of trapped bacteria in liver in partially hepatectomized rats was lower than intact controls at 24 hours, but was similar at 72 hours, 2 1/2 weeks, and 6 weeks. Partial hepatectomy increased the amount of bacterial trapping in the lung at 24 hours and 72 hours and returned to normal at 2 1/2 weeks and 6 weeks. Splenic activity was increased following hepatectomy at 2 1/2 weeks. Renal clearance was increased at 72 hours and 2 1/2 weeks

  15. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    Science.gov (United States)

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  16. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

    Directory of Open Access Journals (Sweden)

    Yo Saito

    Full Text Available Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S; therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

  17. Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

    Science.gov (United States)

    Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

    2006-01-01

    This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

  18. Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates.

    OpenAIRE

    Leroux, A; London, I M

    1982-01-01

    Studies in intact rabbit reticulocytes and reticulocyte lysates provide further evidence of a functional role for the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) in the regulation of initiation of protein synthesis in eukaryotic cells. In intact reticulocytes treated with isonicotinic acid hydrazide to inhibit heme synthesis, the phosphorylation of eIF-2 alpha was significantly greater than in control cells. In heme-deficient reticulocyte lysates and in lysates treat...

  19. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Benjamin A Neely

    Full Text Available Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC. This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened

  20. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

    Science.gov (United States)

    Wilkins, Christopher E.; Marlow, Laura A.; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O.; Kislinger, Thomas; Copland, John A.; Drake, Richard R.

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  1. A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

    Science.gov (United States)

    Song, Jiazhe; Xue, Kai; She, Ji; Ding, Fangrong; Li, Song; Shangguan, Rulan; Dai, Yunping; Du, Liying; Li, Ning

    2014-09-10

    The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment. PMID:24954407

  2. Direct glycan structure determination of intact N-linked glycopeptides by low-energy collision-induced dissociation tandem mass spectrometry and predicted spectral library searching.

    Science.gov (United States)

    Pai, Pei-Jing; Hu, Yingwei; Lam, Henry

    2016-08-31

    Intact glycopeptide MS analysis to reveal site-specific protein glycosylation is an important frontier of proteomics. However, computational tools for analyzing MS/MS spectra of intact glycopeptides are still limited and not well-integrated into existing workflows. In this work, a new computational tool which combines the spectral library building/searching tool, SpectraST (Lam et al. Nat. Methods2008, 5, 873-875), and the glycopeptide fragmentation prediction tool, MassAnalyzer (Zhang et al. Anal. Chem.2010, 82, 10194-10202) for intact glycopeptide analysis has been developed. Specifically, this tool enables the determination of the glycan structure directly from low-energy collision-induced dissociation (CID) spectra of intact glycopeptides. Given a list of possible glycopeptide sequences as input, a sample-specific spectral library of MassAnalyzer-predicted spectra is built using SpectraST. Glycan identification from CID spectra is achieved by spectral library searching against this library, in which both m/z and intensity information of the possible fragmentation ions are taken into consideration for improved accuracy. We validated our method using a standard glycoprotein, human transferrin, and evaluated its potential to be used in site-specific glycosylation profiling of glycoprotein datasets from LC-MS/MS. In addition, we further applied our method to reveal, for the first time, the site-specific N-glycosylation profile of recombinant human acetylcholinesterase expressed in HEK293 cells. For maximum usability, SpectraST is developed as part of the Trans-Proteomic Pipeline (TPP), a freely available and open-source software suite for MS data analysis. PMID:27506355

  3. Digested BLG can induce tolerance when co-administered with intact BLG in Brown Norway rats

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Barkholt, Vibeke; Madsen, Charlotte Bernhard

    Background: Milk is a major constituent of small children’s diet. Milk allergy is also one of the most common allergies in small children. Prevention, treatment and general understanding of this allergy are therefore important. Methods: Intact BLG was digested in an in vitro model simulating...... to complexes of larger sizes. Specific antibody responses revealed that both the high (200 µg) and low (30 µg) amount of intact BLG had both immunogenic and allergenic sensitising capacity, while digested BLG had no sensitizing capacity. In contrast digested BLG and the fraction of large complexes retained...

  4. agged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis

    OpenAIRE

    Culp, L A; Lin, W.C.

    1999-01-01

    Histochemical marker genes were used to "tag" mouse fibrosarcoma or human neuroblastoma cells, providing a better understanding of their subsequent progression and metastasis mechanisms in nude mice. Micrometastases in the lung were initiated from clusters of 2-6 cells rather than single cells in most cases; tumor cells were also visualized binding to the endothelium of small blood vessels to initiate these micrometastases. Shortterm, these mechanisms relied ...

  5. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

    OpenAIRE

    Horowitz, Amir; Strauss-Albee, Dara M.; Leipold, Michael; Kubo, Jessica; Nemat-Gorgani, Neda; DOGAN, Ozge C.; Dekker, Cornelia L; Mackey, Sally; Maecker, Holden; Swan, Gary E.; Davis, Mark M.; Norman, Paul J.; Guethlein, Lisbeth A.; Desai, Manisha; Parham, Peter

    2013-01-01

    Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on perip...

  6. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  7. Computational models reveal a passive mechanism for cell migration in the crypt.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt.

  8. The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise

    NARCIS (Netherlands)

    H. Kempe; A. Schwabe; F. Crémazy; P.J. Verschure; F.J. Bruggeman

    2015-01-01

    Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with single-cel

  9. A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy.

    Science.gov (United States)

    Hingtgen, Shawn D; Kasmieh, Randa; van de Water, Jeroen; Weissleder, Ralph; Shah, Khalid

    2010-04-01

    Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles. PMID:20127797

  10. Lineage relationship of CD8+ T cell subsets is revealed by progressive changes in the epigenetic landscape

    Science.gov (United States)

    Crompton, Joseph G.; Narayanan, Manikandan; Cuddapah, Suresh; Roychoudhuri, Rahul; Ji, Yun; Yang, Wenjing; Patel, Shashank J.; Sukumar, Madhusudhanan; Palmer, Douglas C.; Peng, Weiqun; Wang, Ena; Marincola, Francesco M.; Klebanoff, Christopher A.; Zhao, Keji; Tsang, John S.; Gattinoni, Luca; Restifo, Nicholas P.

    2016-01-01

    To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8+ T-cell subsets. Taken together, our results suggest that CD8+ lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory. PMID:25914936

  11. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  12. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

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

    Directory of Open Access Journals (Sweden)

    Burleigh Barbara A

    2009-05-01

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

  14. Protein methylation reactions in intact pea chloroplasts

    International Nuclear Information System (INIS)

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with (3H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

  15. Expression weighted cell type enrichments reveal genetic and cellular nature of major brain disorders

    Directory of Open Access Journals (Sweden)

    Nathan Gerald Skene

    2016-01-01

    Full Text Available The cell types that trigger the primary pathology in many brain diseases remain largely unknown. One route to understanding the primary pathological cell type for a particular disease is to identify the cells expressing susceptibility genes. Although this is straightforward for monogenic conditions where the causative mutation may alter expression of a cell type specific marker, methods are required for the common polygenic disorders. We developed the Expression Weighted Cell Type Enrichment (EWCE method that uses single cell transcriptomes to generate the probability distribution associated with a gene list having an average level of expression within a cell type. Following validation, we applied EWCE to human genetic data from cases of epilepsy, Schizophrenia, Autism, Intellectual Disability, Alzheimer’s disease, Multiple Sclerosis and anxiety disorders. Genetic susceptibility primarily affected microglia in Alzheimer’s and Multiple Sclerosis; was shared between interneurons and pyramidal neurons in Autism and Schizophrenia; while intellectual disabilities and epilepsy were attributable to a range of cell-types, with the strongest enrichment in interneurons. We hypothesised that the primary cell type pathology could trigger secondary changes in other cell types and these could be detected by applying EWCE to transcriptome data from diseased tissue. In Autism, Schizophrenia and Alzheimer’s disease we find evidence of pathological changes in all of the major brain cell types. These findings give novel insight into the cellular origins and progression in common brain disorders. The methods can be applied to any tissue and disorder and have applications in validating mouse models.

  16. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    Science.gov (United States)

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Ramadan, Abdulraouf; Zhang, Qing; Choi, Sung W.; Zhang, Peng; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir; Paczesny, Sophie

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  17. A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.

    Science.gov (United States)

    Wong, Michael Thomas; Ong, David Eng Hui; Lim, Frances Sheau Huei; Teng, Karen Wei Weng; McGovern, Naomi; Narayanan, Sriram; Ho, Wen Qi; Cerny, Daniela; Tan, Henry Kun Kiaang; Anicete, Rosslyn; Tan, Bien Keem; Lim, Tony Kiat Hon; Chan, Chung Yip; Cheow, Peng Chung; Lee, Ser Yee; Takano, Angela; Tan, Eng-Huat; Tam, John Kit Chung; Tan, Ern Yu; Chan, Jerry Kok Yen; Fink, Katja; Bertoletti, Antonio; Ginhoux, Florent; Curotto de Lafaille, Maria Alicia; Newell, Evan William

    2016-08-16

    Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body. PMID:27521270

  18. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

    Science.gov (United States)

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-06-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  19. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  20. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    Directory of Open Access Journals (Sweden)

    Jyuhn-Huarng Juang

    2015-02-01

    Full Text Available The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  1. NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.

    Science.gov (United States)

    Murayama, Emi; Sarris, Milka; Redd, Michael; Le Guyader, Dorothée; Vivier, Catherine; Horsley, Wyatt; Trede, Nikolaus; Herbomel, Philippe

    2015-01-01

    The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation. PMID:26411530

  2. Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling

    Directory of Open Access Journals (Sweden)

    Kei Sugihara

    2015-12-01

    Full Text Available Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon.

  3. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

    Science.gov (United States)

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J; Brown, Lewis M; Valenzuela, Carlos A; Wolpaw, Adam J; Stockwell, Brent R

    2016-07-01

    Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes. PMID:27159577

  4. Effects of Paclitaxel on EGFR Endocytic Trafficking Revealed Using Quantum Dot Tracking in Single Cells

    OpenAIRE

    Li, Hui; Duan, Zhao-Wen; Xie, Ping; Liu, Yu-Ru; Wang, Wei-Chi; Dou, Shuo-Xing; Wang, Peng-Ye

    2012-01-01

    Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introd...

  5. Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas

    OpenAIRE

    Kopinke, Daniel; Brailsford, Marisa; Shea, Jill E; Leavitt, Rebecca; Scaife, Courtney L.; Murtaugh, L. Charles

    2011-01-01

    Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pan...

  6. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria

    OpenAIRE

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q.

    2008-01-01

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize ...

  7. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  8. Kinome sequencing reveals RET G691S polymorphism in human neuroendocrine lung cancer cell lines

    OpenAIRE

    Sosonkina, Nadiya; HONG, SEUNG-KEUN; Starenki, Dmytro; Park, Jong-In

    2014-01-01

    Neuroendocrine (NE) lung tumors comprise 20–25% of all invasive lung malignancies. Currently, no effective treatments are available to cure these tumors, and it is necessary to identify a molecular alteration(s) that characterizes NE lung tumor cells. We aimed to identify a kinase mutation(s) associated with NE lung tumor by screening 517 kinase-encoding genes in human lung cancer cell lines. Our next-generation sequencing analysis of six NE lung tumor cell lines (four small cell lung cancer ...

  9. Nanotube action between human mesothelial cells reveals novel aspects of inflammatory responses.

    Directory of Open Access Journals (Sweden)

    Julia Ranzinger

    Full Text Available A well-known role of human peritoneal mesothelial cells (HPMCs, the resident cells of the peritoneal cavity, is the generation of an immune response during peritonitis by activation of T-cells via antigen presentation. Recent findings have shown that intercellular nanotubes (NTs mediate functional connectivity between various cell types including immune cells - such as T-cells, natural killer (NK cells or macrophages - by facilitating a spectrum of long range cell-cell interactions. Although of medical interest, the relevance of NT-related findings for human medical conditions and treatment, e.g. in relation to inflammatory processes, remains elusive, particularly due to a lack of appropriate in vivo data. Here, we show for the first time that primary cultures of patient derived HPMCs are functionally connected via membranous nanotubes. NT formation appears to be actin cytoskeleton dependent, mediated by the action of filopodia. Importantly, significant variances in NT numbers between different donors as a consequence of pathophysiological alterations were observable. Furthermore, we show that TNF-α induces nanotube formation and demonstrate a strong correlation of NT connectivity in accordance with the cellular cholesterol level and distribution, pointing to a complex involvement of NTs in inflammatory processes with potential impact for clinical treatment.

