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Sample records for yeast mammalian cells

  1. Rapid and serial quantification of adhesion forces of yeast and Mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eva Potthoff

    Full Text Available Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM. In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

  2. Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

    International Nuclear Information System (INIS)

    Singh, Bhag; Gupta, Radhey S.

    2006-01-01

    Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase

  3. Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

    Science.gov (United States)

    Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

    2016-01-01

    "pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.

  4. Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells

    OpenAIRE

    Zhang, Yunfei; Robertson, J. Brian; Xie, Qiguang; Johnson, Carl Hirschie

    2016-01-01

    “pHlash” is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characteri...

  5. A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

    Directory of Open Access Journals (Sweden)

    Zhang Tingting

    2012-12-01

    Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

  6. Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

    Science.gov (United States)

    Todorova, Atanaska; Pesheva, Margarita; Iliev, Ivan; Bardarov, Krum; Todorova, Teodora

    2017-04-01

    Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 μg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 μg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

  7. Mammalian amyloidogenic proteins promote prion nucleation in yeast.

    Science.gov (United States)

    Chandramowlishwaran, Pavithra; Sun, Meng; Casey, Kristin L; Romanyuk, Andrey V; Grizel, Anastasiya V; Sopova, Julia V; Rubel, Aleksandr A; Nussbaum-Krammer, Carmen; Vorberg, Ina M; Chernoff, Yury O

    2018-03-02

    Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Endoplasmic reticulum involvement in yeast cell death

    International Nuclear Information System (INIS)

    Nicanor Austriaco, O.

    2012-01-01

    Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

  9. Mammalian cell biology

    International Nuclear Information System (INIS)

    Elkind, M.M.

    1979-01-01

    This section contains summaries of research on mechanisms of lethality and radioinduced changes in mammalian cell properties, new cell systems for the study of the biology of mutation and neoplastic transformation, and comparative properties of ionizing radiations

  10. Mammalian cell biology

    International Nuclear Information System (INIS)

    Elkind, M.M.

    1975-01-01

    Progress is reported on the following research projects: the effects of N-ethyl-maleimide and hydroxyurea on hamster cells in culture; sensitization of synchronized human cells to x rays by N-ethylmaleimide; sensitization of hypoxic mammalian cells with a sulfhydryl inhibitor; damage interaction due to ionizing and nonionizing radiation in mammalian cells; DNA damage relative to radioinduced cell killing; spurious photolability of DNA labeled with methyl- 14 C-thymidine; radioinduced malignant transformation of cultured mouse cells; a comparison of properties of uv and near uv light relative to cell function and DNA damage; Monte Carlo simulation of DNA damage and repair mechanisms; and radiobiology of fast neutrons

  11. Propagation of Mammalian Prions in Yeast

    National Research Council Canada - National Science Library

    Harris, David A

    2006-01-01

    ...: the budding yeast Saccharomyces cerevisiae. This unicellular organism offers a number of potential advantages for the study of prion biology, including rapid generation time, ease of culturing, and facile genetics...

  12. Homogenization of Mammalian Cells.

    Science.gov (United States)

    de Araújo, Mariana E G; Lamberti, Giorgia; Huber, Lukas A

    2015-11-02

    Homogenization is the name given to the methodological steps necessary for releasing organelles and other cellular constituents as a free suspension of intact individual components. Most homogenization procedures used for mammalian cells (e.g., cavitation pump and Dounce homogenizer) rely on mechanical force to break the plasma membrane and may be supplemented with osmotic or temperature alterations to facilitate membrane disruption. In this protocol, we describe a syringe-based homogenization method that does not require specialized equipment, is easy to handle, and gives reproducible results. The method may be adapted for cells that require hypotonic shock before homogenization. We routinely use it as part of our workflow to isolate endocytic organelles from mammalian cells. © 2015 Cold Spring Harbor Laboratory Press.

  13. Mammalian cell biology

    International Nuclear Information System (INIS)

    Elkind, M.M.

    1975-01-01

    Studies of the action of N-ethylmaleimide (NEM), as an inhibitor of repair of x radioinduced injuries were extended from synchronous Chinese hamster cells to synchronous human HeLa cells. These studies showed a similar mode of action in both cell types lending support to the notion that conclusions may be extracted from such observations that are of fairly general applicability to mammalian cells. Radiation studies with NEM are being extended to hypoxic cells to inquire if NEM is effective relative to oxygen-independent damage. Observations relative to survival, DNA synthesis, and DNA strand elongation resulting from the addition products to DNA when cells were exposed to near uv in the presence of psoralen were extended. (U.S.)

  14. Structural differences between yeast and mammalian microtubules revealed by cryo-EM

    Energy Technology Data Exchange (ETDEWEB)

    Howes, Stuart C. [Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; Geyer, Elisabeth A. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; LaFrance, Benjamin [Univ. of California, Berkeley, CA (United States). Molecular and Cell Biology Graduate Program; Zhang, Rui [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Kellogg, Elizabeth H. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Westermann, Stefan [Univ. of Duisburg-Essen, Essen (Germany). Dept. of Molecular Genetics, Center for Medical Biotechnology; Rice, Luke M. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; Nogales, Eva [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Molecular Biology and California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

    2017-06-26

    Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

  15. Mitochondrial fission proteins regulate programmed cell death in yeast

    OpenAIRE

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J.; Qi, Bing; Pevsner, Jonathan; McCaffery, J. Michael; Hill, R. Blake; Basañez, Gorka; Hardwick, J. Marie

    2004-01-01

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we fo...

  16. Mammalian cell biology

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Progress is reported on studies of the molecular biology and functional changes in cultured mammalian cells following exposure to x radiation, uv radiation, fission neutrons, or various chemical environmental pollutants alone or in combinations. Emphasis was placed on the separate and combined effects of polycyclic aromatic hydrocarbons released during combustion of fossil fuels and ionizing and nonionizing radiations. Sun lamps, which emit a continuous spectrum of near ultraviolet light of 290 nm to 315 nm were used for studies of predictive cell killing due to sunlight. Results showed that exposure to uv light (254 nm) may not be adequate to predict effects produced by sunlight. Data are included from studies on single-strand breaks and repair in DNA of cultured hamster cells exposed to uv or nearultraviolet light. The possible interactions of the polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)-anthracene (DmBA) alone or combined with exposure to x radiation, uv radiation (254 nm) or near ultraviolet simulating sunlight were compared for effects on cell survival

  17. Mitochondrial fission proteins regulate programmed cell death in yeast.

    Science.gov (United States)

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  18. Mutagenesis in mammalian cells

    International Nuclear Information System (INIS)

    Burki, H.J.

    1981-01-01

    Mutagenic processes in synchronous cultures of Chinese hamster ovary cells have been studied. There is a difference in the induction of mutants by ultraviolet light during the cell cycle. There appears to be a sensitive period in the middle of the G1 stage of the cell cycle suggesting some mutagenic mechanism is present at that time. Studies indicate that mutation induction during the cell cycle is also mutagen specific since exposure to ethyl nitrosourea in the same system produces different results. Two clones have been isolated which are ultrasensitive to ultraviolet light. These cells are being used to determine if this hypermutability is cell-cycle dependent, related to cell cycle biochemistry, or to repair processes independent of cell cycle. Tritium and bromodeoxyuridine induced damage to synchronously dividing cell cultures are also being studied in relation to DNA replication. Cell killing by ionizing radiation is also related to the cell cycle. Sensitive times in the cell cycle for mutation induction by ionization radiation are identified

  19. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  20. Technology of mammalian cell encapsulation

    NARCIS (Netherlands)

    Uludag, H; De Vos, P; Tresco, PA

    2000-01-01

    Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates

  1. Lipid raft involvement in yeast cell growth and death

    Energy Technology Data Exchange (ETDEWEB)

    Mollinedo, Faustino, E-mail: fmollin@usal.es [Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas - Universidad de Salamanca, Salamanca (Spain)

    2012-10-10

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na{sup +}, K{sup +}, and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  2. Lipid raft involvement in yeast cell growth and death

    International Nuclear Information System (INIS)

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na + , K + , and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  3. Producing Newborn Synchronous Mammalian Cells

    Science.gov (United States)

    Gonda, Steve R.; Helmstetter, Charles E.; Thornton, Maureen

    2008-01-01

    A method and bioreactor for the continuous production of synchronous (same age) population of mammalian cells have been invented. The invention involves the attachment and growth of cells on an adhesive-coated porous membrane immersed in a perfused liquid culture medium in a microgravity analog bioreactor. When cells attach to the surface divide, newborn cells are released into the flowing culture medium. The released cells, consisting of a uniform population of synchronous cells are then collected from the effluent culture medium. This invention could be of interest to researchers investigating the effects of the geneotoxic effects of the space environment (microgravity, radiation, chemicals, gases) and to pharmaceutical and biotechnology companies involved in research on aging and cancer, and in new drug development and testing.

  4. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  5. The role of mitochondria in yeast programmed cell death

    International Nuclear Information System (INIS)

    Guaragnella, Nicoletta; Ždralević, Maša; Antonacci, Lucia; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2012-01-01

    Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this mini-review we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

  6. Heavy ion induced genetic effects in mammalian cells. Final report

    International Nuclear Information System (INIS)

    Kiefer, J.; Brend'amour, M.; Casares, A.; Egenolf, R.; Gutermuth, F.; Ikpeme, S.E.; Koch, S.; Kost, M.; Loebrich, M.; Pross, H.D.; Russmann, C.; Schmidt, P.; Schneider, E.; Stoll, U.; Weber, K.J.

    2001-01-01

    DNA double-strand breaks (DSBs) are generally assumed to be the most relevant initial event producing radiation-induced cellular lethality, as well as mutations and transformations. The dependence of their formation on radiation quality has been recently reviewed. Contrary to earlier observations there seems to be now agreement that the RBE does not increase above unity with increasing LET in mammalian cells when conventional techniques are applied which are not able to resolve smaller fragments. If they are, however, included in the analysis maximum RBE values around 2 are obtained. The situation is different with yeast: An increased effectiveness for DSB induction has been reported with alpha particles, as well as for heavy ions. This may be due to differences in methods or to chromosomal structure, as discussed in more detail in this paper. DSB induction was measured for a LET range of 100 to 11500 keV/? m in yeast cells using pulsed field gel electrophoresis. Under the conditions applied the chromosomes of the yeast cells could be separated according to size allowing the direct quantification of the DSB yield by measuring the intensity of the largest chromosomes. The results demonstrate clearly that DSB induction in yeast depends on radiation quality. The derived cross-sections for DSB induction were also compared to those for cell inactivation determined in parallel experiments under identical irradiation conditions. (orig.)