  10. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    Science.gov (United States)

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines. PMID:27362860

  11. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Pakiza

    2016-06-30

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  12. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    Science.gov (United States)

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage. PMID:18832014

  13. Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells

    NARCIS (Netherlands)

    de Haan, Gerald; Gerrits, Alice; Bystrykh, Leonid

    2006-01-01

    Purpose of review The clinical use of hematopoietic stem cells, which produce all mature blood cell lineages in the circulation, is continuously increasing. Identification of genes and gene networks specifying either sternness or commitment will not only be of major relevance for a fundamental under

  14. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

    Science.gov (United States)

    Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

    2016-08-01

    Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. PMID:27085557

  15. Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells

    International Nuclear Information System (INIS)

    Both Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer. The total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data. Taxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents. Taxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer

  16. Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation

    Science.gov (United States)

    Marhavý, Peter; Montesinos, Juan Carlos; Abuzeineh, Anas; Van Damme, Daniel; Vermeer, Joop E.M.; Duclercq, Jerôme; Rakusová, Hana; Nováková, Petra; Friml, Jiři; Geldner, Niko; Benková, Eva

    2016-01-01

    To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots. PMID:26883363

  17. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G;

    2011-01-01

    (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...

  18. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    Directory of Open Access Journals (Sweden)

    Yevgeniy A Grigoryev

    Full Text Available A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+CD62L(- effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  19. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang; Nagarajan, Harish; Yerganian, George; O'Brien, Edward; Bordbar, Aarash; Roth, Anne M; Rosenbloom, Jeffrey; Bian, Chao; Xie, Min; Chen, Wenbin; Li, Ning; Baycin-Hizal, Deniz; Latif, Haythem; Förster, Jochen; Betenbaugh, Michael; Famili, Iman; Xu, Xun; Wang, Jun; Palsson, Bernhard O

    2013-01-01

    Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been...... stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This...... analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this...

  20. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vancini, Ricardo [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Kramer, Laura D. [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY (United States); Ribeiro, Mariana; Hernandez, Raquel [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Brown, Dennis, E-mail: dennis_brown@ncsu.edu [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States)

    2013-01-20

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  1. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    International Nuclear Information System (INIS)

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  2. A method to investigate protein association with intact sealed mycobacterial membrane vesicles.

    Science.gov (United States)

    D'Lima, Nadia G; Teschke, Carolyn M

    2015-09-15

    In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins. PMID:26099936

  3. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist; Lorenzen, Janne Kunchel; Gomes, Sisse;

    2014-01-01

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC) and...... intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic...... dietary treatments that varied in protein source. The study was conducted in a respiration chamber, where EE, substrate oxidation and subjective appetite were measured over 24 h at three independent visits. Moreover, blood and urine samples were collected from the participants. The results showed no...

  4. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

    OpenAIRE

    Hua, Wei; Sheff, David; Toomre, Derek; Mellman, Ira

    2006-01-01

    Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi n...

  5. Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

    Directory of Open Access Journals (Sweden)

    Bidesh Mahata

    2014-05-01

    Full Text Available T helper 2 (Th2 cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.

  6. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    Science.gov (United States)

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line. PMID:27046137

  7. A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division.

    Directory of Open Access Journals (Sweden)

    Scott A Hoose

    Full Text Available Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.

  8. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    Directory of Open Access Journals (Sweden)

    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  9. Radioligand binding assay of β-adrenoceptor on human intact lymphocytes

    International Nuclear Information System (INIS)

    A method for measuring β-adrenoceptor on human blood intact lymphocytes was developed with [3H] DHA as a radioligand. The results showed: (1) The saturable β-adrenoceptors with a binding capacity of 56 +- 8.65 fmol/106 cells and KD value of 6.96 +- 1.62 nmol (X +- S) existed in human blood intact lymphocytes (n = 13); (2) The kinetics of interaction of [3H] DHA with β-adrenoceptors was rapid and reversible without cooperativity between receptors; (3) According to potency for inhibiting the binding by β-adrenergic antigonist of agonists, the order of (-) ALP > (-) ISO > (-) E > (-) NE was found, which was paralleled to the physiological and pharmacological effect of these druge. Assay of β-adrenoceptor on intact lymphocytes will be helpful to study of cardiovascular diseases

  10. Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

    DEFF Research Database (Denmark)

    Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria;

    2015-01-01

    Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell...... identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a...

  11. Proteomic Profiling of Iron Overload-Induced Human Hepatic Cells Reveals Activation of TLR2-Mediated Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2016-03-01

    Full Text Available Background: Hepatic iron overload is common in patients who have undergone hematopoietic cell transplantation (HCT and may predispose to peri- and post-HCT toxicity. To better reveal more molecules that might be involved in iron overload-induced liver injury, we utilized proteomics to investigate differentially expressed proteins in iron overload-induced hepatocytes vs. untreated hepatocytes. Methods and Results: HH4 hepatocytes were exposed to ferric ammonium citrate (FAC to establish an in vitro iron overload model. Differentially expressed proteins initiated by the iron overload were studied by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS analysis. We identified 93 proteins whose quantity statistically significantly changes under excess hepatocyte iron conditions. Gene Ontology (GO analysis showed that these differentially expressed proteins in HH4 cells are involved in various biological process including endocytosis, response to wounding, di-, trivalent inorganic cation homeostasis, inflammatory response, positive regulation of cytokine production, and etc. Meanwhile, proteomics data revealed protein level of TLR2 and IL6ST significantly increased 7 times and 2.9 times, respectively, in iron overloaded HH4 cells. Our subsequent experiments detected that FAC-treated HH4 cells can activate IL6 expression through TLR2-mediated inflammatory responses via the NF-κB pathway. Conclusions: In this study, we demonstrated that iron overload induced hepatocytes triggering TLR2-mediated inflammatory response via NF-κB signaling pathway in HH4 cells.

  12. Papain-gel Degrades Intact Nonmineralized Type I Collagen Fibrils

    OpenAIRE

    BERTASSONI, L. E.; Marshall, G.W.

    2009-01-01

    Papain-gel has been utilized as a chemomechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papaingel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a p...

  13. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Hoei-Hansen, Christina E; Wirkner, Ute;

    2004-01-01

    their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and......Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly...

  14. Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas

    OpenAIRE

    Toru Furukawa; Hitomi Sakamoto; Shoko Takeuchi; Mitra Ameri; Yuko Kuboki; Toshiyuki Yamamoto; Takashi Hatori; Masakazu Yamamoto; Masanori Sugiyama; Nobuyuki Ohike; Hiroshi Yamaguchi; Michio Shimizu; Noriyuki Shibata; Kyoko Shimizu; Keiko Shiratori

    2015-01-01

    Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we ide...

  15. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  16. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target

    Science.gov (United States)

    Toman, O.; Kabickova, T.; Vit, O.; Fiser, R.; Polakova, K. Machova; Zach, J.; Linhartova, J.; Vyoral, D.; Petrak, J.

    2016-01-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib-resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  17. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching.

    Science.gov (United States)

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. PMID:27481325

  18. Id4 knockdown during zebrafish development revealed its functional role in neural stem cell survival

    OpenAIRE

    Patlola, Santosh

    2010-01-01

    Id4 (Inhibitor of DNA binding 4 / Inhibitor of Differentiation 4) is one of the four members of Id protein family that antagonise the function of basic helix-loop-helix (bHLH) transcriptional regulators. In the mouse it has been shown that Id4 plays an important role in the timing of neural stem and progenitor cell differentiation and knockout mice exhibited premature neural stem cell differentiation resulting in significantly smaller brains. To further establish the molecular mechanism under...

  19. Dynamics between Cancer Cell Subpopulations Reveals a Model Coordinating with Both Hierarchical and Stochastic Concepts

    OpenAIRE

    Wang, Weikang; Quan, Yi; Fu, Qibin; Liu, Yu; Liang, Ying; Wu, Jingwen; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2014-01-01

    Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities ...

  20. Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness

    OpenAIRE

    Azeloglu, Evren U.; Bhattacharya, Jahar; COSTA, KEVIN D.

    2008-01-01

    To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation wa...

  1. Combined In Silico and In Vivo Analyses Reveal Role of Hes1 in Taste Cell Differentiation

    OpenAIRE

    Ota, Masato S.; Yoshiyuki Kaneko; Kaori Kondo; Soichi Ogishima; Hiroshi Tanaka; Kazuhiro Eto; Takashi Kondo

    2009-01-01

    Author Summary The sensation of taste is composed of five basic modalities: sweet, bitter, umami, sour, and salty. Specialized taste cells perceive the various chemical cues within food. About 100 taste cells assemble into onion-shaped clusters called taste buds, which are located on taste papillae in the tongue epithelium and on oral mucosa. Of the five taste modalities, the taste stimulants responsible for sweet, bitter, and umami tastes are recognized by a group of G protein–coupled taste ...

  2. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    Science.gov (United States)

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  3. Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

    Science.gov (United States)

    Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

    2011-03-01

    Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

  4. Phenotype-based cell-specific metabolic modeling reveals metabolic liabilities of cancer.

    Science.gov (United States)

    Yizhak, Keren; Gaude, Edoardo; Le Dévédec, Sylvia; Waldman, Yedael Y; Stein, Gideon Y; van de Water, Bob; Frezza, Christian; Ruppin, Eytan

    2014-01-01

    Utilizing molecular data to derive functional physiological models tailored for specific cancer cells can facilitate the use of individually tailored therapies. To this end we present an approach termed PRIME for generating cell-specific genome-scale metabolic models (GSMMs) based on molecular and phenotypic data. We build >280 models of normal and cancer cell-lines that successfully predict metabolic phenotypes in an individual manner. We utilize this set of cell-specific models to predict drug targets that selectively inhibit cancerous but not normal cell proliferation. The top predicted target, MLYCD, is experimentally validated and the metabolic effects of MLYCD depletion investigated. Furthermore, we tested cell-specific predicted responses to the inhibition of metabolic enzymes, and successfully inferred the prognosis of cancer patients based on their PRIME-derived individual GSMMs. These results lay a computational basis and a counterpart experimental proof of concept for future personalized metabolic modeling applications, enhancing the search for novel selective anticancer therapies. PMID:25415239

  5. Pyramidal cells in prefrontal cortex: comparative observations reveal unparalleled specializations in neuronal structure among primate species.

    Directory of Open Access Journals (Sweden)

    Guy eElston

    2011-02-01

    Full Text Available The most ubiquitous neuron in the cerebral cortex, the pyramidal cell, is characterised by markedly different dendritic structure among different cortical areas. The complex pyramidal cell phenotype in granular prefrontal cortex (gPFC of higher primates endows specific biophysical properties and patterns of connectivity, which differ to those in other cortical regions. However, within the gPFC, data have been sampled from only a select few cortical areas. The gPFC of species such as human and macaque monkey includes more than 10 cortical areas. It remains unknown as to what degree pyramidal cell structure may vary among these cortical areas. Here we undertook a survey of pyramidal cells in the dorsolateral, medial and orbital gPFC of cercopethicid primates. We found marked heterogeneity in pyramidal cell structure within and between these regions. Moreover, trends for gradients in neuronal complexity varied among species. As neuron structure determines it’s computational abilities and memory storage capacity and connectivity, we propose that these specializations in the pyramidal cell phenotype are an important determinant of species specific executive cortical functions in primates.

  6. Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks.

    Science.gov (United States)

    Schiarea, Silvia; Solinas, Graziella; Allavena, Paola; Scigliuolo, Graziana Maria; Bagnati, Renzo; Fanelli, Roberto; Chiabrando, Chiara

    2010-09-01

    The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating. PMID:20687567

  7. Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo-Electron Tomography

    OpenAIRE

    Liu, Jun; Howell, Jerrilyn K.; Bradley, Sherille D.; Zheng, Yesha; Zhou, Z. Hong; Norris, Steven J

    2010-01-01

    High resolution cryo-electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3-D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member in the spirochetal family. High resolution cryo-ET reconstructions provided the detailed structures of the cell envelope, which is significantly different from that of gram-negative bacteria. The 4 nm lipid ...

  8. Lipidation of intact proteins produces highly immunogenic vaccine candidates.

    Science.gov (United States)

    Zeng, Weiguang; Eriksson, Emily M; Lew, Andrew; Jackson, David C

    2011-01-01

    In this study we investigate the feasibility of generating self-adjuvanting vaccines capable of inducing high titre antibody responses following the covalent attachment of the TLR2 agonist Pam(2)Cys to intact proteins. Three Pam(2)Cys-based lipid moieties were prepared which contain a solubilising spacer composed of either lysine residues or polyethyleneglycol. A model protein, hen egg white lysozyme (HEL), was lipidated individually with each of these lipid modules and the immunogenicity of the lipidated species studied in mice by measuring antibody responses. We found that lipidated HEL elicited antibodies which is much stronger than the responses obtained when the HEL was administered in Freund's adjuvant or in Alum. Little or no antibody was elicited by the lipidated HEL in CD4 T cell-deficient mice indicating that the antibody response is T cell dependent. Furthermore, the lipidated protein elicited similar antibody responses in two different strains of mice indicating that sufficient helper T cell epitopes are available to enable antibody production across the histocompatability barrier. In a similar way, lipidated bovine insulin was found to be highly immunogenic in mice despite the largely conserved sequences of bovine and murine insulin. The results provide evidence that lipidation of proteins provides a simple and safe method for the manufacture of soluble self-adjuvanting protein-based vaccines. PMID:21056473

  9. Metabolism of sucrose during storage in intact sorghum stalk

    International Nuclear Information System (INIS)

    Grasses of the Andropogoneae, such as sorghum (Sorghum bicolor [L.] Moench) and sugarcane (Saccharum spp.), accumulate large amounts of harvestable sucrose in the stalk. Sucrose is translocated from the leaves, yet the metabolic fate of sucrose up to and during storage in the stalk is uncertain. This study determined whether sucrose is necessarily routed through inversion and hexose-phosphate isomerization for storage in cells of the intact stalk. Sorghum stalks were dual-infused with [U-14C]sucrose and [fructose-1-3H(N)]sucrose during stages of sucrose accumulation. Unperturbed tissue was used for subsequent sugar analyses. Solutes in cell extracts were separated sequentially through enzymatic conversion and ion-exchange removal of the reaction products. The hexose moieties of sucrose were analyzed separately. A mean of 95% of recovered 14C was in sucrose. In sucrose, 46% of 14C and 77% of 3H were recovered in the fructose moiety. The significant retention of asymmetry in tritium labeling of sucrose indicates that a cycle of cleavage and resynthesis did not dominate sucrose accumulation in cells of the sorghum stalk. (author)

  10. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

    Directory of Open Access Journals (Sweden)

    Hartung Odelya

    2007-12-01

    Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

  11. Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association

    Science.gov (United States)

    Cenens, William; Makumi, Angela; Govers, Sander K.; Lavigne, Rob; Aertsen, Abram

    2015-01-01

    Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host. PMID:26720743

  12. Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

    Science.gov (United States)

    Huebinger, Jan; Han, Hong-Mei; Hofnagel, Oliver; Vetter, Ingrid R; Bastiaens, Philippe I H; Grabenbauer, Markus

    2016-02-23

    Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth. PMID:26541066

  13. Gene Regulatory Network Analysis Reveals Differences in Site-specific Cell Fate Determination in Mammalian Brain

    Directory of Open Access Journals (Sweden)

    Gokhan eErtaylan

    2014-12-01

    Full Text Available Neurogenesis - the generation of new neurons - is an ongoing process that persists in the adult mammalian brain of several species, including humans. In this work we analyze two discrete brain regions: the subventricular zone (SVZ lining the walls of the lateral ventricles; and the subgranular zone (SGZ of the dentate gyrus of the hippocampus in mice and shed light on the SVZ and SGZ specific neurogenesis. We propose a computational model that relies on the construction and analysis of region specific gene regulatory networks from the publicly available data on these two regions. Using this model a number of putative factors involved in neuronal stem cell (NSC identity and maintenance were identified. We also demonstrate potential gender and niche-derived differences based on cell surface and nuclear receptors via Ar, Hif1a and Nr3c1.We have also conducted cell fate determinant analysis for SVZ NSC populations to Olfactory Bulb interneurons and SGZ NSC populations to the granule cells of the Granular Cell Layer. We report thirty-one candidate cell fate determinant gene pairs, ready to be validated. We focus on Ar - Pax6 in SVZ and Sox2 - Ncor1 in SGZ. Both pairs are expressed and localized in the suggested anatomical structures as shown by in situ hybridization and found to physically interact.Finally, we conclude that there are fundamental differences between SGZ and SVZ neurogenesis. We argue that these regulatory mechanisms are linked to the observed differential neurogenic potential of these regions. The presence of nuclear and cell surface receptors in the region specific regulatory circuits indicate the significance of niche derived extracellular factors, hormones and region specific factors such as the oxygen sensitivity, dictating SGZ and SVZ specific neurogenesis.

  14. Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

    DEFF Research Database (Denmark)

    Hattori, Naoko; Niwa, Tohru; Kimura, Kana;

    2013-01-01

    Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which....... Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell...

  15. Optical nanoscopy to reveal structural and functional properties of liver cells (Presentation Recording)

    Science.gov (United States)

    McCourt, Peter; Huser, Thomas R.; Sørensen, Karen K.; Øie, Cristina I.; Mönkemöller, Viola; Ahluwalia, Balpreet S.

    2015-08-01

    The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy.

  16. Tracking of plus-ends reveals microtubule functional diversity in different cell types

    Science.gov (United States)

    Shaebani, M. Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-07-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics.

  17. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    Science.gov (United States)

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells. PMID:24973589

  18. Tracking of plus-ends reveals microtubule functional diversity in different cell types.

    Science.gov (United States)

    Shaebani, M Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-01-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics. PMID:27461361

  19. Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

    Science.gov (United States)

    Tortiglione, Claudia; Quarta, Alessandra; Malvindi, Maria Ada; Tino, Angela; Pellegrino, Teresa

    2009-01-01

    Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism. PMID:19888325

  20. Gene expression profiling reveals novel protective effects of Aminaphtone on ECV304 endothelial cells.

    Science.gov (United States)

    Salazar, Giulia; Bellocchi, Chiara; Todoerti, Katia; Saporiti, Federica; Piacentini, Luca; Scorza, Raffaella; Colombo, Gualtiero I

    2016-07-01

    Aminaphtone, a drug used in the treatment of chronic venous insufficiency (CVI), showed a remarkable role in the modulation of several vasoactive factors, like endothelin-1 and adhesion molecules. We analysed in vitro the effects of Aminaphtone on whole-genome gene expression and production of different inflammatory proteins. ECV-304 endothelial cells were stimulated with IL-1β 100U/ml in the presence or absence of Aminaphtone 6μg/ml. Gene expression profiles were compared at 1, 3, and 6h after stimulation by microarray. Supernatants of ECV-304 cultures were analysed at 3, 6, 12, and 24h by multiplex ELISA for production of several cytokine and chemokines. Microarrays showed a significant down-regulation at all times of a wide range of inflammatory genes. Aminaphtone appeared also able to modulate the regulation of immune response process (down-regulating cytokine biosynthesis, transcripts involved in lymphocyte differentiation and cell proliferation, and cytokine-cytokine receptor interaction) and to regulate genes engaged in homeostasis, secretion, body fluid levels, response to hypoxia, cell division, and cell-to-cell communication and signalling. Results were confirmed and extended analysing the secretome, which showed significant reduction of the release of 14 cytokines and chemokines. These effects are predicted to be mediated by interaction with different transcription factors. Aminaphtone was able to modulate the expression of inflammatory molecules relevant to the pathogenesis of several conditions in which the endothelial dysfunction is the main player and early event, like scleroderma, lung fibrosis, or atherosclerosis. PMID:27083548

  1. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria.

    Science.gov (United States)

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q

    2008-04-22

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize the dormant state of single persister bacteria. Surprisingly, we observe that protein production does take place in supposedly dormant bacteria, over a narrow time window after the exit from stationary phase. Only thereafter does protein production stop, suggesting that differentiation into persisters fully develops over this time window and not during starvation, as previously believed. In effect, we observe that exposure of bacteria to antibiotics during this time window significantly reduces persistence. Our results point to new strategies to fight persistent bacterial infections. The quantitative measurement of single-cell induction presented in this study should shed light on the processes leading to the dormancy of subpopulations in different systems, such as in subpopulations of viable but nonculturable bacteria, or those of quiescent cancer cells. PMID:18427112

  2. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Mark R. Winter

    2015-10-01

    Full Text Available Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex.

  3. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells.

    Science.gov (United States)

    Winter, Mark R; Liu, Mo; Monteleone, David; Melunis, Justin; Hershberg, Uri; Goderie, Susan K; Temple, Sally; Cohen, Andrew R

    2015-10-13

    Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex. PMID:26344906

  4. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    Directory of Open Access Journals (Sweden)

    Kerry Routenberg Love

    Full Text Available Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  5. RhizoFlowCell system reveals early effects of micropollutants on aquatic plant rhizosphere.

    Science.gov (United States)

    Mynampati, Kalyan Chakravarthy; Lee, Yong Jian; Wijdeveld, Arjan; Reuben, Sheela; Samavedham, Lakshminarayanan; Kjelleberg, Staffan; Swarup, Sanjay

    2015-12-01

    In aquatic systems, one of the non-destructive ways to quantify toxicity of contaminants to plants is to monitor changes in root exudation patterns. In aquatic conditions, monitoring and quantifying such changes are currently challenging because of dilution of root exudates in water phase and lack of suitable instrumentation to measure them. Exposure to pollutants would not only change the plant exudation, but also affect the microbial communities that surround the root zone, thereby changing the metabolic profiles of the rhizosphere. This study aims at developing a device, the RhizoFlowCell, which can quantify metabolic response of plants, as well as changes in the microbial communities, to give an estimate of the stress to which the rhizosphere is exposed. The usefulness of RhizoFlowCell is demonstrated using naphthalene as a test pollutant. Results show that RhizoFlowCell system is useful in quantifying the dynamic metabolic response of aquatic rhizosphere to determine ecosystem health. PMID:26386206

  6. Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.

    Science.gov (United States)

    Caldwell, Brittany; Ustione, Alessandro; Piston, David W

    2016-08-01

    Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release. PMID:27508444

  7. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  8. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    Science.gov (United States)

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells. PMID:23733942

  9. Neural activation differences in amputees during imitation of intact versus amputee movements

    Directory of Open Access Journals (Sweden)

    William F Cusack

    2012-06-01

    Full Text Available The mirror neuron system has been attributed with increased activation in motor-related cortical areas upon viewing of another’s actions. Recent work suggests that limb movements that are similar and dissimilar in appearance to that of the viewer equivalently activate the mirror neuron system. It is unclear if this result can be observed in the action encoding areas in amputees who use prosthetic devices. Intact subjects and upper extremity amputee prosthesis users were recruited to view video demonstrations of tools being used by an intact actor and a prosthetic device user. All subjects were asked to pantomime the movements seen in the video while recording electroencephalography. Intact subjects showed equivalent left parietofrontal activity during imitation after watching the intact or prosthetic arm. Likewise, when prosthesis users imitated prosthesis demonstrations, typical left parietofrontal activation was observed during planning. When prosthesis users imitated intact actors, a new pattern was revealed which showed greater bilateral parietal and occipital activity during movement planning (p<0.001. This change may be required for prosthesis users to imitate movements in which the limb states between the observed and the observer do not match. The finding that prosthesis users imitating other prosthesis users showed typical left parietofrontal activation suggests that these subjects engage normal planning related activity when they are able to imitate a limb matching their own. This result has significant implications on rehabilitation, as standard therapy involves training with an intact occupational therapist, which could necessitate atypical planning mechanisms in amputees when learning to use their prosthesis.

  10. Molecular Stratification of Clear Cell Renal Cell Carcinoma by Consensus Clustering Reveals Distinct Subtypes and Survival Patterns

    OpenAIRE

    Brannon, A. Rose; Reddy, Anupama; Seiler, Michael; Arreola, Alexandra; Moore, Dominic T.; Pruthi, Raj S.; Wallen, Eric M.; Nielsen, Matthew E; Liu, Huiqing; Nathanson, Katherine L.; Ljungberg, Börje; Zhao, Hongjuan; BROOKS, JAMES D.; Ganesan, Shridar; Bhanot, Gyan

    2010-01-01

    Clear cell renal cell carcinoma (ccRCC) is the predominant RCC subtype, but even within this classification, the natural history is heterogeneous and difficult to predict. A sophisticated understanding of the molecular features most discriminatory for the underlying tumor heterogeneity should be predicated on identifiable and biologically meaningful patterns of gene expression. Gene expression microarray data were analyzed using software that implements iterative unsupervised consensus cluste...

  11. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  12. Neutron scattering reveals extremely slow cell water in a Dead Sea organism

    OpenAIRE

    Tehei, Moeava; Franzetti, Bruno; Wood, Kathleen; Gabel, Frank; Fabiani, Elisa; Jasnin, Marion; Zamponi, Michaela; Oesterhelt, Dieter; Zaccai, Giuseppe; Ginzburg, Margaret; Ginzburg, Ben-Zion

    2007-01-01

    Intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The water in centrifuged cell pellets was examined by means of two spectrometers, IN6 and IN16, sensitive to motions with time scales of 10 ps and 1 ns, respectively. From IN6 data, a translational diffusion constant of 1.3 × 10−5 cm2 s−1 was determined at 285 K. This value is close to that found previously for other cells and close...