  7. Acoustophoretic Synchronization of Mammalian Cells in Microchannels

    DEFF Research Database (Denmark)

    Thévoz, P.; Adams, J.D.; Shea, H.

    2010-01-01

    We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel to selec......We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel...

  8. The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

    DEFF Research Database (Denmark)

    Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro

    2011-01-01

    with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES...

  9. Reactivation of neutron killed mammalian cells by gamma irradiation: The observations, possible mechanism and implication

    International Nuclear Information System (INIS)

    Calkins, J.; Harrison, W.; Einspenner, M.

    1990-01-01

    We have observed that combinations of neutron plus gamma ray exposure can significantly increase the colony forming ability of monkey and human cell cultures over the neutron dose alone. The 'reactivation' of neutron killed mammalian cells by gamma rays is analogous to observations made in lower eukaryotic organisms and fits the pattern termed 'T repair' previously postulated for yeast and protozoans. (orig.)

  10. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  11. Rheological behavior of mammalian cells.

    Science.gov (United States)

    Stamenović, D

    2008-11-01

    Rheological properties of living cells determine how cells interact with their mechanical microenvironment and influence their physiological functions. Numerous experimental studies have show that mechanical contractile stress borne by the cytoskeleton and weak power-law viscoelasticity are governing principles of cell rheology, and that the controlling physics is at the level of integrative cytoskeletal lattice properties. Based on these observations, two concepts have emerged as leading models of cytoskeletal mechanics. One is the tensegrity model, which explains the role of the contractile stress in cytoskeletal mechanics, and the other is the soft glass rheology model, which explains the weak power-law viscoelasticity of cells. While these two models are conceptually disparate, the phenomena that they describe are often closely associated in living cells for reasons that are largely unknown. In this review, we discuss current understanding of cell rheology by emphasizing the underlying biophysical mechanism and critically evaluating the existing rheological models.

  12. Nutrient acquisition strategies of mammalian cells.

    Science.gov (United States)

    Palm, Wilhelm; Thompson, Craig B

    2017-06-07

    Mammalian cells are surrounded by diverse nutrients, such as glucose, amino acids, various macromolecules and micronutrients, which they can import through transmembrane transporters and endolysosomal pathways. By using different nutrient sources, cells gain metabolic flexibility to survive periods of starvation. Quiescent cells take up sufficient nutrients to sustain homeostasis. However, proliferating cells depend on growth-factor-induced increases in nutrient uptake to support biomass formation. Here, we review cellular nutrient acquisition strategies and their regulation by growth factors and cell-intrinsic nutrient sensors. We also discuss how oncogenes and tumour suppressors promote nutrient uptake and thereby support the survival and growth of cancer cells.

  13. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  14. Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

    Science.gov (United States)

    Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

    2014-06-03

    Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

  15. Radiation effects in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Hill, C.K.; Han, A.; Elkind, M.M.; Wells, R.L.; Buess, E.M.; Lin, C.M.

    1985-01-01

    The purpose of this research effort is to elucidate the mechanisms for the radiation-induced changes in mammalian cells that lead to cell death, mutation, neoplastic transformation, DNA damage, and chromosomal alterations. Of particular interest are the effects of low-dose-rate and fractionated irradiation on these end points with respect to the mechanisms whereby these effects are influenced by cellular repair processes, inhibitors, and promoters that act at the genetic or biochemical level. 17 refs

  16. Regulation of Autophagy by Glucose in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Erwin Knecht

    2012-07-01

    Full Text Available Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

  17. Radiation sensitivity of mammalian cells

    International Nuclear Information System (INIS)

    Koch, C.J.

    1985-01-01

    The authors tested various aspects of the so-called ''competition'' model for radiation sensitization/protection. In this model, sensitizers and/or protectors react in first order chemical reactions with radiation-induced target radicals in the cell, producing damage fixation or repair respectively. It is only because of these parallel, first-order competing reactions that they may assign net amounts of damage on the basis of the chemical reactivity of the sentiziers/protectors with the radicals. It might be expected that such a simple model could not explain all aspects of cellular radiosensitivity and this has indeed been found to be true. However, one is able, with the simple model, to pose quite specific questions, and obtain quantitative information with respect to the relative agreement between experiment and theory. Many experiments by several investigators have found areas of disagreement with the competition theory, particularly with respect to the follow items: 1) role of cellular glutathione as the most important endogeneous radiation protector 2) characteristics of various sensitizers which cause them to behave differently from each other 3) methods relating to the quantitative kinetic analysis of experimenal results. This paper addresses these specific areas of disagreement from both an experimental and theoretical basis

  18. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel

    2006-01-01

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  19. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  20. A universal mammalian vaccine cell line substrate.

    Directory of Open Access Journals (Sweden)

    Jackelyn Murray

    Full Text Available Using genome-wide small interfering RNA (siRNA screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205 showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

  1. X-rays sensitive mammalian cell mutant

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi

    1982-01-01

    A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

  2. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    Energy Technology Data Exchange (ETDEWEB)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D' Urso, Gennaro; Huberman, Joel A

    2003-11-27

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

  3. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    International Nuclear Information System (INIS)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D'Urso, Gennaro; Huberman, Joel A.

    2003-01-01

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication

  4. Immobilization of yeast cells by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Fujimura, T.; Kaetsu, I.

    1982-01-01

    Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system. (orig.)

  5. Fungal cell gigantism during mammalian infection.

    Directory of Open Access Journals (Sweden)

    Oscar Zaragoza

    2010-06-01

    Full Text Available The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  6. Fungal cell gigantism during mammalian infection.

    Science.gov (United States)

    Zaragoza, Oscar; García-Rodas, Rocío; Nosanchuk, Joshua D; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

    2010-06-17

    The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  7. Mammalian synthetic biology for studying the cell.

    Science.gov (United States)

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  8. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

    Science.gov (United States)

    Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

    2014-10-16

    Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Focusing on RISC assembly in mammalian cells.

    Science.gov (United States)

    Hong, Junmei; Wei, Na; Chalk, Alistair; Wang, Jue; Song, Yutong; Yi, Fan; Qiao, Ren-Ping; Sonnhammer, Erik L L; Wahlestedt, Claes; Liang, Zicai; Du, Quan

    2008-04-11

    RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.

  10. Focusing on RISC assembly in mammalian cells

    International Nuclear Information System (INIS)

    Hong Junmei; Wei Na; Chalk, Alistair; Wang Jue; Song, Yutong; Yi Fan; Qiao Renping; Sonnhammer, Erik L.L.; Wahlestedt, Claes; Liang Zicai; Du, Quan

    2008-01-01

    RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi

  11. Engineered Trehalose Permeable to Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Alireza Abazari

    Full Text Available Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre and trehalose tetraacetate (4-O-Ac-Tre. Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

  12. Redox signaling during hypoxia in mammalian cells

    Directory of Open Access Journals (Sweden)

    Kimberly A. Smith

    2017-10-01

    Full Text Available Hypoxia triggers a wide range of protective responses in mammalian cells, which are mediated through transcriptional and post-translational mechanisms. Redox signaling in cells by reactive oxygen species (ROS such as hydrogen peroxide (H2O2 occurs through the reversible oxidation of cysteine thiol groups, resulting in structural modifications that can change protein function profoundly. Mitochondria are an important source of ROS generation, and studies reveal that superoxide generation by the electron transport chain increases during hypoxia. Other sources of ROS, such as the NAD(PH oxidases, may also generate oxidant signals in hypoxia. This review considers the growing body of work indicating that increased ROS signals during hypoxia are responsible for regulating the activation of protective mechanisms in diverse cell types.

  13. Synthetic RNA Controllers for Programming Mammalian Cell Fate and Function

    Science.gov (United States)

    2015-11-04

    Final report for “Synthetic RNA controllers for programming mammalian cell fate and function” Principal Investigator: Christina D. Smolke...SUBTITLE Synthetic RNA controllers for programming mammalian cell fate and function 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18   2 Synthetic RNA controllers for programming mammalian cell fate and function Task 1

  14. Host cell reactivation in mammalian cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Benane, S.G.; Stafford, J.E.

    1976-01-01

    The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promoted photereactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7 to 0.8 for ovary cells and 0.5 to 0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more effecient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survival curves of herpes virus in Potoroo cells indicated a high level of 'dark' host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (lambda>600 nm) and human cells with normal repair and with cells deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreactivating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps. (author)

  15. DNA synthesis in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Painter, R.B.; California Univ., San Francisco; Young, B.R.

    1987-01-01

    One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v. -induced inhibition of replicon initiation. (author

  16. A hybrid mammalian cell cycle model

    Directory of Open Access Journals (Sweden)

    Vincent Noël

    2013-08-01

    Full Text Available Hybrid modeling provides an effective solution to cope with multiple time scales dynamics in systems biology. Among the applications of this method, one of the most important is the cell cycle regulation. The machinery of the cell cycle, leading to cell division and proliferation, combines slow growth, spatio-temporal re-organisation of the cell, and rapid changes of regulatory proteins concentrations induced by post-translational modifications. The advancement through the cell cycle comprises a well defined sequence of stages, separated by checkpoint transitions. The combination of continuous and discrete changes justifies hybrid modelling approaches to cell cycle dynamics. We present a piecewise-smooth version of a mammalian cell cycle model, obtained by hybridization from a smooth biochemical model. The approximate hybridization scheme, leading to simplified reaction rates and binary event location functions, is based on learning from a training set of trajectories of the smooth model. We discuss several learning strategies for the parameters of the hybrid model.