  13. High-throughput sequencing reveals an altered T cell repertoire in X-linked agammaglobulinemia

    OpenAIRE

    Ramesh, Manish; Simchoni, Noa; Hamm, David; Cunningham-Rundles, Charlotte

    2015-01-01

    To examine the T cell receptor structure in the absence of B cells, the TCR β CDR3 was sequenced from DNA of 15 X-linked agammaglobulinemia (XLA) subjects and 18 male controls, using the Illumina HiSeq platform and the ImmunoSEQ analyzer. V gene usage and the V–J combinations, derived from both productive and nonproductive sequences, were significantly different between XLA samples and controls. Although the CDR3 length was similar for XLA and control samples, the CDR3 region of the XLA T cel...

  14. Fossilization and degradation of archaeal intact polar tetraether lipids in deeply buried marine sediments (Peru Margin)

    OpenAIRE

    Lengger, S. K.; Hopmans, E.C.; Sinninghe Damsté, J.S.; Schouten, S.

    2014-01-01

    Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX86. GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL-GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death, but it has been suggested that some of these IPL-GDGTs can, just ...

  15. Evidence that the central canal lining of the spinal cord contributes to oligodendrogenesis during postnatal development and adulthood in intact rats.

    Science.gov (United States)

    Sevc, Juraj; Matiašová, Anna; Kútna, Viera; Daxnerová, Zuzana

    2014-10-01

    Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14-E18) and E20-E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2(+) /Sox-10(+) cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2(+) /Sox-10(+) cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2(+) /Sox-10(+) cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2(+) /Sox-10(+) cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2(+) /Sox-10(+) cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120-P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats. PMID:24659141

  16. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

    DEFF Research Database (Denmark)

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga;

    2015-01-01

    heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for...

  17. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

    Science.gov (United States)

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. DOI: http://dx.doi.org/10.7554/eLife.16578.001 PMID:27481325

  18. Mucus-secreting 'signet-ring' cells in CSF revealing the site of primary cancer.

    OpenAIRE

    Agnelli, G.; Gresele, P.

    1980-01-01

    A case is reported of leptomeningeal carcinomatosis in which identification of mucus-secreting 'signet-ring' carcinoma cells in the CSF allowed diagnosis of an otherwise asymptomatic gastric cancer. When lumbar puncture is performed, careful cytological examination of the CSF should be carried out in any undiagnosed patient with neurological symptoms and signs.

  19. Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

    Science.gov (United States)

    Wang, Weikang; Quan, Yi; Fu, Qibin; Liu, Yu; Liang, Ying; Wu, Jingwen; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2014-01-01

    Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs) can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy. PMID:24416258

  20. Networks of neuroblastoma cells on porous silicon substrates reveal a small world topology

    KAUST Repository

    Marinaro, Giovanni

    2015-01-01

    The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized. This journal is

  1. Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Alice Nemajerova

    Full Text Available Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.

  2. Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

    Directory of Open Access Journals (Sweden)

    Roberto Plebani

    2012-11-01

    Full Text Available mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (≈2 x 10-5 of all mRNA. Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

  3. Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

    Directory of Open Access Journals (Sweden)

    Weikang Wang

    Full Text Available Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy.

  4. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  5. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies

    NARCIS (Netherlands)

    Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E.; Porubsky, David; Spierings, Diana C. J.; de Jong, Tristan V.; Halsema, Nancy; Kazemier, Hinke G.; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S. J. M.; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M.; Colome-Tatche, Maria; Foijer, Floris

    2016-01-01

    Background: Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number cha

  6. Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain

    Directory of Open Access Journals (Sweden)

    Anna-Lena Hallmann

    2016-05-01

    Full Text Available Reprogramming technology enables the production of neural progenitor cells (NPCs from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.

  7. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Holger A. Russ

    2016-01-01

    Full Text Available Current approaches in human embryonic stem cell (hESC to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1, Neuroplastin (NPTN, and the Laminin α-2 subunit (LAMA2. Two of the three factors (LGALS1 and LAMA2 increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

  8. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

    Science.gov (United States)

    Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

    2016-01-01

    Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

  9. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

    OpenAIRE

    Ellen, Albert F.; Albers, Sonja-Verena; Huibers, Wim; Pitcher, Angela; Hobel, Cedric F. V.; Schwarz, Heinz; Folea, Mihaela; Schouten, Stefan; Boekema, Egbert J.; Poolman, Bert; Driessen, Arnold J. M.

    2009-01-01

    The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles ...

  10. Integrated analysis of breast cancer cell lines reveals unique signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.; Laderoute, Keith R.; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L.; Laquerre, Sylvie; Jackson, Jeffrey R.; Wooster, Richard F.; Kuo, Wen-Lin; Gray, Joe W.; Spellman, Paul T.

    2009-03-31

    Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

  11. Gene expression profiling of dendritic cells reveals important mechanisms associated with predisposition to Staphylococcus infections.

    Directory of Open Access Journals (Sweden)

    Mehdi Toufeer

    Full Text Available BACKGROUND: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. CONCLUSION/SIGNIFICANCE: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment

  12. Gene Expression Profiling of Dendritic Cells Reveals Important Mechanisms Associated with Predisposition to Staphylococcus Infections

    Science.gov (United States)

    Toufeer, Mehdi; Bonnefont, Cécile M. D.; Foulon, Eliane; Caubet, Cécile; Tasca, Christian; Aurel, Marie-Rose; Robert-Granié, Christèle; Rupp, Rachel; Foucras, Gilles

    2011-01-01

    Background Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. Methodology/Principal Findings We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. Conclusion/Significance We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct

  13. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    Energy Technology Data Exchange (ETDEWEB)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  14. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  15. 46 CFR 28.570 - Intact righting energy.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  16. An Epigenetic Mechanism of High Gdnf Transcription in Glioma Cells Revealed by Specific Sequence Methylation.

    Science.gov (United States)

    Zhang, Bao-Le; Liu, Jie; Lei, Yu; Xiong, Ye; Li, Heng; Lin, Xiaoqian; Yao, Rui-Qin; Gao, Dian-Shuai

    2016-09-01

    Glioma cells express high levels of GDNF. When investigating its transcriptional regulation mechanism, we observed increased or decreased methylation of different cis-acting elements in the gdnf promoter II. However, it is difficult to determine the contributions of methylation changes of each cis-acting element to the abnormally high transcription of gdnf gene. To elucidate the contributions of methylation changes of specific cis-acting elements to the regulation of gdnf transcription, we combined gene site-directed mutation, molecular cloning, and dual luciferase assay to develop the "specific sequence methylation followed by plasmid recircularization" method to alter methylation levels of specific cis-acting elements in the gdnf promoter in living cells and assess gene transcriptional activity. This method successfully introduced artificial changes in the methylation of different cis-acting elements in the gdnf promoter II. Moreover, compared with unmethylated gdnf promoter II, both silencer II hypermethylation plus enhancer II unmethylation and hypermethylation of the entire promoter II (containing enhancer II and silencer II) significantly enhanced gdnf transcriptional activity (P  0.05). Enhancer II hypermethylation plus silencer II unmethylation did not significantly affect gene transcription (P > 0.05). Furthermore, we found significantly increased DNA methylation in the silencer II of the gdnf gene in high-grade astroglioma cells with abnormally high gdnf gene expression (P < 0.01). The absence of silencer II significantly increased gdnf promoter II activity in U251 cells (P < 0.01). In conclusion, our specific sequence methylation followed by plasmid recircularization method successfully altered the methylation levels of a specific cis-acting element in a gene promoter in living cells. This method allows in-depth investigation of the impact of methylation changes of different cis-acting elements in the same promoter on gene transcriptional

  17. Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells.

    OpenAIRE

    Ellman, M; Bikel, I; Figge, J; Roberts, T; Schlossman, R; Livingston, D M

    1984-01-01

    Monkey and mouse cells producing simian virus 40 small t antigen in the absence of clearly detectable intact or truncated large T antigens were subjected to indirect immunofluorescence and biochemical cell compartment analyses. Results revealed specific immunofluorescence and small t polypeptide in both the nucleus and cytoplasm of these cells.

  18. Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts

    Directory of Open Access Journals (Sweden)

    Yonatan Stelzer

    2015-04-01

    Full Text Available Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5′ RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.

  19. Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

    Directory of Open Access Journals (Sweden)

    Roberto Weigert

    2012-11-01

    Full Text Available Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events.

  20. Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells

    DEFF Research Database (Denmark)

    Lauber, Chris; Vieyres, Gabrielle; Terczynska-Dyla, Ewa;

    2015-01-01

    4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using...... transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified...... genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium....

  1. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase....... mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  2. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    International Nuclear Information System (INIS)

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

  3. Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina.

    Science.gov (United States)

    Kombrink, E; Wöber, G

    1980-07-01

    A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,L-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm(-3). Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm(-3). Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each. PMID:24306242

  4. Cryo-STEM Tomography of Intact Vitrified Fibroblasts

    Directory of Open Access Journals (Sweden)

    David Kirchenbuechler

    2015-07-01

    Full Text Available Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET. Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.

  5. Genome-Wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells

    OpenAIRE

    Chiarle, Roberto; Zhang, Yu; Frock, Richard L.; Lewis, Susanna M.; Molinie, Benoit; Ho, Yu-Jui; Myers, Darienne R; Choi, Vivian W.; Compagno, Mara; Malkin, Daniel J.; Neuberg, Donna; Monti, Stefano; Giallourakis, Cosmas C.; Gostissa, Monica; Alt, Frederick W.

    2011-01-01

    While chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for Act...

  6. Astrocyte morphology after cortical stab wound revealed by single-cell confocal 3D morphometry

    Czech Academy of Sciences Publication Activity Database

    Chvátal, Alexandr; Anděrová, Miroslava; Petřík, David; Syková, Eva

    č. 2 (2003), s. 63. ISSN 0894-1491. [European Meeting on Glial Cell Function in Health and Disease /6./. Berlín, 03.09.2003-06.09.2003] R&D Projects: GA ČR GA305/02/1528; GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5039906; CEZ:MSM 111300004 Keywords : cortical stab wound * morphometry Subject RIV: FH - Neurology Impact factor: 4.677, year: 2003

  7. Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

    OpenAIRE

    Durand, Charles; Robin, Catherine; Bollerot, Karine; Baron, Margaret H.; Ottersbach, Katrin; Dzierzak, Elaine

    2007-01-01

    Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identif...

  8. Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

    OpenAIRE

    Nayar, Asha S.; Dougherty, Thomas J.; Ferguson, Keith E.; Granger, Brett A.; McWilliams, Lisa; Stacey, Clare; Leach, Lindsey J.; Narita, Shin-ichiro; Tokuda, Hajime; Miller, Alita A.; Brown, Dean G.; McLeod, Sarah M.

    2015-01-01

    A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. ...

  9. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity

    OpenAIRE

    Modlich, Ute; Bohne, Jens; Schmidt, Manfred; von Kalle, Christof; Knöss, Sabine; Schambach, Axel; Baum, Christopher

    2006-01-01

    Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhance...

  10. Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling

    OpenAIRE

    Brand Andrea H; Egger Boris; von Trotha Jakob W

    2009-01-01

    Abstract Background The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain. Results We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU) labelling, that cell proliferation does occur in the Droso...

  11. An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development

    OpenAIRE

    Benitez, Cecil M.; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T.; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H. Efsun; Zhang, Jiajing; Dekker, Joseph D.; Tucker, Haley O; Chang, Howard Y.; Kim, Seung K.

    2014-01-01

    Author Summary Discovery of specific pancreas developmental regulators has accelerated in recent years. In contrast, the global regulatory programs controlling pancreas development are poorly understood compared to other organs or tissues like heart or blood. Decoding this regulatory logic may accelerate development of replacement organs from renewable sources like stem cells, but this goal requires identification of regulators and assessment of their functions on a global scale. To address t...

  12. Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis*

    OpenAIRE

    Chornoguz, Olesya; Grmai, Lydia; Sinha, Pratima; Artemenko, Konstantin A.; Zubarev, Roman A.; Ostrand-Rosenberg, Suzanne

    2010-01-01

    Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in...

  13. Signal Inhibition Reveals JAK/STAT3 Pathway as Critical for Bovine Inner Cell Mass Development.

    Science.gov (United States)

    Meng, Fanli; Forrester-Gauntlett, Blaise; Turner, Pavla; Henderson, Harold; Oback, Björn

    2015-12-01

    The inner cell mass (ICM) of mammalian blastocysts consists of pluripotent epiblast and hypoblast lineages, which develop into embryonic and extraembryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine Day 8 blastocysts. From the morula stage onward, in vitro fertilized embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signaling pathway components, including TGFbeta1, BMP, EGF, VEGF, PDGF, FGF, cAMP, PI3K, and JAK signals. Using 1) blastocyst quality (by morphological grading), 2) cell numbers (by differential stain), and 3) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR), we identified positive and negative regulators of ICM development and pluripotency. TGFbeta1, BMP, and cAMP and combined VEGF/PDGF/FGF signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF/PDGF/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signaling with AZD1480. This treatment interfered with ICM formation, but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts as well as naive pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3(Y705)) was restricted to ICM nuclei, where it colocalized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3(Y705) signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naive pluripotency markers. PMID:26510863

  14. Three Distinct Modes of Exocytosis Revealed by Amperometry in Neuroendocrine Cells

    OpenAIRE

    van Kempen, G. Th. H.; vanderLeest, H.T.; Van den Berg, R J; Eilers, P.; Westerink, R.H.S.

    2011-01-01

    Neurotransmission requires Ca2+-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quanta...

  15. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

    OpenAIRE

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E.

    2016-01-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry prope...

  16. Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

    OpenAIRE

    Roberto Weigert; Myo-Pale' Aye; Kamil Rechache; Natalie Porat-Shliom; Andrius Masedunskas

    2012-01-01

    Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. ...

  17. Convective cell structures revealed by Mie laser radar observations and image data processing.

    Science.gov (United States)

    Sasano, Y; Shimizu, H; Takeuchi, N

    1982-09-01

    Mie scattering laser radar has been used to study the structure of the convective mixed layer in optically clear air. Two-dimensional image data obtained by scanning measurements (PPI and RHI modes) have been processed using edge-enhancement techniques to display distinct structures within the convective layer. The structures of convective cells penetrating into upper clean air were very similar to those obtained previously by sodar, radar, and FM-cw radar. PMID:20396196

  18. Atomic force microscopy reveals differences in cell membrane properties in nuclear myosin I mutant

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Rohožková, Jana; Kalendová, Alžběta; Hozák, Pavel

    Praha : ČSMS, 2013. s. 25-25. [Mikroskopie 2013. 13.05.2013-14.05.2013, Lednice] R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020118; GA MŠk LH12143 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : atomic force microscopy * cell membrane * myosin 1C * NM1 * nuclear myosin I

  19. Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks.

    Science.gov (United States)

    Vidyasekar, Prasanna; Shyamsunder, Pavithra; Arun, Rajpranap; Santhakumar, Rajalakshmi; Kapadia, Nand Kishore; Kumar, Ravi; Verma, Rama Shanker

    2015-01-01

    Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2. PMID:26295583

  20. X-rays Reveal the Internal Structure of Keratin Bundles in Whole Cells.

    Science.gov (United States)

    Hémonnot, Clément Y J; Reinhardt, Juliane; Saldanha, Oliva; Patommel, Jens; Graceffa, Rita; Weinhausen, Britta; Burghammer, Manfred; Schroer, Christian G; Köster, Sarah

    2016-03-22

    In recent years, X-ray imaging of biological cells has emerged as a complementary alternative to fluorescence and electron microscopy. Different techniques were established and successfully applied to macromolecular assemblies and structures in cells. However, while the resolution is reaching the nanometer scale, the dose is increasing. It is essential to develop strategies to overcome or reduce radiation damage. Here we approach this intrinsic problem by combing two different X-ray techniques, namely ptychography and nanodiffraction, in one experiment and on the same sample. We acquire low dose ptychography overview images of whole cells at a resolution of 65 nm. We subsequently record high-resolution nanodiffraction data from regions of interest. By comparing images from the two modalities, we can exclude strong effects of radiation damage on the specimen. From the diffraction data we retrieve quantitative structural information from intracellular bundles of keratin intermediate filaments such as a filament radius of 5 nm, hexagonal geometric arrangement with an interfilament distance of 14 nm and bundle diameters on the order of 70 nm. Thus, we present an appealing combined approach to answer a broad range of questions in soft-matter physics, biophysics and biology. PMID:26905642

  1. Metabolomics reveals that carnitine palmitoyltransferase-1 is a novel target for oxidative inactivation in human cells.

    Science.gov (United States)

    Setoyama, Daiki; Fujimura, Yoshinori; Miura, Daisuke

    2013-12-01

    Oxidative dysfunction in the metabolism has long been implicated in diverse biological disorders. Although a substantial number of metabolic enzymes are targeted for inactivation by oxidative stress, identifying those targets remains difficult due to a lack of comprehensive observations of the metabolism acting through the stress response. We herein developed a metabolomics strategy using integrative liquid chromatography-mass spectrometry (LC-MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H2 O2 )-induced oxidative stress in HeLa cells. Among the many metabolite changes detected, the most characteristic metabolites uniquely indicated carnitine palmitoyltransferase-1 (CPT1), the critical enzyme for mitochondrial β-oxidation of long-chain fatty acids, to be a target for oxidative inactivation. We showed that the enzymatic activity of CPT1 significantly declined by H2 O2 in several human cells. Interestingly, the inactivation was shown to be a direct effect of H2 O2 in vitro, but substantially occurred when cells were cultured with some reagents that generate reactive oxygen species (ROS). Thus, our results suggest the generality of CPT1 inhibition under various stress conditions associated with ROS generation, providing an insight into a mechanism for oxidative dysfunction in mitochondrial metabolism. Our metabolome data additionally suggest that certain methyltransferase(s) may be targets of oxidative stress as well. PMID:24118240

  2. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    Science.gov (United States)

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  3. Equilibrium physics breakdown reveals the active nature of red blood cell flickering

    Science.gov (United States)

    Turlier, H.; Fedosov, D. A.; Audoly, B.; Auth, T.; Gov, N. S.; Sykes, C.; Joanny, J.-F.; Gompper, G.; Betz, T.

    2016-05-01

    Red blood cells, or erythrocytes, are seen to flicker under optical microscopy, a phenomenon initially described as thermal fluctuations of the cell membrane. But recent studies have suggested the involvement of non-equilibrium processes, without definitively ruling out equilibrium interpretations. Using active and passive microrheology to directly compare the membrane response and fluctuations on single erythrocytes, we report here a violation of the fluctuation-dissipation relation, which is a direct demonstration of the non-equilibrium nature of flickering. With an analytical model of the composite erythrocyte membrane and realistic stochastic simulations, we show that several molecular mechanisms may explain the active fluctuations, and we predict their kinetics. We demonstrate that tangential metabolic activity in the network formed by spectrin, a cytoskeletal protein, can generate curvature-mediated active membrane motions. We also show that other active membrane processes represented by direct normal force dipoles may explain the observed membrane activity. Our findings provide solid experimental and theoretical frameworks for future investigations of the origin and function of active motion in cells.

  4. Reversible membrane reorganizations during photosynthesis in vivo: revealed by small-angle neutron scattering.

    Science.gov (United States)

    Nagy, Gergely; Posselt, Dorthe; Kovács, László; Holm, Jens K; Szabó, Milán; Ughy, Bettina; Rosta, László; Peters, Judith; Timmins, Peter; Garab, Gyozo

    2011-06-01

    In the present study, we determined characteristic repeat distances of the photosynthetic membranes in living cyanobacterial and eukaryotic algal cells, and in intact thylakoid membranes isolated from higher plants with time-resolved small-angle neutron scattering. This non-invasive technique reveals light-induced reversible reorganizations in the seconds-to-minutes time scale, which appear to be associated with functional changes in vivo. PMID:21473741

  5. Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid

    Directory of Open Access Journals (Sweden)

    Daniel Fraher

    2016-02-01

    Full Text Available The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

  6. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

    Science.gov (United States)

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  7. Primary B-cell deficiencies reveal a link between human IL-17-producing CD4 T-cell homeostasis and B-cell differentiation.

    Directory of Open Access Journals (Sweden)

    Rita R Barbosa

    Full Text Available IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17 share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID, defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21(lowCD38(low. Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies.

  8. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

    International Nuclear Information System (INIS)

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

  9. Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation

    Directory of Open Access Journals (Sweden)

    Treuner-Lange Anke

    2010-04-01

    Full Text Available Abstract Background Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. Results Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate

  10. Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

    Science.gov (United States)

    Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

    2008-01-01

    Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress. PMID:18436547

  11. Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

    DEFF Research Database (Denmark)

    Brünner, N; Svenstrup, B; Spang-Thomsen, M; Bennett, P; Nielsen, A; Nielsen, J

    1986-01-01

    An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those of t...

  12. HIGH-SPEED SINGLE QUANTUM DOT IMAGING OF IN LIVE CELLS REVEAL HOP DIFFUSION

    DEFF Research Database (Denmark)

    Lagerholm, B. Christoffer; Clausen, Mathias P.

    2011-01-01

    have yet to be independently confirmed. In this work, we show that high-speed single particle tracking with quantum dots (QDs) and using a standard wide-field fluorescence microscope and an EMCCD is possible at image acquisition rates of up to ~2000 Hz. The spatial precision in these experiments is ~40...... nm (as determined from the standard deviation of repeated position measurements of an immobile QD on a cell). Using this system, we show that membrane proteins and lipids, which have been exogenously labeled with functionalized QDs, show examples of three types of motion in the plasma membrane of...

  13. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.

    OpenAIRE

    Krämer, A

    1987-01-01

    Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ri...

  14. Atomic force microscopy reveals differences in cell membrane properties in nuclear myosin I mutant

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Rohožková, Jana; Hozák, Pavel

    Praha : Society for Histochemistry, 2013. [55th Symposium of the Society for Histochemistry. 11.06.2013-14.06.2013, Praha] R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GAP305/11/2232; GA TA ČR TE01020118; GA MŠk LH12143 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : atomic force microscopy * cell membrane * myosin 1C * NM1 * nuclear myosin I Subject RIV: EB - Genetics ; Molecular Biology

  15. DNA methylation profiling reveals novel diagnostic biomarkers in renal cell carcinoma

    OpenAIRE

    Lasseigne, Brittany N.; Burwell, Todd C; Patil, Mohini A; Absher, Devin M.; BROOKS, JAMES D.; Myers, Richard M.

    2014-01-01

    Background Renal cell carcinoma (RCC) is the tenth most commonly diagnosed cancer in the United States. While it is usually lethal when metastatic, RCC is successfully treated with surgery when tumors are confined to the kidney and have low tumor volume. Because most early stage renal tumors do not result in symptoms, there is a strong need for biomarkers that can be used to detect the presence of the cancer as well as to monitor patients during and after therapy. Methods We examined genome-w...

  16. Strong HIV-1-Specific T Cell Responses in HIV-1-Exposed Uninfected Infants and Neonates Revealed after Regulatory T Cell Removal

    OpenAIRE

    Legrand, Fatema A.; Nixon, Douglas F.; Loo, Christopher P.; Erika Ono; Chapman, Joan M; Maristela Miyamoto; Diaz, Ricardo S.; Amélia M N Santos; Succi, Regina C. M.; Jacob Abadi; Rosenberg, Michael G.; Maria Isabel de Moraes-Pinto; Esper G Kallas

    2006-01-01

    BACKGROUND: In utero transmission of HIV-1 occurs on average in only 3%-15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4(+) CD25(+) T-regulatory (Treg) cells can reveal strong HIV-1...

  17. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging

    Science.gov (United States)

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-01

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes.Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the

  18. Activation and deactivation of vibronic channels in intact phycocyanin rods

    Science.gov (United States)

    Nganou, C.; David, L.; Meinke, R.; Adir, N.; Maultzsch, J.; Mkandawire, M.; Pouhè, D.; Thomsen, C.

    2014-02-01

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm-1 is assigned to the C-C stretching vibration while the mode at 454 cm-1 is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm-1 does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm-1 rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  19. Intact implicit processing of facial threat cues in schizophrenia.

    Science.gov (United States)

    Shasteen, Jonathon R; Pinkham, Amy E; Kelsven, Skylar; Ludwig, Kelsey; Payne, B Keith; Penn, David L

    2016-01-01

    An emerging body of research suggests that people with schizophrenia retain the ability to implicitly perceive facial affect, despite well-documented difficulty explicitly identifying emotional expressions. It remains unclear, however, whether such functional implicit processing extends beyond emotion to other socially relevant facial cues. Here, we constructed two novel versions of the Affect Misattribution Procedure, a paradigm in which affective responses to primes are projected onto neutral targets. The first version included three face primes previously validated to elicit varying inferences of threat from healthy individuals via emotion-independent structural modification (e.g., nose and eye size). The second version included the threat-relevant emotional primes of angry, neutral, and happy faces. Data from 126 participants with schizophrenia and 84 healthy controls revealed that although performing more poorly on an assessment of explicit emotion recognition, patients showed normative implicit threat processing for both non-emotional and emotional facial cues. Collectively, these results support recent hypotheses postulating that the initial perception of salient facial information remains intact in schizophrenia, but that deficits arise at subsequent stages of contextual integration and appraisal. Such a breakdown in the stream of face processing has important implications for mechanistic models of social cognitive impairment in schizophrenia and treatment strategies aiming to improve functional outcome. PMID:26673971

  20. Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

    Science.gov (United States)

    Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shujing; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2015-01-01

    Summary Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states; and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. PMID:26804902

  1. Metabolomics reveals metabolic targets and biphasic responses in breast cancer cells treated by curcumin alone and in association with docetaxel.