  17. Physiological significance of polyploidization in mammalian cells.

    Science.gov (United States)

    Pandit, Shusil K; Westendorp, Bart; de Bruin, Alain

    2013-11-01

    Programmed polyploidization occurs in all mammalian species during development and aging in selected tissues, but the biological properties of polyploid cells remain obscure. Spontaneous polyploidization arises during stress and has been observed in a variety of pathological conditions, such as cancer and degenerative diseases. A major challenge in the field is to test the predicted functions of polyploidization in vivo. However, recent genetic mouse models with diminished polyploidization phenotypes represent novel, powerful tools to unravel the biological function of polyploidization. Contrary to a longstanding hypothesis, polyploidization appears to not be required for differentiation and has no obvious impact on proliferation. Instead, polyploidization leads to increased cell size and genetic diversity, which could promote better adaptation to chronic injury or stress. We discuss here the consequences of reducing polyploidization in mice and review which stress responses and molecular signals trigger polyploidization during development and disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Plasma treatment of mammalian vascular cells : A quantitative description

    NARCIS (Netherlands)

    Kieft, IE; Darios, D; Roks, AJM; Stoffels, E

    For the first time, quantitative data was obtained on plasma treatment of living mammalian cells. The nonthermal atmospheric discharge produced by the plasma needle was used for treatment of mammalian endothelial and smooth muscle cells. The influence of several experimental parameters on cell

  19. Plasma treatment of mammalian vascular cells: a quantitative description

    NARCIS (Netherlands)

    Kieft, I.E.; Darios, D.; Roks, A.J.M.; Stoffels - Adamowicz, E.

    2005-01-01

    For the first time, quantitative data was obtained on plasma treatment of living mammalian cells. The nonthermal atmospheric discharge produced by the plasma needle was used for treatment of mammalian endothelial and smooth muscle cells. The influence of several experimental parameters on cell

  20. Repair of radiation damage in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R.B.

    1981-01-01

    The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis.

  1. Repair of radiation damage in mammalian cells

    International Nuclear Information System (INIS)

    Setlow, R.B.

    1981-01-01

    The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis

  2. Repair of furocoumarin adducts in mammalian cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Smith, C.A.; Hanawalt, P.C.

    1984-01-01

    DNA repair was studied in cultured mammalian cells treated with the furocoumarins 8-methoxypsoralen (8-MOP), aminomethyl trioxsalen, or angelicin and irradiated with near UV light. The amount of DNA cross-linked by 8-MOP in normal human cells decreased by about one-half in 24 hours after treatment; no decrease was observed in xeroderma pigmentosum cells, group A. At present, it is not known to what extent this decrease represents complete repair events at the sites of cross-links. Furocoumarin adducts elicited excision repair in normal human and monkey cells but not in xeroderma pigmentosum group A cells. This excision repair resembled in several aspects that elicited by pyrimidine dimers, formed in DNA by irradiation with 254-nm UV light; however, it appeared that for at least 8-MOP and aminomethyl trioxsalen, removal of adducts was not as efficient as was the removal of pyrimidine dimers. A comparison was also made of repair in the 172-base-pair repetitive alpha-DNA component of monkey cells to repair in the bulk of the genome. Although repair elicited by pyrimidine dimers in alpha-DNA was the same as in the bulk DNA, that following treatment of cells with either aminomethyl trioxsalen or angelicin and near UV was markedly deficient in alpha-DNA. This deficiency reflected the removal of fewer adducts from alpha-DNA after the same initial adduct frequencies. These results could mean that each furocoumarin may produce several structurally distinct adducts to DNA in cells and that the capacity of cellular repair systems to remove these various adducts may vary greatly

  3. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  4. Protection of cultured mammalian cells by rebamipide

    International Nuclear Information System (INIS)

    Antoku, Shigetoshi; Aramaki, Ryoji; Tanaka, Hisashi; Kusumoto, Naotoshi.

    1997-01-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO 2 incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with 14 C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  5. Radiation- induced aneuploidy in mammalian germ cells

    International Nuclear Information System (INIS)

    Tease, C.

    1989-01-01

    The ability of ionizing radiation to induce aneuploidy in mammalian germ cells has been investigated experimentally in the laboratory mouse using a variety of cytogenetic and genetic methods. These studies have provided unambiguous evidence of induced nondisjunction in both male and female germ cells when the effect of irradiation is screened in meiotic cells or preimplantation embryos. In contrast, however, cytogenetic analyses of post-implantation embryos and genetic assays for induced chromosome gains have not found a significant radiation effect. These apparently contradictory findings may be reconciled if (a) radiation induces tertiary rather than primary trisomy, or (b) induces embryo-lethal genetic damage, such as deletions, in addition to numerical anomalies. Either or both of these explanations may account for the apparent loss during gestation of radiation-induced trisomic embryos. Extrapolating from the information so far available, it seems unlikely that environmental exposure to low doses if low dose rate radiation will result in a detectable increase in the rate of aneuploidy in the human population. (author)

  6. The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nishimura, Akira; Kawahara, Nobuhiro [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan); Takagi, Hiroshi, E-mail: hiro@bs.naist.jp [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer NO is produced from L-arginine in response to elevated temperature in yeast. Black-Right-Pointing-Pointer Tah18 was first identified as the yeast protein involved in NO synthesis. Black-Right-Pointing-Pointer Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.

  7. The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

    International Nuclear Information System (INIS)

    Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi

    2013-01-01

    Highlights: ► NO is produced from L-arginine in response to elevated temperature in yeast. ► Tah18 was first identified as the yeast protein involved in NO synthesis. ► Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe–S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.

  8. Does autophagy have a license to kill mammalian cells?

    NARCIS (Netherlands)

    Scarlatti, F.; Granata, R.; Meijer, A. J.; Codogno, P.

    2009-01-01

    Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the

  9. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  10. Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

    Science.gov (United States)

    Lebar, Tina; Jerala, Roman

    2018-01-01

    Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

  11. Guidelines and recommendations on yeast cell death nomenclature

    OpenAIRE

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andres; Austriaco, Nicanor; Sigrist, Stephan J.

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of mor...

  12. Introduction of the yeast DNA repair gene PHR1 into normal and xeroderma pigmentosum human cells

    International Nuclear Information System (INIS)

    Whyte, D.B.

    1988-01-01

    The goal of the work described herein is to determine how UV light kills and mutates human cells. Specifically, the hypothesis to be tested states that the major cause of cell death is the cyclobutane dimer. The yeast (S. cerevisiae) enzyme photolyase provides an elegant means of dissecting the biological effects of the two lesions. Photolyase, the product of the PHR1 gene, catalyzes the visible light-dependent reversal of cyclobutane pyrimidine dimers. Introducing the gene for photolyase into human cells, which do not have a functional photoreactivation mechanism, should allow specific repair of cyclobutane pyrimidine dimers. To express the yeast DNA repair gene in human cells, the yeast PHR1 coding sequence was cloned into the mammalian expression vector pRSV4NEO-I. The resulting plasmid, pRSVPHR1, contains the coding sequence of the yeast gene, under control of transcription signals recognized by mammalian cells, and the dominant selectable gene neo. pRSVPHR1 was introduced into normal and XP SV40-transformed fibroblasts by the calcium phosphate coprecipitation technique, and G418-resistant clones were isolated. The level of PHR1 expression was determined by cytoplasmic RNA dot blots. Two clones, XP-3B and GM-20A, had high levels of expression

  13. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  14. Engineered mammalian cells for production of recombinant proteins

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

  15. Yeast CUP1 protects HeLa cells against copper-induced stress

    Energy Technology Data Exchange (ETDEWEB)

    Xie, X.X. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China); College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou (China); Ma, Y.F.; Wang, Q.S.; Chen, Z.L.; Liao, R.R.; Pan, Y.C. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China)

    2015-06-12

    As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. YeastCUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study,CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

  16. Amino acids in the cultivation of mammalian cells.

    Science.gov (United States)

    Salazar, Andrew; Keusgen, Michael; von Hagen, Jörg

    2016-05-01

    Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

  17. Guidelines and recommendations on yeast cell death nomenclature

    NARCIS (Netherlands)

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J; Breitenbach, Michael; Burhans, William C; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W; Grant, Chris M; Greenwood, Michael T; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D; Outeiro, Tiago F; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F; Sharon, Amir; Sigrist, Stephan J; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B; Tuite, Mick; Vögtle, F-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J; Zhao, Richard Y; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely

  18. Mammalian cell culture capacity for biopharmaceutical manufacturing.

    Science.gov (United States)

    Ecker, Dawn M; Ransohoff, Thomas C

    2014-01-01

    : With worldwide sales of biopharmaceuticals increasing each year and continuing growth on the horizon, the manufacture of mammalian biopharmaceuticals has become a major global enterprise. We describe the current and future industry wide supply of manufacturing capacity with regard to capacity type, distribution, and geographic location. Bioreactor capacity and the use of single-use products for biomanufacturing are also profiled. An analysis of the use of this capacity is performed, including a discussion of current trends that will influence capacity growth, availability, and utilization in the coming years.

  19. Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

    Science.gov (United States)

    Lakkaraju, Asvin K. K.; Thankappan, Ratheeshkumar; Mary, Camille; Garrison, Jennifer L.; Taunton, Jack; Strub, Katharina

    2012-01-01

    Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120–160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway. PMID:22648169

  20. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-08-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after ..gamma..-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation.

  1. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    International Nuclear Information System (INIS)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-01-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after γ-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation. (author)

  2. Dielectric modelling of cell division for budding and fission yeast

    International Nuclear Information System (INIS)

    Asami, Koji; Sekine, Katsuhisa

    2007-01-01

    The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast

  3. Optical sorting and photo-transfection of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-02-01

    Full Text Available and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human...

  4. Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

    Science.gov (United States)

    Palková, Zdena; Váchová, Libuše

    2016-09-01

    Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Regeneration of hair cells in the mammalian vestibular system.

    Science.gov (United States)

    Li, Wenyan; You, Dan; Chen, Yan; Chai, Renjie; Li, Huawei

    2016-06-01

    Hair cells regenerate throughout the lifetime of non-mammalian vertebrates, allowing these animals to recover from hearing and balance deficits. Such regeneration does not occur efficiently in humans and other mammals. Thus, balance deficits become permanent and is a common sensory disorder all over the world. Since Forge and Warchol discovered the limited spontaneous regeneration of vestibular hair cells after gentamicininduced damage in mature mammals, significant efforts have been exerted to trace the origin of the limited vestibular regeneration in mammals after hair cell loss. Moreover, recently many strategies have been developed to promote the hair cell regeneration and subsequent functional recovery of the vestibular system, including manipulating the Wnt, Notch and Atoh1. This article provides an overview of the recent advances in hair cell regeneration in mammalian vestibular epithelia. Furthermore, this review highlights the current limitations of hair cell regeneration and provides the possible solutions to regenerate functional hair cells and to partially restore vestibular function.