    Directory of Open Access Journals (Sweden)

    Mathilde Bayet-Robert

    Full Text Available BACKGROUND: Curcumin (CUR has deserved extensive research due to its anti-inflammatory properties, of interest in human diseases including cancer. However, pleiotropic even paradoxical responses of tumor cells have been reported, and the mechanisms of action of CUR remain uncompletely elucidated. METHODOLOGY/PRINCIPAL FINDINGS: (1H-NMR spectroscopy-based metabolomics was applied to get novel insight into responses of MCF7 and MDA-MB-231 breast cancer cells to CUR alone, and MCF7 cells to CUR in cotreatment with docetaxel (DTX. In both cell types, a major target of CUR was glutathione metabolism. Total glutathione (GSx increased at low dose CUR (≤ 10 mg.l(-1-28 µM- (up to +121% in MCF7 cells, P<0.01, and +138% in MDA-MB-231 cells, P<0.01, but decreased at high dose (≥ 25 mg.l(-1 -70 µM- (-49%, in MCF7 cells, P<0.02, and -56% in MDA-MB-231 cells, P<0.025. At high dose, in both cell types, GSx-related metabolites decreased, including homocystein, creatine and taurine (-60 to -80%, all, P<0.05. Together with glutathione-S-transferase actvity, data established that GSx biosynthesis was upregulated at low dose, and GSx consumption activated at high dose. Another major target, in both cell types, was lipid metabolism involving, at high doses, accumulation of polyunsaturated and total free fatty acids (between ×4.5 and ×11, P<0.025, and decrease of glycerophospho-ethanolamine and -choline (about -60%, P<0.025. Multivariate statistical analyses showed a metabolic transition, even a biphasic behavior of some metabolites including GSx, between low and high doses. In addition, CUR at 10 mg.l(-1 in cotreatment with DTX induced modifications in glutathione metabolism, lipid metabolism, and glucose utilization. Some of these changes were biphasic depending on the duration of exposure to CUR. CONCLUSIONS/SIGNIFICANCE: Metabolomics reveals major metabolic targets of CUR in breast cancer cells, and biphasic responses that challenge the widely accepted

  2. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

    Science.gov (United States)

    Toman, O; Kabickova, T; Vit, O; Fiser, R; Polakova, K Machova; Zach, J; Linhartova, J; Vyoral, D; Petrak, J

    2016-09-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  3. Systems model of T cell receptor proximal signaling reveals emergent ultrasensitivity.

    Directory of Open Access Journals (Sweden)

    Himadri Mukhopadhyay

    Full Text Available Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs. Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy.

  4. A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

    Science.gov (United States)

    Mentrup, Torben; Häsler, Robert; Fluhrer, Regina; Saftig, Paul; Schröder, Bernd

    2015-08-01

    During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes. PMID:25824657

  5. Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing

    Directory of Open Access Journals (Sweden)

    Muammar eMansor

    2015-08-01

    Full Text Available Large, sulfur-cycling, calcite-precipitating bacteria in the genus Achromatium represent a significant proportion of bacterial communities near sediment-water interfaces throughout the world. Our understanding of their potentially crucial roles in calcium, carbon, sulfur, nitrogen, and iron cycling is limited because they have not been cultured or sequenced using environmental genomics approaches to date. We utilized single-cell genomic sequencing to obtain one incomplete and two nearly complete draft genomes for Achromatium collected at Warm Mineral Springs, FL. Based on 16S rRNA gene sequences, the three cells represent distinct and relatively distant Achromatium populations (91-92% identity. The draft genomes encode key genes involved in sulfur and hydrogen oxidation; oxygen, nitrogen and polysulfide respiration; carbon and nitrogen fixation; organic carbon assimilation and storage; chemotaxis; twitching motility; antibiotic resistance; and membrane transport. Known genes for iron and manganese energy metabolism were not detected. The presence of pyrophosphatase and vacuolar (V-type ATPases, which are generally rare in bacterial genomes, suggests a role for these enzymes in calcium transport, proton pumping, and/or energy generation in the membranes of calcite-containing inclusions.

  6. Comparative genomics as a tool to reveal functional equivalences between human and mouse dendritic cell subsets.

    Science.gov (United States)

    Crozat, Karine; Guiton, Rachel; Guilliams, Martin; Henri, Sandrine; Baranek, Thomas; Schwartz-Cornil, Isabelle; Malissen, Bernard; Dalod, Marc

    2010-03-01

    During evolution, vertebrates have developed an adaptive immune system able to cope with a variety of pathogens. Dendritic cells (DCs) are central to this process. DCs integrate information derived from pathogens or endogenous danger signals and convey them to T lymphocytes. Most of the present knowledge on DCs was generated in mice or by using human DCs differentiated in vitro from monocytes. In both species, several DC subsets have been identified in vivo based on differences in their phenotypes, anatomical locations or functions. In mice, protective immunity against intracellular pathogens or tumors can be induced most efficiently by targeting antigens to the CD8 alpha(+) DCs, a subset of DCs which resides in lymphoid tissues and is especially efficient at cross-presenting exogenous antigens to CD8(+) T lymphocytes. In contrary, harnessing human DC subsets for medical purposes is currently hampered by insufficient knowledge about these cells. To overcome this cognitive gap, we are using comparative genomics as a tool for designing hypotheses and experiments to further characterize DC subset functions and their molecular control, including the investigation of the functional equivalences that might exist between human and mouse DC subsets. PMID:20193019

  7. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Directory of Open Access Journals (Sweden)

    Shufeng Liu

    Full Text Available Hepatitis C virus (HCV poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP, UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1 and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.

  8. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Science.gov (United States)

    Liu, Shufeng; Zhao, Ting; Song, BenBen; Zhou, Jianhua; Wang, Tony T

    2016-01-01

    Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. PMID:26808496

  9. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  10. Prospective monitoring reveals dynamic levels of T cell immunity to Mycobacterium tuberculosis in HIV infected individuals.

    Directory of Open Access Journals (Sweden)

    Jessica E Mitchell

    Full Text Available Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG and IFN-γ inducible protein-10 (IP10 (MIG: r=0.6527, p=0.0114; IP-10: r=0.6967, p=0.0056; IP-10+MIG: r=0.7055, p=0.0048. During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.

  11. Transcriptomic gene-network analysis of exposure to silver nanoparticle reveals potentially neurodegenerative progression in mouse brain neural cells.

    Science.gov (United States)

    Lin, Ho-Chen; Huang, Chin-Lin; Huang, Yuh-Jeen; Hsiao, I-Lun; Yang, Chung-Wei; Chuang, Chun-Yu

    2016-08-01

    Silver nanoparticles (AgNPs) are commonly used in daily living products. AgNPs can induce inflammatory response in neuronal cells, and potentially develop neurological disorders. The gene networks in response to AgNPs-induced neurodegenerative progression have not been clarified in various brain neural cells. This study found that 3-5nm AgNPs were detectable to enter the nuclei of mouse neuronal cells after 24-h of exposure. The differentially expressed genes in mouse brain neural cells exposure to AgNPs were further identified using Phalanx Mouse OneArray® chip, and permitted to explore the gene network pathway regulating in neurodegenerative progression according to Cytoscape analysis. In focal adhesion pathway of ALT astrocytes, AgNPs induced the gene expression of RasGRF1 and reduced its downstream BCL2 gene for apoptosis. In cytosolic DNA sensing pathway of microglial BV2 cells, AgNPs reduced the gene expression of TREX1 and decreased IRF7 to release pro-inflammatory cytokines for inflammation and cellular activation. In MAPK pathway of neuronal N2a cells, AgNPs elevated GADD45α gene expression, and attenuated its downstream PTPRR gene to interfere with neuron growth and differentiation. Moreover, AgNPs induced beta amyloid deposition in N2a cells, and decreased PSEN1 and PSEN2, which may disrupt calcium homeostasis and presynaptic dysfunction for Alzheimer's disease development. These findings suggested that AgNPs exposure reveals the potency to induce the progression of neurodegenerative disorder. PMID:27131904

  12. Ex Vivo Modeling of Multidomain Peptide Hydrogels with Intact Dental Pulp.

    Science.gov (United States)

    Moore, A N; Perez, S C; Hartgerink, J D; D'Souza, R N; Colombo, J S

    2015-12-01

    Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP

  13. High-Speed Single Quantum Dot Imaging of Artificial Lipids in Live Cells Reveal Partial Hop Diffusion

    DEFF Research Database (Denmark)

    Lagerholm, B. Christoffer; Clausen, Mathias P.; Christensen, Eva Arnspang

    2010-01-01

    . The spatial precision in these experiments is ~40 nm (as determined from the standard deviation of repeated position measurements of an immobile QD on a cell). Using this system, we further show that an artificial lipid, biotin-cap-DPPE, inserted in a mouse embryo fibroblast (MEF), labeled with s......Av-QD655, and imaged as described reveals examples of three types of motion, 1) approximately free diffusion, 2) confined (immobile) diffusion, and 3) hop diffusion between compartments with a size of ~100 nm diameter, and a lifetime of ~100-200 milliseconds. In these experiments, we have used sAv-QD655s...

  14. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells

    Science.gov (United States)

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-01

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  15. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells.

    Science.gov (United States)

    Fang, Yi; Zhang, Qian; Wang, Xin; Yang, Xue; Wang, Xiangyu; Huang, Zhen; Jiao, Yuchen; Wang, Jing

    2016-03-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  16. A simple cell-based assay reveals that diverse neuropsychiatric risk genes converge on primary cilia.

    Directory of Open Access Journals (Sweden)

    Aaron Marley

    Full Text Available Human genetic studies are beginning to identify a large number of genes linked to neuropsychiatric disorders. It is increasingly evident that different genes contribute to risk for similar syndromes and, conversely, the same genes or even the same alleles cross over traditional diagnostic categories. A current challenge is to understand the cellular biology of identified risk genes. However, most genes associated with complex neuropsychiatric phenotypes are not related through a known biochemical pathway, and many have an entirely unknown cellular function. One possibility is that diverse disease-linked genes converge at a higher-level cellular structure. The synapse is already known to be one such convergence, and emerging evidence suggests the primary cilium as another. Because many genes associated with neuropsychiatric illness are expressed also outside the nervous system, as are cilia, we tested the hypothesis that such genes affect conserved features of the primary cilium. Using RNA interference to test 41 broadly expressed candidate genes associated with schizophrenia, bipolar affective disorder, autism spectrum disorder and intellectual disability, we found 20 candidates that reduce ciliation in NIH3T3 cells when knocked down, and three whose manipulation increases cilia length. Three of the candidate genes were previously implicated in cilia formation and, altogether, approximately half of the candidates tested produced a ciliary phenotype. Our results support the hypothesis that primary cilia indeed represent a conserved cellular structure at which the effects of diverse neuropsychiatric risk genes converge. More broadly, they suggest a relatively simple cell-based approach that may be useful for exploring the complex biological underpinnings of neuropsychiatric disease.

  17. Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons.

    Science.gov (United States)

    Grealish, Shane; Heuer, Andreas; Cardoso, Tiago; Kirkeby, Agnete; Jönsson, Marie; Johansson, Jenny; Björklund, Anders; Jakobsson, Johan; Parmar, Malin

    2015-06-01

    Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson's disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models. PMID:26004633

  18. A genome-wide systematic analysis reveals different and predictive proliferation expression signatures of cancerous vs. non-cancerous cells.