  6. Cytolethal Distending Toxin Demonstrates Genotoxic Activity in a Yeast Model

    OpenAIRE

    Hassane, Duane C.; Lee, Robert B.; Mendenhall, Michael D.; Pickett, Carol L.

    2001-01-01

    Cytolethal distending toxins (CDTs) are multisubunit proteins produced by a variety of bacterial pathogens that cause enlargement, cell cycle arrest, and apoptosis in mammalian cells. While their function remains uncertain, recent studies suggest that they can act as intracellular DNases in mammalian cells. Here we establish a novel yeast model for understanding CDT-associated disease. Expression of the CdtB subunit in yeast causes a G2/M arrest, as seen in mammalian cells. CdtB toxicity is n...

  7. Measurement of Heme Synthesis Levels in Mammalian Cells.

    Science.gov (United States)

    Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

    2015-07-09

    Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

  8. Polyamine metabolism in synchronously growing mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Heby, O.; Marton, L.J.; Gray, J.W.; Lindl, P.A.; Wilson, C.B.

    1976-03-02

    The times of synthesis of the polyamines putrescine, spermidine and spermine in relation to the cell cycle have been examined in Chinese hamster ovary (CHO) cells synchronized by selective detachment of mitotic cells. This technique produced cell populations with narrow age distributions. Following plating, the cells grew with high synchrony for more than one cell cycle in monolayer culture. At various times after plating, the distribution of cells among the G1, S and G2M phases of the cell cycle was calculated from DNA histograms obtained by flow microfluorometric analysis. At these same times L-ornithine decarboxylase assays and polyamine determinations showed that the synthesis of the polyamines was initiated in mid-G1 and that the polyamines started to accumulate towards the end of the G1 phase. Maximal rate of synthesis was obtained as the cells started to synthesize DNA and the highest polyamine content was obtained in the beginning of the S phase. Synthesis and accumulation of the polyamines decreased significantly during mid-S but towards the end of the S phase they increased again. The polyamine biosynthetic activity and the concentration of the polyamines reached a second maximum prior to cell division. The role of the polyamines in the traverse of the cell cycle and especially in the initiation or continuation of DNA synthesis is indicated also by the fact that fewer cells were found in the S phase when spermidine and spermine synthesis was inhibited by methylglyoxal-bis(guanylhydrazone).

  9. Induced DNA repair pathway in mammalian cells

    International Nuclear Information System (INIS)

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

  10. Effect of salt hyperosmotic stress on yeast cell viability

    Directory of Open Access Journals (Sweden)

    Logothetis Stelios

    2007-01-01

    Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

  11. Intracellular dielectric tagging for improved optical manipulation of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-05-01

    Full Text Available review the application of optical forces for cellmanipulation and sorting, highlighting some of the key experiments over the last twenty years.We then introduce a new technique for enhancing the dielectric contrast of mammalian cells, which is a result...

  12. Reasons of reproductive death of mammalian cells

    International Nuclear Information System (INIS)

    Obaturov, G.M.

    1988-01-01

    According to its functional-structural organization the cell is rather a difficult object. It contains many various components, which essentially differ from the another according to their significance for its normal functioning, as well as sizes and number. When analyzing damage different types in cell sensitive target, that is - DNA, the author concludes, that it is most probable, that chromosomal aberrations are, mainly the reasons of cell reproduction death, rather than DNA unrepaired breaks

  13. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  14. Paraquat-induced radiosensitization of mammalian cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Fujikura, Toshio; Hiraoka, Toshio; Tenou, Hiromi.

    1983-06-01

    The herbicide, paraquat (methyl viologen, 1-1' dimethy1-4, 4'-bipyridinium dichloride), stimulates the production of superoxide anion (O 2 sup(-.)) in aerobic cells and therefore mimics some effects of ionizing radiation. In addition, concentrations of cellular glutathione are reduced by reaction with O 2 sup(-.). It is reported here that paraquat, toxic in its own right to aerobic cells, acts as a radiosensitizer when cells are exposed to nontoxic concentrations of the drug prior to and during irradiation. The radiomimetic effect of paraquat, alone and in combination with X-rays, was examined. Paraquat affects aerated cells (hamster lung V79 cells) in a dose-dependent manner. Doses in excess of 1 mM for two hours cause significant cell killing. In combination with radiation, sublethal doses of paraquat, given for two hours prior to irradiation, enhance the lethal effects of radiation. However, if cells are exposed to the same concentration of paraquat following irradiation, no additional lethal effect is observed. Paraquat is a useful tool to study the effects of O 2 sup(-.) and may lead to better understanding of the mechanisms of radiation-induced energy deposition in cells. (author)

  15. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  16. Negative pion irradiation of mammalian cells

    International Nuclear Information System (INIS)

    Dertinger, H.; Luecke-Huhle, C.; Schlag, H.; Weibezahn, K.F.

    1976-01-01

    Monolayers and spheroids of Chinese hamster cells (V79) were subjected to negative pion irradiation under aerobic conditions. R.b.e. values in the pion peak of 1.8 and 1.5 were obtained for monolayers and spheroids, respectively, whereas the r.b.e. for the plateau was found to be slightly higher than 1. In addition, it was observed that the higher resistance of the V79 spheroid cells than the monolayers to γ-irradiation is not diminished in the pion peak, suggesting that the underlying phenomenon of intercellular communication influences cell survival even after high-LET irradiation. (author)

  17. Radiosensitivity of yeast cells as a function of radiation LET

    International Nuclear Information System (INIS)

    Lobachevskij, P.N.; Krasavin, E.A.

    1988-01-01

    A model is proposed for interpreting the radiosensitivity of yeast cells as a function of linear energy transfer (LET) of ionizing radiation. The model takes into account the role of repair processes in sensitivity of yeast cells to ionizing radiation of different LET. Two types of repair are discussed: (1) a nonspecific repair (characteristic of both haploid and diploid cells), and (2) a diploid - soecific repair (characteristic of diploid cells only)

  18. Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later

    Science.gov (United States)

    Bond, Allison M.; Ming, Guo-li; Song, Hongjun

    2015-01-01

    Summary Adult somatic stem cells in various organs maintain homeostatic tissue regeneration and enhance plasticity. Since its initial discovery five decades ago, investigations of adult neurogenesis and neural stem cells have led to an established and expanding field that has significantly influenced many facets of neuroscience, developmental biology and regenerative medicine. Here we review recent progress and focus on questions related to adult mammalian neural stem cells that also apply to other somatic stem cells. We further discuss emerging topics that are guiding the field toward better understanding adult neural stem cells and ultimately applying these principles to improve human health. PMID:26431181

  19. Regulation of Autophagy by Glucose in Mammalian Cells

    OpenAIRE

    Moruno, Félix; Pérez-Jiménez, Eva; Knecht, Erwin

    2012-01-01

    Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focu...

  20. Photooxidative damage to mammalian cells and proteins by visible light

    International Nuclear Information System (INIS)

    Packer, L.; Kellogg, E.W. III

    1980-01-01

    In the present article, studies carried out in our laboratory on the effects of visible irradiation and O 2 in a variety of target systems ranging from cultured mammalian cells to purified catalase are reviewed. We will relate these studies of photooxidative damage to a scheme for the propagation of intracellular damage which traces a number of the possible pro-oxidant and anti-oxidant pathways found in the cell

  1. Intracellular transport of cholesterol in mammalian cells

    International Nuclear Information System (INIS)

    Brasaemle, D.L.

    1989-01-01

    The erythrocyte was selected as a simple cell for the study of transbilayer movement of cholesterol. Cholesterol oxidase was used to measure the distribution of [ 3 H]cholesterol across the erythrocyte membrane. Cholesterol oxidase was also used to estimate the rate of transport of low density lipoprotein (LDL) cholesterol to the plasma membrane of cultured Chinese hamster ovary (CHO) fibroblasts; the half-time of this process was 42 minutes. The rate of transport of LDL cholesterol to the plasma membrane was confirmed by a second procedure using amphotericin B. Amphotericin B was also used to estimate the rate of transport of endogenously synthesized cholesterol to the plasma membrane of CHO cells. New methodology was developed including improvements of the previously published cholesterol oxidase assay for plasma membrane cholesterol. A new method for detecting transport of cholesterol to the plasma membrane in cultured cells was developed using amphotericin B. Preliminary studies investigated the use of fluorescent polyenes, pimaricin and etruscomycin, as probes for plasma membrane cholesterol in transport studies. Finally, a modification of a previously published cell staining protocol yielded a simple, quantitative assay for cell growth

  2. Lipid map of the mammalian cell

    NARCIS (Netherlands)

    van Meer, G.; de Kroon, A.I.P.M.

    2010-01-01

    Technological developments, especially in mass spectrometry and bioinformatics, have revealed that living cells contain thousands rather than dozens of different lipids [for classification and nomenclature, see Fahy et al. (Fahy et al., 2009)]. Now, the resulting questions are what is the relevance

  3. X-ray irradiation of yeast cells

    Science.gov (United States)

    Masini, Alessandra; Batani, Dimitri; Previdi, Fabio; Conti, Aldo; Pisani, Francesca; Botto, Cesare; Bortolotto, Fulvia; Torsiello, Flavia; Turcu, I. C. Edmond; Allott, Ric M.; Lisi, Nicola; Milani, Marziale; Costato, Michele; Pozzi, Achille; Koenig, Michel

    1997-10-01

    Saccharomyces Cerevisiae yeast cells were irradiated using the soft X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterized by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency.

  4. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  5. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

  6. Sensing the Heat Stress by Mammalian Cells

    OpenAIRE

    Cates Jordan; Graham Garrett C; Omattage Natalie; Pavesich Elizabeth; Setliff Ian; Shaw Jack; Smith Caitlin; Lipan Ovidiu

    2011-01-01

    Abstract Background The heat-shock response network controls the adaptation and survival of the cell against environmental stress. This network is highly conserved and is connected with many other signaling pathways. A key element of the heat-shock network is the heat-shock transcription factor-1 (HSF), which is transiently activated by elevated temperatures. HSF translocates to the nucleus upon elevated temperatures, forming homotrimeric complexes. The HSF homotrimers bind to the heat shock ...

  7. DNA repair studies in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.; Owens, J.G.

    1984-01-01

    In submammalian test systems, nitrosocarbamates (NEC) are 100-fold more mutagenic than are their corresponding nitrosourea homologues. To learn more about its interaction with germ-cell DNA in the mouse testis, male mice were given i.p. injections of NEC. Testicular injections of [ 3 H]dThd were given along with the NEC. Sixteen days after treatment, sperm were recovered from the caudal epididymides and assayed for an unscheduled-DNA-synthesis

  8. Bartonella entry mechanisms into mammalian host cells.

    Science.gov (United States)

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment. © 2012 Blackwell Publishing Ltd.

  9. Sensing the Heat Stress by Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Cates Jordan

    2011-08-01

    Full Text Available Abstract Background The heat-shock response network controls the adaptation and survival of the cell against environmental stress. This network is highly conserved and is connected with many other signaling pathways. A key element of the heat-shock network is the heat-shock transcription factor-1 (HSF, which is transiently activated by elevated temperatures. HSF translocates to the nucleus upon elevated temperatures, forming homotrimeric complexes. The HSF homotrimers bind to the heat shock element on the DNA and control the expression of the hsp70 gene. The Hsp70 proteins protect cells from thermal stress. Thermal stress causes the unfolding of proteins, perturbing thus the pathways under their control. By binding to these proteins, Hsp70 allows them to refold and prevents their aggregation. The modulation of the activity of the hsp70-promoter by the intensity of the input stress is thus critical for cell's survival. The promoter activity starts from a basal level and rapidly increases once the stress is applied, reaches a maximum level and attenuates slowely back to the basal level. This phenomenon is the hallmark of many experimental studies and of all computational network analysis. Results The molecular construct used as a measure of the response to thermal stress is a Hsp70-GFP fusion gene transfected in Chinese hamster ovary (CHO cells. The time profile of the GFP protein depends on the transient activity, Transient(t, of the heat shock system. The function Transient(t depends on hsp70 promoter activity, transcriptional regulation and the translation initiation effects elicited by the heat stress. The GFP time profile is recorded using flow cytometry measurements, a technique that allows a quantitative measurement of the fluorescence of a large number of cells (104. The GFP responses to one and two heat shocks were measured for 261 conditions of different temperatures and durations. We found that: (i the response of the cell to two

  10. Saturation of DNA repair in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F E; Setlow, R B

    1979-01-01

    Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

  11. Carbamazepine induces mitotic arrest in mammalian Vero cells

    International Nuclear Information System (INIS)

    Perez Martin, J.M.; Fernandez Freire, P.; Labrador, V.; Hazen, M.J.

    2008-01-01

    We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells

  12. Carbamazepine induces mitotic arrest in mammalian Vero cells

    Energy Technology Data Exchange (ETDEWEB)

    Perez Martin, J.M.; Fernandez Freire, P.; Labrador, V. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Hazen, M.J. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)], E-mail: mariajose.hazen@uam.es

    2008-01-01

    We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells.

  13. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    International Nuclear Information System (INIS)

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

    2012-01-01

    -apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.

  14. Generation of Induced Pluripotent Stem Cells from Mammalian Endangered Species.

    Science.gov (United States)

    Ben-Nun, Inbar Friedrich; Montague, Susanne C; Houck, Marlys L; Ryder, Oliver; Loring, Jeanne F

    2015-01-01

    For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent their extinction. Cellular reprogramming is a means to capture the genomes of individual animals as induced pluripotent stem cells (iPSCs), which may eventually facilitate reintroduction of genetic material into breeding populations. Here, we describe a method for generating iPSCs from fibroblasts of mammalian endangered species.

  15. Yeast cell factories on the horizon

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2015-01-01

    For thousands of years, yeast has been used for making beer, bread, and wine. In modern times, it has become a commercial workhorse for producing fuels, chemicals, and pharmaceuticals such as insulin, human serum albumin, and vaccines against hepatitis virus and human papillomavirus. Yeast has also...... been engineered to make chemicals at industrial scale (e.g., succinic acid, lactic acid, resveratrol) and advanced biofuels (e.g., isobutanol) (1). On page 1095 of this issue, Galanie et al. (2) demonstrate that yeast can now be engineered to produce opioids (2), a major class of compounds used...

  16. Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

    Science.gov (United States)

    Yoshikawa, Yuki; Nasuno, Ryo; Kawahara, Nobuhiro; Nishimura, Akira; Watanabe, Daisuke; Takagi, Hiroshi

    2016-07-01

    Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

  18. Plasticity within stem cell hierarchies in mammalian epithelia.

    Science.gov (United States)

    Tetteh, Paul W; Farin, Henner F; Clevers, Hans

    2015-02-01

    Tissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur unidirectionally, with the arrows 'pointing away' from the stem cell. Recent studies, all based on genetic lineage tracing, describe various strategies employed by epithelial stem cell hierarchies to replace damaged or lost cells. While transdifferentiation from one tissue type into another ('metaplasia') appears to be generally forbidden in nonpathological contexts, plasticity within an individual tissue stem cell hierarchy may be much more common than previously appreciated. In this review, we discuss recent examples of such plasticity in selected mammalian epithelia, highlighting the different modes of regeneration and their implications for our understanding of cellular hierarchy and tissue self-renewal. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Action spectra in mammalian cells exposed to ultraviolet radiation

    International Nuclear Information System (INIS)

    Anon.

    1981-01-01

    A review is given of the literature published since 1977 on action spectra in mammalian cells exposed to ultraviolet radiation in the wavelength region above 220 nm. Action spectra for lethal events are discussed for cell inactivation in normal cells, growth arrested cells and photosensitive cells. Action spectra for non-lethal events are also discussed in relation to pyrimidine dimer formation, photoreactivation and the use of photosensitisers. It was concluded from these studies that damage to the DNA, and the extent of the repair of this damage, seems to determine a cell's response to such parameters as inactivation, mutation, transformation, latent viral activation, cellular viral capacity and ultraviolet enhanced viral reactivation. In addition to the direct effects of UV on DNA, photosensitization of cellular responses with chemicals such as 8-MOP extend the wavelength range at which damage can be demonstrated. (U.K.)

  1. Correlating yeast cell stress physiology to changes in the cell surface morphology: atomic force microscopic studies.

    Science.gov (United States)

    Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

    2006-07-06

    Atomic Force Microscopy (AFM) has emerged as a powerful biophysical tool in biotechnology and medicine to investigate the morphological, physical, and mechanical properties of yeasts and other biological systems. However, properties such as, yeasts' response to environmental stresses, metabolic activities of pathogenic yeasts, cell-cell/cell-substrate adhesion, and cell-flocculation have rarely been investigated so far by using biophysical tools. Our recent results obtained by AFM on one strain each of Saccharomyces cerevisiae and Schizosaccharomyces pombe show a clear correlation between the physiology of environmentally stressed yeasts and the changes in their surface morphology. The future directions of the AFM related techniques in relation to yeasts are also discussed.

  2. Mammalian designer cells: Engineering principles and biomedical applications.

    Science.gov (United States)

    Xie, Mingqi; Fussenegger, Martin

    2015-07-01

    Biotechnology is a widely interdisciplinary field focusing on the use of living cells or organisms to solve established problems in medicine, food production and agriculture. Synthetic biology, the science of engineering complex biological systems that do not exist in nature, continues to provide the biotechnology industry with tools, technologies and intellectual property leading to improved cellular performance. One key aspect of synthetic biology is the engineering of deliberately reprogrammed designer cells whose behavior can be controlled over time and space. This review discusses the most commonly used techniques to engineer mammalian designer cells; while control elements acting on the transcriptional and translational levels of target gene expression determine the kinetic and dynamic profiles, coupling them to a variety of extracellular stimuli permits their remote control with user-defined trigger signals. Designer mammalian cells with novel or improved biological functions not only directly improve the production efficiency during biopharmaceutical manufacturing but also open the door for cell-based treatment strategies in molecular and translational medicine. In the future, the rational combination of multiple sets of designer cells could permit the construction and regulation of higher-order systems with increased complexity, thereby enabling the molecular reprogramming of tissues, organisms or even populations with highest precision. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  4. Oxidative Stress and Programmed Cell Death in Yeast

    International Nuclear Information System (INIS)

    Farrugia, Gianluca; Balzan, Rena

    2012-01-01

    Yeasts, such as Saccharomyces cerevisiae, have long served as useful models for the study of oxidative stress, an event associated with cell death and severe human pathologies. This review will discuss oxidative stress in yeast, in terms of sources of reactive oxygen species (ROS), their molecular targets, and the metabolic responses elicited by cellular ROS accumulation. Responses of yeast to accumulated ROS include upregulation of antioxidants mediated by complex transcriptional changes, activation of pro-survival pathways such as mitophagy, and programmed cell death (PCD) which, apart from apoptosis, includes pathways such as autophagy and necrosis, a form of cell death long considered accidental and uncoordinated. The role of ROS in yeast aging will also be discussed.

  5. Mammalian Cochlear Hair Cell Regeneration and Ribbon Synapse Reformation

    Directory of Open Access Journals (Sweden)

    Xiaoling Lu

    2016-01-01

    Full Text Available Hair cells (HCs are the sensory preceptor cells in the inner ear, which play an important role in hearing and balance. The HCs of organ of Corti are susceptible to noise, ototoxic drugs, and infections, thus resulting in permanent hearing loss. Recent approaches of HCs regeneration provide new directions for finding the treatment of sensor neural deafness. To have normal hearing function, the regenerated HCs must be reinnervated by nerve fibers and reform ribbon synapse with the dendrite of spiral ganglion neuron through nerve regeneration. In this review, we discuss the research progress in HC regeneration, the synaptic plasticity, and the reinnervation of new regenerated HCs in mammalian inner ear.

  6. Guidelines and recommendations on yeast cell death nomenclature

    Directory of Open Access Journals (Sweden)

    Didac Carmona-Gutierrez

    2018-01-01

    Full Text Available Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

  7. Immobilisation increases yeast cells' resistance to dehydration-rehydration treatment.

    Science.gov (United States)

    Borovikova, Diana; Rozenfelde, Linda; Pavlovska, Ilona; Rapoport, Alexander

    2014-08-20

    This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Guidelines and recommendations on yeast cell death nomenclature

    Science.gov (United States)

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J.; Breitenbach, Michael; Burhans, William C.; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F.; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B.; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W.; Grant, Chris M.; Greenwood, Michael T.; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M.; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P.; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A.; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D.; Outeiro, Tiago F.; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F.; Sharon, Amir; Sigrist, Stephan J.; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M.; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B.; Tuite, Mick; Vögtle, F.-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J.; Zhao, Richard Y.; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research. PMID:29354647

  9. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  10. Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

    Science.gov (United States)

    Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

    2016-08-01

    The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig, E-mail: blackstc@ninds.nih.gov

    2016-11-15

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  12. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    International Nuclear Information System (INIS)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

    2016-01-01

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  13. Labeling proteins on live mammalian cells using click chemistry.