    Directory of Open Access Journals (Sweden)

    Yedael Y Waldman

    Full Text Available Understanding cell proliferation mechanisms has been a long-lasting goal of the scientific community and specifically of cancer researchers. Previous genome-scale studies of cancer proliferation determinants have mainly relied on knockdown screens aimed to gauge their effects on cancer growth. This powerful approach has several limitations such as off-target effects, partial knockdown, and masking effects due to functional backups. Here we employ a complementary approach and assign each gene a cancer Proliferation Index (cPI that quantifies the association between its expression levels and growth rate measurements across 60 cancer cell lines. Reassuringly, genes found essential in cancer gene knockdown screens exhibit significant positive cPI values, while tumor suppressors exhibit significant negative cPI values. Cell cycle, DNA replication, splicing and protein production related processes are positively associated with cancer proliferation, while cellular migration is negatively associated with it - in accordance with the well known "go or grow" dichotomy. A parallel analysis of genes' non-cancerous proliferation indices (nPI across 224 lymphoblastoid cell lines reveals surprisingly marked differences between cancerous and non-cancerous proliferation. These differences highlight genes in the translation and spliceosome machineries as selective cancer proliferation-associated proteins. A cross species comparison reveals that cancer proliferation resembles that of microorganisms while non-cancerous proliferation does not. Furthermore, combining cancerous and non-cancerous proliferation signatures leads to enhanced prediction of patient outcome and gene essentiality in cancer. Overall, these results point to an inherent difference between cancerous and non-cancerous proliferation determinants, whose understanding may contribute to the future development of novel cancer-specific anti-proliferative drugs.

  19. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas

    OpenAIRE

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S.; Furness, Andrew JS; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H.; McGranahan, Nicholas; Rowan, Andrew J.; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L.

    2013-01-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples...

  20. Single cell analysis reveals gametic and tissue-specific instability of the SCA1 CAG repeat

    Energy Technology Data Exchange (ETDEWEB)

    Chong, S.S.; McCall, A.E.; Cota, J. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Spinocerebellar ataxia type 1 is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat within the SCA1 gene on chromosome 6p22-23. We performed a comparative analysis of the SCA1 CAG repeat from blood and sperm of an affected male. Genomic amplification revealed a broader smear of the SCA1 allele product from sperm compared to that from peripheral blood leukocytes (PBL). To resolve this observed difference, we analyzed single sperm directly and demonstrate that the SCA1 allele in PBL is also heterogeneous, although the range of variability in allele sizes is much less than that observed in sperm. Limited genome analysis was also performed on PBL DNA from an unaffected individual with an upper normal allele of 36 repeats in parallel with an affected individual with an expanded allele of 40 repeats. The 36 repeat normal allele, which contains a CAT interruption, was completely stable compared to the uninterrupted repeat of the SCA1 allele, demonstrating a direct correlation between absence of a CAT interruption and somatic instability of the repeat. We also analyzed the size of the CAG repeat in tissues derived from various brain regions from a patient with juvenile-onset disease to determine if the size of the expansion correlated with the site of neuropathology. The results clearly show tissue-specific differences in mosaicism of repeat length. More importantly, the pattern of tissue-specific differences in repeat-length mosaicism in SCA1 within the brain parallels those seen in Huntington disease. In both disorders the expanded alleles are smaller in cerebellar tissue. These results suggest that the observed tissue-specific differences in instability of the SCA1 CAG repeat, either within the brain or between blood and sperm, are a function of the intracellular milieu or the intrinsic replicative potential of the various celltypes.

  1. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    Science.gov (United States)

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  2. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    Science.gov (United States)

    Barker, Ed; Abercromby, Kira J.; Abell, Paul

    2009-01-01

    A key objective of NASA s Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the 0.6 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectra is flat and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar panels with a strong band gap feature near 1 micron. The two spacecraft are spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but with fewer

  3. The effects of silver nanoparticles on intact wastewater biofilms

    Directory of Open Access Journals (Sweden)

    Zhiya eSheng

    2015-07-01

    Full Text Available Silver nanoparticles (Ag-NPs have strong antibacterial properties, which may adversely affect biological wastewater treatment processes. To determine the overall effect, intact biofilm samples were collected from the rotating biological contactor (RBC at the local wastewater treatment plant and treated with 200 mg Ag/L Ag-NPs for 24 h. The biofilm uptake of Ag-NPs was monitored with transmission electron microscopy (TEM. Forty-five min after Ag-NP application, Ag-NPs were seen in the biofilm extracellular polymeric substances (EPS. After 24 h, Ag-NPs had entered certain microbial cells, while other cells contained no observable Ag-NPs. Some cells were dying after the uptake of Ag-NPs. However, there was no significant reduction in cultivable bacteria in the biofilms, based on heterotrophic plate counts (HPC. While this may indicate that wastewater biofilms are highly resistant to Ag-NPs, the HPC represents only a small portion of the total microbial population. To further investigate the effects of Ag-NPs, a GeoChip microarray was used to directly detect changes in the functional gene structure of the microbial community in the biofilm. A clear decrease (34.6% decrease in gene number in gene diversity was evident in the GeoChip analysis. However, the complete loss of any specific gene was rare. Some gene families present in both treated and untreated biofilms. However, this doesn’t necessarily mean that there was no change in these families. Signal intensity decreased in certain variants in each family while other variants increased to compensate the effects of Ag-NPs. The results indicate that Ag-NP treatment decreased microbial community diversity but did not significantly affect the microbial community function. This provides direct evidence for the functional redundancy of microbial community in engineered ecosystems such as wastewater biofilms.

  4. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Xuewei Wang

    Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

  5. Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR)

    Science.gov (United States)

    Holtrup, Frank; Bauer, Andrea; Fellenberg, Kurt; Hilger, Ralf A; Wink, Michael; Hoheisel, Jörg D

    2011-01-01

    BACKGROUND AND PURPOSE Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s). PMID:21091652

  6. Open pipelines for integrated tumor genome profiles reveal differences between pancreatic cancer tumors and cell lines

    International Nuclear Information System (INIS)

    We describe open, reproducible pipelines that create an integrated genomic profile of a cancer and use the profile to find mutations associated with disease and potentially useful drugs. These pipelines analyze high-throughput cancer exome and transcriptome sequence data together with public databases to find relevant mutations and drugs. The three pipelines that we have developed are: (1) an exome analysis pipeline, which uses whole or targeted tumor exome sequence data to produce a list of putative variants (no matched normal data are needed); (2) a transcriptome analysis pipeline that processes whole tumor transcriptome sequence (RNA-seq) data to compute gene expression and find potential gene fusions; and (3) an integrated variant analysis pipeline that uses the tumor variants from the exome pipeline and tumor gene expression from the transcriptome pipeline to identify deleterious and druggable mutations in all genes and in highly expressed genes. These pipelines are integrated into the popular Web platform Galaxy at #http://usegalaxy.org/cancer# to make them accessible and reproducible, thereby providing an approach for doing standardized, distributed analyses in clinical studies. We have used our pipeline to identify similarities and differences between pancreatic adenocarcinoma cancer cell lines and primary tumors

  7. Evolutionary strategies of cells and viruses in deep-sea hydrothermal systems revealed through comparative metagenomics

    Science.gov (United States)

    Anderson, R.; Sogin, M. L.; Baross, J. A.

    2013-12-01

    The deep-sea hydrothermal vent habitat hosts a diverse community of archaea and bacteria that withstand extreme fluctuations in environmental conditions. Abundant viruses in these systems must also withstand these environmental extremes, and a high proportion of viruses in these systems are lysogenic. Comparative analysis of a cellular and viral metagenome from a diffuse flow hydrothermal vent has provided insights into the evolutionary strategies of both cells and viruses in hydrothermal systems. We detected numerous mobile elements in the viral and cellular gene pools as well as a large number of prophage in the cellular fraction. We show that the hydrothermal vent viral gene pool is relatively enriched in genes related to energy metabolism, a feature that is unique to the hydrothermal vent viral gene pool compared to viral gene pools from other environments, indicating a potential for integrated prophage to enhance host metabolic flexibility. We also detected stronger purifying selection in the viral versus cellular gene pool, indicating selection pressures that promote prolonged viral integration in the host. Our results support the hypothesis that viruses enhance host genomic plasticity and adaptability in this extreme and dynamic environment. Finally, we will discuss general implications of this work for understanding the viral impact on biogeochemical cycles and evolutionary trajectories of microbial populations in the deep subsurface biosphere.

  8. An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

    Science.gov (United States)

    Cirilo, Priscila Daniele Ramos; Marchi, Fábio Albuquerque; Barros Filho, Mateus de Camargo; Rocha, Rafael Malagoli; Domingues, Maria Aparecida Custódio; Jurisica, Igor; Pontes, Anagloria; Rogatto, Silvia Regina

    2013-01-01

    Background Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40–50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings The integrated analysis identified the top 30 significant genes (PTranscriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. PMID:23483937

  9. Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo-Electron Tomography

    Science.gov (United States)

    Liu, Jun; Howell, Jerrilyn K.; Bradley, Sherille D.; Zheng, Yesha; Zhou, Z. Hong; Norris, Steven J.

    2010-01-01

    High resolution cryo-electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3-D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member in the spirochetal family. High resolution cryo-ET reconstructions provided the detailed structures of the cell envelope, which is significantly different from that of gram-negative bacteria. The 4 nm lipid bilayer of both outer and cytoplasmic membranes resolved in 3-D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located, cone-shaped structure at both ends of bacterium. Furthermore, 3-D subvolume averages of the periplasmic flagellar motors and filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Together, our findings provide the most detailed structural understanding of the periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and escape host immune responses. PMID:20850455

  10. Phase Boundary Propagation in Li-Alloying Battery Electrodes Revealed by Liquid-Cell Transmission Electron Microscopy.

    Science.gov (United States)

    Leenheer, Andrew J; Jungjohann, Katherine L; Zavadil, Kevin R; Harris, Charles T

    2016-06-28

    Battery cycle life is directly influenced by the microstructural changes occurring in the electrodes during charge and discharge cycles. Here, we image in situ the nanoscale phase evolution in negative electrode materials for Li-ion batteries using a fully enclosed liquid cell in a transmission electron microscope (TEM) to reveal early degradation that is not evident in the charge-discharge curves. To compare the electrochemical phase transformation behavior between three model materials, thin films of amorphous Si, crystalline Al, and crystalline Au were lithiated and delithiated at controlled rates while immersed in a commercial liquid electrolyte. This method allowed for the direct observation of lithiation mechanisms in nanoscale negative electrodes, revealing that a simplistic model of a surface-to-interior lithiation front is insufficient. For the crystalline films, a lithiation front spread laterally from a few initial nucleation points, with continued grain nucleation along the growing interface. The intermediate lithiated phases were identified using electron diffraction, and high-resolution postmortem imaging revealed the details of the final microstructure. Our results show that electrochemically induced solid-solid phase transformations can lead to highly concentrated stresses at the laterally propagating phase boundary which should be considered for future designs of nanostructured electrodes for Li-ion batteries. PMID:27243921

  11. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

    International Nuclear Information System (INIS)

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-κB (NF-κB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-α and NF-κB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30 μg/ml CSC significantly activated PKC-α, while CSC doses above 20 μg/ml CSC significantly activated NF-κB. As NF-κB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30 μg/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30 μg/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-α, NF-κB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  12. IntAct: an open source molecular interaction database.

    Science.gov (United States)

    Hermjakob, Henning; Montecchi-Palazzi, Luisa; Lewington, Chris; Mudali, Sugath; Kerrien, Samuel; Orchard, Sandra; Vingron, Martin; Roechert, Bernd; Roepstorff, Peter; Valencia, Alfonso; Margalit, Hanah; Armstrong, John; Bairoch, Amos; Cesareni, Gianni; Sherman, David; Apweiler, Rolf

    2004-01-01

    IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains approximately 2200 binary and complex interactions imported from the literature and curated in collaboration with the Swiss-Prot team, making intensive use of controlled vocabularies to ensure data consistency. All IntAct software, data and controlled vocabularies are available at http://www.ebi.ac.uk/intact. PMID:14681455

  13. Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation.

    Directory of Open Access Journals (Sweden)

    Emma M Quinn

    Full Text Available Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; P(adjusted = 2.40x10(-11 in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (P(adjusted = 0.002, and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10(-16 and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; P(adjusted = 3.6x10(-3 as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10(-16 indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis.

  14. Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

    OpenAIRE

    Jung, Song-Yi; Willard, Scott T

    2014-01-01

    Background The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investi...

  15. A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

    Directory of Open Access Journals (Sweden)

    Amanda Scherer

    Full Text Available We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity, and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

  16. IntAct: an open source molecular interaction database

    OpenAIRE

    Hermjakob, Henning; Montecchi-Palazzi, Luisa; Lewington, Chris; Mudali, Sugath; Kerrien, Samuel; Orchard, Sandra; Vingron, Martin; Roechert, Bernd; Roepstorff, Peter; Valencia, Alfonso; Margalit, Hanah; Armstrong, John; Bairoch, Amos Marc; Cesareni, Gianni; Sherman, David

    2004-01-01

    IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains approximately 2200 binary and complex interactions ...