    Science.gov (United States)

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

  14. Monitoring of yeast cell concentration using a micromachined impedance sensor

    NARCIS (Netherlands)

    Krommenhoek, E.E.; Gardeniers, Johannes G.E.; Bomer, Johan G.; van den Berg, Albert; Li, X.; Ottens, M.; van der Wielen, L.A.M.; van Dedem, G.W.K.; van Leeuwen, M.; van Gulik, W.M.; Heijnen, J.J.

    2005-01-01

    The paper describes the design, modelling and experimental characterization of a micromachined impedance sensor for on-line monitoring of the viable yeast cell concentration (biomass) in a miniaturized cell assay. Measurements in a Saccharomyces cerevisiae cell culture show that the permittivity of

  15. Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts

    Czech Academy of Sciences Publication Activity Database

    Pálková, Z.; Váchová, Libuše

    2016-01-01

    Roč. 57, SEP (2016), s. 110-119 ISSN 1084-9521 R&D Projects: GA ČR GA13-08605S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Pathogenic yeasts * Biofilms and colonies * Cell differentiation Subject RIV: EE - Microbiology, Virology Impact factor: 6.614, year: 2016

  16. Inertial picobalance reveals fast mass fluctuations in mammalian cells

    Science.gov (United States)

    Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

    2017-10-01

    The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

  17. Yeast cell wall chitin reduces wine haze formation.

    Science.gov (United States)

    Ndlovu, Thulile; Divol, Benoit; Bauer, Florian F

    2018-04-27

    Protein haze formation in bottled wines is a significant concern for the global wine industry and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels indicating differences in haze protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli -produced GFP-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and propose a strategy for optimizing wine yeast strains to improve wine clarification. Importance In this study, we establish a new mechanism by which wine yeast strains can impact on the protein haze formation of wines, and demonstrate that yeast cell wall chitin binds grape chitinase in a chitin-concentration dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also

  18. Membrane phospholipids and radiation-induced death of mammalian cells

    International Nuclear Information System (INIS)

    Wolters, H.

    1987-01-01

    Radiation-induced cell killing is generally believed to be a consequence of residual DNA damage or damage that is mis-repaired. However, besides this DNA damage, damage to other molecules or structures of the cell may be involved in the killing. Especially membranes have been suggested as a determinant in cellular radiosensitivity. In this thesis experiments are described, dealing with the possible involvement of membranes in radiation-induced killing of mammalian cells. A general treatise of membrane structure is followed by information concerning deleterious effects of radiation on membranes. Consequences of damage to structure and function of membranes are reviewed. Thereafter evidence relating to the possible involvement of membranes in radiation-induced cell killing is presented. (Auth.)

  19. Application of recombinant fluorescent mammalian cells as a toxicity biosensor.

    Science.gov (United States)

    Kim, E J; Lee, Y; Lee, J E; Gu, M B

    2002-01-01

    With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

  20. Differential biological effects of iodoacetate in mammalian cell lines; radio sensitization and radio protection

    International Nuclear Information System (INIS)

    Yadav, Usha; Anjaria, K.B.; Desai, Utkarsha N.; Chaurasia, Rajesh K.; Shirsath, K.B.; Bhat, Nagesh N.; Balakrishnan, Sreedevi; Sapra, B.K.; Nairy, Rajesha

    2014-01-01

    There are several studies where it has been shown that Iodoacetate (IA) possesses in vivo anti-tumor activity. The fact that it is a model glycolytic inhibitor makes it more interesting. As seen in recent trends, glycolytic inhibitors are emerging as new strategy for cancer therapeutic research taking advantage of glycolytic phenotype of cancerous tissues. IA has been reported to have radioprotective effects in yeast cells and human lymphocytes. Biological effects of IA in response to radiation in mammalian cell lines are not well documented. We screened IA for cytotoxicity using clonogenic assay at different concentrations ranging from 0.1 to 2.5 μg/ml using three different mammalian cell lines; A-549 (human lung carcinoma cell line), MCF-7 (human mammary cancer cell line) and a noncancerous CHO (Chinese hamster ovary cell line). For studying radioprotective/radio sensitizing efficacy, cells were exposed to 4 Gy of 60 Co-γ radiation using a teletherapy source at a dose rate of 1 Gy/min, following which IA post-treatment was carried out. Clonogenic and micronucleus assay were performed to assess radioprotection/sensitization. The results indicated that IA was highly cytotoxic in cancerous cell lines A-549 (IC 50 =1.25 μg/ml) and MCF-7 (IC 50 = 1.9 μg/ml). In contrast, it was totally non-toxic in non-cancerous cell line, viz. CHO, in the same concentration range. In addition, IA exhibited radio protective effect in CHO cell line, whereas in other two cancer cell lines, viz. A-549 and MCF-7, radio sensitizing effect was seen as judged by induction of cell killing and micronuclei. In conclusion, lA, a model glycolytic inhibitor, was found to be selectively cytotoxic in cancer cells as compared to normal cells. Further, it reduced radiation induced damage (micronuclei and cell killing) in normal cells but increased it in cancer cells indicating its potential use in cancer therapy. (author)

  1. Regulation of gene expression in mammalian cells following ionizing radiation

    International Nuclear Information System (INIS)

    Boothman, D.A.; Lee, S.W

    1991-01-01

    Mammalian cells use a variety of mechanisms to control the expression of new gene transcrips elicited in response to ionizing radiation. Damage-induced proteins have been found which contain DNA binding sites located within the promoter regions of SV40 and human thymidine kinase genes. DNA binding proteins as well as proteins which bind to specific DNA lesions (e.g., XIP bp 175 binds specifically to X-ray-damaged DNA) may play a role in the initial recognition of DNA damage and may initiate DNA repair processes, along with new transcription. Mammalian gene expression after DNA damage is also regulated via the stabilization of preexisting mRNA transcripts. Stabilized mRNA transcripts are translated into protein products not previously present in the cell due to undefined posttranscriptional modifications. Thus far, the only example of mRNA stabilization following X-irradiation is the immediate induction of tissue-type plasminogen activator. Mammalian cells synthesize new mRNA transcripts indirect response to DNA damage. Using cDNA cloning, Northern RNA blotting and nuclear run-on techniques, the levels of a variety of known and previously unknown genes dramatically increase following X-irradiation. These genes/proteins now include; a) DNA binding transcripts factors, such as the UV-responsive element binding factors, ionizing radiation-induced DNA-binding proteins, and XIP bP 175; b) proto-oncogenes, such as c-fos, c-jun, and c-myc; c) several growth-related genes, (e.g., the gadd genes, protein kinase C, IL-1, and thymidine kinase); and d) a variety of other genes, including proteases, tumor necrosis factor-alpha, and DT diaphorase. Mammalian cells respond to X-irradiation by eliciting a very complex series of events resulting in the appearance of new genes and proteins. These gene products may affect DNA repair, adaptive responses, apoptosis, SOS-type mutagenic response, and/or carcinogenesis. (J.P.N.)

  2. Does autophagy have a license to kill mammalian cells?

    Science.gov (United States)

    Scarlatti, F; Granata, R; Meijer, A J; Codogno, P

    2009-01-01

    Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.

  3. Yeast mother cell-specific aging

    Czech Academy of Sciences Publication Activity Database

    Breitenbach, M.; Laun, P.; Pichová, Alena; Madeo, F.; Heeren, G.; Kohlwein, S. D.; Froehlich, K. U.; Dawes, I.

    2001-01-01

    Roč. 18, - (2001), s. 21 ISSN 0749-503X. [International Conference on Yeast Genetics and Molecular Biology /20./. 26.08.2001-31.08.2001, Prague] Institutional research plan: CEZ:AV0Z5020903 Subject RIV: EB - Genetics ; Molecular Biology

  4. Sensitization of ultraviolet radiation damage in bacteria and mammalian cells

    International Nuclear Information System (INIS)

    Fisher, G.J.; Watts, M.E.; Patel, K.B.; Adams, G.E.

    1978-01-01

    Bacteria (Serratia marcescens) and mammalian cells (Chinese hamsters V79-379A) were irradiated in monolayers with ultraviolet light at 254 nm or 365 nm in the presence or absence of radiosensitizing drugs. At 254 nm, killing is very efficient (Dsub(37) approximately equal 1 J m -2 exposure, or approximately equal 6 x 10 4 photons absorbed by DNA per bacterium), and sensitizers have no effect. At 365 nm, cells are not killed in buffer, but are inactivated in the presence of nifurpipone or misonidazole. Lethal exposures (approximately equal 5 x 10 3 J m -2 at 10 nM misonidazole) correspond to about 10 7 photons absorbed by sensitizer molecules per bacterium. Toxicity of stable photoproducts of the drugs is not involved, nor is oxygen required. Hence the transient species formed by photo-excitation of radiosensitizer molecules are capable of killing cells in the absence of other types of radiation damage. (author)

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

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

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

  6. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  7. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

    2013-01-01

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions

  8. Liquid holding recovery kinetics in yeast cells with regard to radiation quality

    International Nuclear Information System (INIS)

    Kim, Jin Kyu; Lee, Byoung Hun; Petin, Vladislav G.

    2004-01-01

    It is widely accepted that the RBE of ionizing radiation with a high linear energy transfer (LET) is dependent both on the increased probability of primary damage production (physical events) and the reduced ability of a cell for post-irradiation recovery (biological events). A relatively unexpected role of the specific repair pathways in the RBE of high-LET radiation was demonstrated for bacterial, yeast and mammalian cells. It seems to exist a common agreement that high-LET radiations produce more portion of damage that are considered to be irreversible compared with low-LET radiation such as photons. Cellular recovery and repair of radiation-induced DNA double-strand breaks (DSB) could be also dependent upon radiation quality. Studies concerning the rate of the recovery and repair from radiation damage produced with low- and high-LET radiations in cells of various origins on the survival and macromolecular level have also revealed that in general at a high ionization density, these processes may be reduced or even absent. When irradiated yeast cells are held in a liquid non-nutrient media at 30 .deg. C before planting on to a growth medium, their survival increases. This phenomena is known as liquid holding recovery (LHR). A quantitative approach describing the LHR kinetics of the yeast cells was described, which enables the estimation of the probability of the recovery per unit time and the fraction of the irreversible damage. The main goals of this study were (i) to answer the question whether or not high-LET radiation affects the recovery process itself or if it only produces a higher level of severe irreversible damage that cannot be repaired at all; (ii) to elucidate the role of irreversible damage and the probability of recovery in some rad mutants of the yeast Saccharomyces cerevisiae. In this study, the liquid-holing recovery will serve as an indicator of the cellular repair activity

  9. Distinctive transforming genes in x-ray-transformed mammalian cells

    International Nuclear Information System (INIS)

    Borek, C.; Ong, A.; Mason, H.

    1987-01-01

    DNAs from hamster embryo cells and mouse C3H/10T1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and in a rat cell line, the Rat-2 cells. DNAs from unirradiated cells or irradiated and visibly untransformed cells do not produce transformed colonies. The transfectant grow in agar and form tumors in nude mice. Treatment of the DNAs with restriction endonucleases prior to transfection indicates that the same transforming gene (oncogene) is present in each of the transformed mouse cells and is the same in each of the transformed hamster cells. Southern blot analysis of 3T3 or Rat-2 transfectants carrying oncogenes from radiation-transformed C3H/10T1/2 or hamster cells indicates that the oncogenes responsible for the transformation of 3T3 cells are not the Ki-ras, Ha-ras, N-ras genes, nor are they neu, trk, raf, abl, or fms. The work demonstrates that DNAs from mammalian cells transformed into malignancy by direct exposure in vitro to radiation contain genetic sequences with detectable transforming activity in three recipient cell lines. The results provide evidence that DNA is the target of radiation carcinogenesis induced at a cellular level in vitro. The experiments indicate that malignant radiogenic transformation in vitro of hamster embryo and mouse C3H/10T1/2 cells involves the activation of unique non-ras transforming genes, which heretofore have not been described

  10. Damage and repair in mammalian cells after ultraviolet and/or visible light treatment

    International Nuclear Information System (INIS)

    Harm, H.

    1976-01-01

    Ultraviolet (uv) light (254 nm or 302 nm) was used to induce lesions in DNA of cultured mammalian cells in vivo, particularly in fibroblasts from potoroo cornea, mouse skin (3T3), cat cornea, human skin (healthy and diseased), and in freshly obtained ox cornea tissue. In addition, white light (WL) from daylight fluorescent lamps, filtered through a plexiglass plate cutting off virtually all photons less than 380 nm and being fully transparent for greater than 400 nm, was applied in vivo either as photoreactivating light after uv irradiation, or as damaging radiation by itself. Completely unirradiated samples under otherwise identical conditions served as controls. DNA from cells exposed to these different radiations was extracted and tested for its capability of competitively inhibiting photoenzymatic repair of uv-irradiated Haemophilus influenzae transforming DNA in vitro in the presence of yeast photoreactivating enzyme (PRE) and photoreactivating light. In several (but not all) of the cases, DNA from cells treated with uv + WL displayed considerably less competitive inhibition than DNA from cells treated with uv alone, even though under certain conditions WL itself caused damage serving as substrate from the PRE in vitro. Cell cultures differing in their origin or in their number of passages varied substantially in this respect

  11. Cell lineage analysis of the mammalian female germline.

    Directory of Open Access Journals (Sweden)

    Yitzhak Reizel

    Full Text Available Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote. We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  12. THE GERMLINE STEM CELL NICHE UNIT IN MAMMALIAN TESTES

    Science.gov (United States)

    Oatley, Jon M.; Brinster, Ralph L.

    2014-01-01

    This review addresses current understanding of the germline stem cell niche unit in mammalian testes. Spermatogenesis is a classic model of tissue-specific stem cell function relying on self-renewal and differentiation of spermatogonial stem cells (SSCs). These fate decisions are influenced by a niche microenvironment composed of a growth factor milieu that is provided by several testis somatic support cell populations. Investigations over the last two decades have identified key determinants of the SSC niche including cytokines that regulate SSC functions and support cells providing these factors, adhesion molecules that influence SSC homing, and developmental heterogeneity of the niche during postnatal aging. Emerging evidence suggests that Sertoli cells are a key support cell population influencing the formation and function of niches by secreting soluble factors and possibly orchestrating contributions of other support cells. Investigations with mice have shown that niche influence on SSC proliferation differs during early postnatal development and adulthood. Moreover, there is mounting evidence of an age-related decline in niche function, which is likely influenced by systemic factors. Defining the attributes of stem cell niches is key to developing methods to utilize these cells for regenerative medicine. The SSC population and associated niche comprise a valuable model system for study that provides fundamental knowledge about the biology of tissue-specific stem cells and their capacity to sustain homeostasis of regenerating tissue lineages. While the stem cell is essential for maintenance of all self-renewing tissues and has received considerable attention, the role of niche cells is at least as important and may prove to be more receptive to modification in regenerative medicine. PMID:22535892

  13. Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

    Science.gov (United States)

    Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

    2018-03-01

    A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

  14. Centriole movements in mammalian epithelial cells during cytokinesis

    Directory of Open Access Journals (Sweden)

    Tanke Hans J

    2010-05-01

    Full Text Available Abstract Background In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. Results Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1 along the nuclear envelope, 2 irregular, and 3 along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. Conclusions These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

  15. Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

    Science.gov (United States)

    Wolf, Moritz K F; Closet, Aurélie; Bzowska, Monika; Bielser, Jean-Marc; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

    2018-05-21

    Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Radiation activation of transcription factors in mammalian cells

    International Nuclear Information System (INIS)

    Kraemer, M.; Stein, B.; Mai, S.; Kunz, E.; Koenig, H.; Ponta, H.; Herrlich, P.; Rahmsdorf, H.J.; Loferer, H.; Grunicke, H.H.

    1990-01-01

    In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s). (orig.)

  17. Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

    Science.gov (United States)

    Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

    2015-09-02

    Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

  18. Influence the oxidant action of selenium in radiosensitivity induction and cell death in yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Porto, Barbara Abranches de Araujo

    2012-01-01

    Ionizing radiations are from both natural sources such as from anthropogenic sources. Recently, radiotherapy has emerged as one of the most common therapies against cancer. Co-60 irradiators (cobalt-60 linear accelerators) are used to treat of malignant tumors routinely in hospitals around the world. Exposure to ionizing radiation can induce changes in cellular macromolecules and affect its functions, because they cause radiolysis of the water molecule generating reactive oxygen species, which can cause damage to virtually all organelles and cell components known as oxidative damage that can culminate in oxidative stress. Oxidative stress is a situation in which the balance between oxidants and antioxidants is broken resulting in excessive production of reactive species, it is not accompanied by the increase in antioxidant capacity, making it impossible to neutralize them. Selenium is a micronutrient considered as antioxidant, antiinflammatory, which could prevent cancer. Selenium in biological system exists as seleno proteins. Nowadays, 25 human seleno proteins have been identified, including glutathione peroxidase, an antioxidant enzyme. Yeasts have the ability to incorporate various metals such as iron, cadmium, zinc and selenium, as well as all biological organisms. The yeast Saccharomyces cerevisiae, unlike mammalian cells is devoid of seleno proteins, being considered as a practical model for studies on the toxicity of selenium, without any interference from the metabolism of seleno proteins. Moreover, yeast cells proliferate through the fermentation, the microbial equivalent of aerobic glycolysis in mammals and the process is also used by tumors. Several reports show that the pro-oxidante effects and induced toxic selenium compounds occur at lower doses and in malignant cells compared with benign cells. Therefore selenium giving a great therapeutic potential in cancer treatment .Our objective was to determine whether selenium is capable to sensitize yeasts

  19. 31P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

    International Nuclear Information System (INIS)

    Brindle, K.; Braddock, P.; Fulton, S.

    1990-01-01

    Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creating kinase activities similar to those found in mammalian heart muscle. 31 P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts

  20. A study on immobilized ethanol yeast cells by radiation technique

    International Nuclear Information System (INIS)

    Li Zhengkui; Zhang Bosen

    1994-01-01

    Hydrophilic monomer 2-hydroxyethyl acrylate (HEA) and a series of polyethylene glycol dimethacrylate monomers were copolymerized by radiation technique at low temperature (-78 degree C) and hydrophilic hydrogels were obtained. The immobilization of yeast cells with these copolymer carriers led to a higher ethanol productivity than free cells. Of all copolymer carriers, the ethanol yield with poly (HEA-14 G) was the highest, about 2.45 times as high as that of free yeast cells. In addition, the ethanol productivity of 12 batch repeated reactions with poly (HEA-14G) carrier was all higher than that of free yeast cells. The ethanol productivity of immobilized yeast cells was dependent on the proportion of hydrophilic monomer to other monomers in copolymer systems, the chain length of the bifunctional monomer, the degree of hydration of copolymer carriers, the structure of copolymer carriers and porosity in the internal structure of carriers. The ethanol yield of immobilized cells depended on swelling ability and porosity of copolymer carriers

  1. Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

    Science.gov (United States)

    Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

    The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.

  2. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  3. Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

    Science.gov (United States)

    Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

    2017-10-01

    The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  4. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

    International Nuclear Information System (INIS)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

  5. The fungicide mancozeb induces toxic effects on mammalian granulosa cells

    International Nuclear Information System (INIS)

    Paro, Rita; Tiboni, Gian Mario; Buccione, Roberto; Rossi, Gianna; Cellini, Valerio; Canipari, Rita; Cecconi, Sandra

    2012-01-01

    The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant. Highlights: ► The fungicide mancozeb affects oocyte spindle morphology and fertilization rate. ► We investigated the toxic effects of mancozeb on mouse and human granulosa cells. ► Granulosa cells modify their morphology and expression level of p53. ► Mancozeb induces a premalignant-like status in exposed cells.