  17. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    Directory of Open Access Journals (Sweden)

    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  18. Quantitative proteomics analysis reveals glutamine deprivation activates fatty acid β-oxidation pathway in HepG2 cells.

    Science.gov (United States)

    Long, Baisheng; Muhamad, Rodiallah; Yan, Guokai; Yu, Jie; Fan, Qiwen; Wang, Zhichang; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-05-01

    Glutamine, a multifunctional amino acid, functions in nutrient metabolism, energy balance, apoptosis, and cell proliferation. Lipid is an important nutrient and controls a broad range of physiological processes. Previous studies have demonstrated that glutamine can affect lipolysis and lipogenesis, but the effect of glutamine on the detailed lipid metabolism remains incompletely understood. Here, we applied the quantitative proteomics approach to estimate the relative abundance of proteins in HepG2 cells treated by glutamine deprivation. The results showed that there were 212 differentially abundant proteins in response to glutamine deprivation, including 150 significantly increased proteins and 62 significantly decreased proteins. Interestingly, functional classification showed that 43 differentially abundant proteins were related to lipid metabolism. Further bioinformatics analysis and western blotting validation revealed that lipid accumulation may be affected by β-oxidation of fatty acid induced by glutamine deprivation in HepG2 cells. Together, our results may provide the potential for regulating lipid metabolism by glutamine in animal production and human nutrition. The MS data have been deposited to the ProteomeXchange Consortium with identifier PXD003387. PMID:26837383

  19. Modeling reveals bistability and low-pass filtering in the network module determining blood stem cell fate.

    Directory of Open Access Journals (Sweden)

    Jatin Narula

    2010-05-01

    Full Text Available Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad-a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models.

  20. In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering

    Directory of Open Access Journals (Sweden)

    T Brune

    2007-12-01

    Full Text Available The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL and cells in an intact ACL (iACL, particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA. Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

  1. Mutations in Traf3ip1 reveal defects in ciliogenesis, embryonic development, and altered cell size regulation.

    Science.gov (United States)

    Berbari, Nicolas F; Kin, Nicholas W; Sharma, Neeraj; Michaud, Edward J; Kesterson, Robert A; Yoder, Bradley K

    2011-12-01

    Tumor necrosis factor alpha receptor 3 interacting protein 1 (Traf3ip1), also known as MIPT3, was initially characterized through its interactions with tubulin, actin, TNFR-associated factor-3 (Traf3), IL-13R1, and DISC1. It functions as an inhibitor of IL-13-mediated phosphorylation of Stat6 and in sequestration of Traf3 and DISC1 to the cytoskeleton. Studies of the Traf3ip1 homologs in C. elegans (DYF-11), Zebrafish (elipsa), and Chlamydomonas (IFT54) revealed that the protein localizes to the cilium and is required for ciliogenesis. Similar localization data has now been reported for mammalian Traf3ip1. This raises the possibility that Traf3ip1 has an evolutionarily conserved role in mammalian ciliogenesis in addition to its previously indicated functions. To evaluate this possibility, a Traf3ip1 mutant mouse line was generated. Traf3ip1 mutant cells are unable to form cilia. Homozygous Traf3ip1 mutant mice are not viable and have both neural developmental defects and polydactyly, phenotypes typical of mouse mutants with ciliary assembly defects. Furthermore, in Traf3ip1 mutants the hedgehog pathway is disrupted, as evidenced by abnormal dorsal-ventral neural tube patterning and diminished expression of a hedgehog reporter. Analysis of the canonical Wnt pathway indicates that it was largely unaffected; however, specific domains in the pharyngeal arches have elevated levels of reporter activity. Interestingly, Traf3ip1 mutant embryos and cells failed to show alterations in IL-13 signaling, one of the pathways associated with its initial discovery. Novel phenotypes observed in Traf3ip1 mutant cells include elevated cytosolic levels of acetylated microtubules and a marked increase in cell size in culture. The enlarged Traf3ip1 mutant cell size was associated with elevated basal mTor pathway activity. Taken together, these data demonstrate that Traf3ip1 function is highly conserved in ciliogenesis and is important for proper regulation of a number of essential

  2. What is the discrepancy between drug permeation into/across intact and diseased skins? Atopic dermatitis as a model.

    Science.gov (United States)

    Fang, Yi-Ping; Yang, Sien-Hung; Lee, Chih-Hung; Aljuffali, Ibrahim A; Kao, Hsiao-Ching; Fang, Jia-You

    2016-01-30

    The discrepancy in drug absorption between healthy and diseased skins is an issue that needs to be elucidated. The present study attempted to explore the percutaneous absorption of drugs via lesional skin by using atopic dermatitis (AD) as a model. Tape-stripping and ovalbumin (OVA) sensitization induced AD-like skin. The lesions were evaluated by physiological parameters, histology, cytokines, and differentiation proteins. The permeants of tacrolimus, 8-methoxypsoralen, methotrexate, and dextran were used to examine in vitro and in vivo cutaneous permeation. Transepidermal water loss (TEWL) increased from 5.2 to 27.4 g/m(2)/h by OVA treatment. AD-like lesions were characterized by hyperplasia, skin redness, desquamation, and infiltration of inflammatory cells. Repeated OVA challenge produced a T-helper 2 (Th2) hypersensitivity accompanied by downregulation of filaggrin, involucrin, and integrin β. Tacrolimus, the most lipophilic permeant, revealed an increase of cutaneous deposition by 2.7-fold in AD-like skin compared to intact skin. The transdermal flux of methotrexate and dextran, the hydrophilic permeants, across AD-like skin increased about 18 times compared to the control skin. Surprisingly, AD-like skin showed less skin deposition of 8-methoxypsoralen than intact skin. This may be because the deficient lipids in the atopic-affected stratum corneum (SC) diminished drug partitioning into the superficial skin layer. The fluorescence and confocal microscopic images demonstrated a broad and deep passage of small-molecular and macromolecular dyes into AD-like skin. The results obtained from this report were advantageous for showing how the lesional skin influenced percutaneous absorption. PMID:26657274

  3. Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

    International Nuclear Information System (INIS)

    The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between -10 and 5 degree C (low-temperature transition), 10 and 22 degree C (middle-temperature transition), and 32 and 40 degree C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degree C). Second, the middle-temperature transition shifts up to the range of about 20-32 degree C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degree C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties

  4. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells.

    Directory of Open Access Journals (Sweden)

    Pitipreya Suauam

    Full Text Available Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway but not Δcnb1 (lacking the calcineurin pathway strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.

  5. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas.

    Science.gov (United States)

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S; Furness, Andrew J S; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H; McGranahan, Nicholas; Rowan, Andrew J; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L; Larkin, James; Swanton, Charles

    2013-12-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367-16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, -0.218 to 0.465). 3-93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. PMID:24122851

  6. An Approach to Breast Cancer Immunotherapy: The Apoptotic Activity of Recombinant Anti-Interleukin-6 Monoclonal Antibodies in Intact Tumour Microenvironment of Breast Carcinoma.

    Science.gov (United States)

    Abou-Shousha, S; Moaaz, M; Sheta, M; Motawea, M A

    2016-06-01

    Current work is one of our comprehensive preclinical studies, a new approach to breast cancer (BC) immunotherapy through induction of tumour cell apoptosis. Tumour growth is not just a result of uncontrolled cell proliferation but also of reduced apoptosis. High levels of interleukin-6 (IL-6) are associated with metastatic BC and correlated with poor survival as it promotes growth of tumour-initiating cells during early tumorigenesis protecting these cells from apoptosis. Therefore, this study aims at investigating the potential of anti-IL-6 monoclonal antibodies to suppress IL-6 proliferative/anti-apoptotic activities in intact tumour microenvironment of BC. Fresh sterile tumour and normal breast tissue specimens were taken from 50 female Egyptian patients with BC undergoing radical mastectomy. A unique tissue culture system designed to provide cells of each intact tumour/normal tissue sample with its proper microenvironment either supplemented or not with anti-IL-6 monoclonal antibodies. To evaluate the apoptotic activity of anti-IL-6 as a novel candidate for BC treatment strategy, we compared its effects with those obtained using tumour necrosis-related apoptosis-inducing ligand TRAIL as an established apoptotic agent. Our results revealed that levels of either anti-IL-6- or TRAIL-induced apoptosis in the tumour or normal tissue cultures were significantly higher than those in their corresponding untreated ones (P Recombinant anti-IL-6 monoclonal antibodies could represent a novel effective element of immunotherapeutic treatment strategy for BC. The selectivity and anti-apoptotic potential of anti-IL-6 is highly hopeful in IL-6- abundant BC tumour microenvironment. PMID:26971879

  7. Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

    International Nuclear Information System (INIS)

    Prostate cancer cells in primary tumors have been typed CD10-/CD13-/CD24hi/CD26+/CD38lo/CD44-/CD104-. This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure. CD26+ cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells. The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines. Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types

  8. Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

    Directory of Open Access Journals (Sweden)

    Anne Rupprecht

    Full Text Available Uncoupling protein 2 (UCP2 is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.

  9. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    Science.gov (United States)

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering. PMID:27002234

  10. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin. PMID:27135362

  11. Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

    Directory of Open Access Journals (Sweden)

    Silvia Rodríguez-Lombardero

    2014-07-01

    Full Text Available Sky1 is the only member of the SR (Serine–Arginine protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

  12. Genomic Analysis Reveals Disruption of Striatal Neuronal Development and Therapeutic Targets in Human Huntington’s Disease Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Karen L. Ring

    2015-12-01

    Full Text Available We utilized induced pluripotent stem cells (iPSCs derived from Huntington’s disease (HD patients as a human model of HD and determined that the disease phenotypes only manifest in the differentiated neural stem cell (NSC stage, not in iPSCs. To understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls. Differential gene expression and pathway analysis pointed to transforming growth factor β (TGF-β and netrin-1 as the top dysregulated pathways. Using data-driven gene coexpression network analysis, we identified seven distinct coexpression modules and focused on two that were correlated with changes in gene expression due to the CAG expansion. Our HD NSC model revealed the dysregulation of genes involved in neuronal development and the formation of the dorsal striatum. The striatal and neuronal networks disrupted could be modulated to correct HD phenotypes and provide therapeutic targets.

  13. Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons

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    Shane Grealish

    2015-06-01

    Full Text Available Human embryonic stem cell (hESC-derived dopamine neurons are currently moving toward clinical use for Parkinson’s disease (PD. However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models.

  14. Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes.

    Science.gov (United States)

    Zhu, Zengrong; Li, Qing V; Lee, Kihyun; Rosen, Bess P; González, Federico; Soh, Chew-Li; Huangfu, Danwei

    2016-06-01

    Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR/Cas-mediated gene editing and hPSC-directed differentiation for a systematic analysis of the roles of eight pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1, and ARX). Our analysis not only verified conserved gene requirements between mice and humans but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic β cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for understanding mechanisms underlying congenital disorders. PMID:27133796

  15. Analysis of endocytic pathways in Drosophila cells reveals a conserved role for GBF1 in internalization via GEECs.

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    Gagan D Gupta

    Full Text Available In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs forms GEECs (GPI-AP enriched early endosomal compartments via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes. Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion.

  16. Lack of association between intact/deletion polymorphisms of the APOBEC3B gene and HIV-1 risk.

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    Mayumi Imahashi

    Full Text Available OBJECTIVE: The human APOBEC3 family of proteins potently restricts HIV-1 replication APOBEC3B, one of the family genes, is frequently deleted in human populations. Two previous studies reached inconsistent conclusions regarding the effects of APOBEC3B loss on HIV-1 acquisition and pathogenesis. Therefore, it was necessary to verify the effects of APOBEC3B on HIV-1 infection in vivo. METHODS: Intact (I and deletion (D polymorphisms of APOBEC3B were analyzed using PCR. The syphilis, HBV and HCV infection rates, as well as CD4(+ T cell counts and viral loads were compared among three APOBEC3B genotype groups (I/I, D/I, and D/D. HIV-1 replication kinetics was assayed in vitro using primary cells derived from PBMCs. RESULTS: A total of 248 HIV-1-infected Japanese men who have sex with men (MSM patients and 207 uninfected Japanese MSM were enrolled in this study. The genotype analysis revealed no significant differences between the APOBEC3B genotype ratios of the infected and the uninfected cohorts (p = 0.66. In addition, HIV-1 disease progression parameters were not associated with the APOBEC3B genotype. Furthermore, the PBMCs from D/D and I/I subjects exhibited comparable HIV-1 susceptibility. CONCLUSION: Our analysis of a population-based matched cohort suggests that the antiviral mechanism of APOBEC3B plays only a negligible role in eliminating HIV-1 in vivo.

  17. A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

    International Nuclear Information System (INIS)

    A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by