  6. The fungicide mancozeb induces toxic effects on mammalian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Paro, Rita [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Tiboni, Gian Mario [Department of Medicine and Aging, Section of Reproductive Sciences, University “G. D' Annunzio”, Chieti-Pescara (Italy); Buccione, Roberto [Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti (Italy); Rossi, Gianna; Cellini, Valerio [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Canipari, Rita [Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Embryology, School of Pharmacy and Medicine, “Sapienza” University of Rome, Rome (Italy); Cecconi, Sandra, E-mail: sandra.cecconi@cc.univaq.it [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy)

    2012-04-15

    The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant. Highlights: ► The fungicide mancozeb affects oocyte spindle morphology and fertilization rate. ► We investigated the toxic effects of mancozeb on mouse and human granulosa cells. ► Granulosa cells modify their morphology and expression level of p53. ► Mancozeb induces a premalignant-like status in exposed cells.

  7. Chromosome aberrations and cell survival in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Tremp, J.

    1981-01-01

    A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of x rays in early G 1 . Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-h intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected

  8. Antioxidation activities of pteridines in mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.; Shen, R. (Univ. of Texas, Galveston (United States))

    1991-03-11

    L-erythro-5,6,7,8-Tetrahydrobiopterin (BH{sub 4}), the cofactor for aromatic amino acid hydroxylases (AAA-H), is a predominant form of pteridines which occur ubiquitously in nature. When BH{sub 4} is oxidized to quinonoid dihydrobiopterin by AAA-H, it is regenerated by dihydropteridine reductase (DHPR) at the expense of NADH. The role of BH{sub 4} other than serving as the hydroxylase cofactor is not clear. The existence of BH{sub 4} and DHPR in tissues which are devoid of AAA-H suggests that BH{sub 4} may play an as yet undiscovered physiological function. This study demonstrates a BH{sub 4}-mediated antioxidation system, which consists of BH{sub 4}, DHPR, peroxidase and NADH in rat pheochromocytoma PC 12 cells and mouse macrophages J774A.1. This system was as effective as catalase and ascorbic acid in protecting cells against H{sub 2}O{sub 2} and xanthine/xanthine oxidase-induced toxicity and was more effective than catalase in defense against nitrofurantoin-induced toxicity. The antioxidation effect of this system was not due to peroxidase and was improved when synthetic pteridines were substituted for BH{sub 4}. Since BH{sub 4}, DHPR, peroxidases and NADH are widely distributed in major organs and blood cells, they may constitute an as yet little known antioxidation system in mammalian cells.

  9. Combined effects of hyperthermia and radiation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Ben-Hur, E.; Elkind, M.M.; Riklis, E.

    1977-01-01

    Hyperthermia (temperatures of 39 0 C or higher) enhances the killing of mammalian cells by ionizing radiation (fission-spectrum neutrons and x-rays). The nature and the magnitude of the enhanced radiation killing varies with temperature and for a fixed temperature during irradiation, the enhanced lethality varies inversely with dose rate. For temperatures up to 41 0 C, dose fractionation measurements indicate that hyperthermia inhibits the repair of sublethal damage. At higher temperatures, the expression of potentially lethal damage is enhanced. Since the effect of heat is greatest in cells irradiated during DNA synthesis, the radiation age-response pattern is flattened by hyperthermia. In addition to the enhanced cell killing described above, three other features of the effect of hyperthermia are important in connection with the radiation treatment of cancer. The first is that heat selectively sensitizes S-phase cells to radiation. The second is that it takes radiation survivors 10 to 20 hrs after a modest heat treatment to recover their ability to repair sublethal damage. And the third is that hyperthermia reduces the magnitude of the oxygen enhancement ratio. Thus, heat if applied selectively, could significantly increase the margin of damage between tumors and normal tissues

  10. Structure and function of stem cell pools in mammalian cell renewal systems

    International Nuclear Information System (INIS)

    Fliedner, T.M.; Nothdurft, W.

    1979-01-01

    Stem cells play a key-role in the maintenance of the equilibrium between cell loss and cell production in cell renewal systems as well as in the understanding of the radiation pathophysiology of mammalian organisms. The integrity of mammalian organisms with the need to maintain a constant ''millieu interior'' is depending on the normal functioning of cell renewal systems, especially those of epithelial surfaces and blood cell forming organs. All cell renewal systems of bodies have a very similar functional structure consisting of functional, proliferative - amplifying and stem cell compartments. They differ in transit and cell cycle times and in the number of amplification division - aside from the difference in their functional and biochemical make-up. The stem cell pools are providing the cells capable of differentiation without depleting their own kind. This can be achieved by symmetrical or assymmetrical stem cell division. In normal steady state, 50% of the stem cell division remain in the stem cell pool, while the other 50% leave it to differentiate, proliferate and mature, hemopoietic system is distributed throughout bodies. This is an important factor in the radiation biology of mammalian organisms since the loss of function in one area can be compensated for by more production in other areas, and locally depleted sites can be reseeded with the stem cells migrating in from blood. (Yamashita, S.)

  11. The digestion of yeast cell wall polysaccharides in veal calves

    NARCIS (Netherlands)

    Gaillard, B.D.E.; Weerden, van E.J.

    1976-01-01

    1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their ‘normal’, milk-substitute

  12. Magnetically responsive yeast cells: methods of preparation and applications

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Maděrová, Zdeňka; Pospišková, K.; Horská, Kateřina; Šafaříková, Miroslava

    2015-01-01

    Roč. 32, č. 1 (2015), s. 227-237 ISSN 0749-503X R&D Projects: GA MŠk(CZ) LD13023; GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : yeast cells * Saccharomyces * Kluyveromyces * Rhodotorula * Yarrowia * magnetic modification Subject RIV: CE - Biochemistry Impact factor: 2.259, year: 2015

  13. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  14. Synchronization of glycolytic oscillations in a yeast cell population

    DEFF Research Database (Denmark)

    Dano, S.; Hynne, F.; De Monte, Silvia

    2001-01-01

    The mechanism of active phase synchronization in a suspension of oscillatory yeast cells has remained a puzzle for almost half a century. The difficulty of the problem stems from the fact that the synchronization phenomenon involves the entire metabolic network of glycolysis and fermentation, and...

  15. The impact of metabolism on aging and cell size in single yeast cells

    NARCIS (Netherlands)

    Huberts, Daphne

    2015-01-01

    The aim of this thesis was to determine how metabolism affects yeast aging in single yeast cells using a novel microfluidic device. We first review how cells are able to sense nutrients in their environment and then describe the use of the microfluidic dissection platform that greatly improves our

  16. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

    Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

  17. ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

    Science.gov (United States)

    Baudier, Jacques

    2018-05-01

    In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

  18. Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

    Science.gov (United States)

    Tissot, Stéphanie; Oberbek, Agata; Reclari, Martino; Dreyer, Matthieu; Hacker, David L; Baldi, Lucia; Farhat, Mohamed; Wurm, Florian M

    2011-07-01

    Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Automatic Control of Gene Expression in Mammalian Cells.

    Science.gov (United States)

    Fracassi, Chiara; Postiglione, Lorena; Fiore, Gianfranco; di Bernardo, Diego

    2016-04-15

    Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

  20. Sharing the cell's bounty - organelle inheritance in yeast.

    Science.gov (United States)

    Knoblach, Barbara; Rachubinski, Richard A

    2015-02-15

    Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.

  1. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

    Science.gov (United States)

    Muller, Nicolas; Piel, Matthieu; Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-04-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.

  2. Genetic changes in Mammalian cells transformed by helium cells

    Energy Technology Data Exchange (ETDEWEB)

    Durante, M.; Grossi, G. (Naples Univ. (Italy). Dipt. di Scienze Fisiche); Yang, T.C.; Roots, R. (Lawrence Berkeley Lab., CA (USA))

    1990-11-01

    Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9--10 keV/{mu}m). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells. 26 refs., 5 figs., 2 tabs.

  3. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.

    Science.gov (United States)

    White, Patricia M; Doetzlhofer, Angelika; Lee, Yun Shain; Groves, Andrew K; Segil, Neil

    2006-06-22

    Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.

  4. Effects of gamma radiation on Sporothrix schenckii yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacerda, Camila M. de Sousa; Martins, Estefania Mara Nascimento; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: cmsl@cdtn.br, e-mail: estefaniabio@yahoo.com.br, e-mail: antero@cdtn.br; Resende, Maria Aparecida de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: maressend@mono.icb.ufmg.br

    2009-07-01

    Sporotrichosis is a subacute or chronic infection caused by the fungus Sporothrix schenckii. Zoonotic transmission can occur after scratches or bites of animals, mainly cats, rodents, and armadillos. Up to the moment, no approved vaccine was reported for S. schenckii or to any important pathogenic fungi infection in humans, indicating the need to expand the research in this field and to explore new alternatives. The aim of this study was to evaluate the effects of gamma radiation in the viability, metabolic activity and reproductive ability of S. schenckii yeast cells for further studies on the development of a vaccine for immunization of cats and dogs. The culture of S. schenckii, in solid medium, was irradiated at doses ranging from 1.0 to 9.0 kGy. After each dose the reproductive capacity, viability and protein synthesis were estimated. The results showed that a reduction of 6 log{sub 10} cycles in the number of colonies was achieved at 6.0 kGy and after 8.0 kGy no colonies could be recovered. The viability analysis indicated that yeast cells remained viable up to 9.0 kGy. The results of protein synthesis analysis showed that the yeast cells, irradiated up to 9.0 kGy, were able to synthesize proteins. Our preliminary results indicated that for the yeast cells of S. schenckii, it is possible to find an absorbed dose in which the pathogen loses its reproductive ability, while retaining its viability, a necessary condition for the development of a radioattenuated yeast vaccine. (author)

  5. Effects of gamma radiation on Sporothrix schenckii yeast cells

    International Nuclear Information System (INIS)

    Lacerda, Camila M. de Sousa; Martins, Estefania Mara Nascimento; Andrade, Antero S.R.; Resende, Maria Aparecida de

    2009-01-01

    Sporotrichosis is a subacute or chronic infection caused by the fungus Sporothrix schenckii. Zoonotic transmission can occur after scratches or bites of animals, mainly cats, rodents, and armadillos. Up to the moment, no approved vaccine was reported for S. schenckii or to any important pathogenic fungi infection in humans, indicating the need to expand the research in this field and to explore new alternatives. The aim of this study was to evaluate the effects of gamma radiation in the viability, metabolic activity and reproductive ability of S. schenckii yeast cells for further studies on the development of a vaccine for immunization of cats and dogs. The culture of S. schenckii, in solid medium, was irradiated at doses ranging from 1.0 to 9.0 kGy. After each dose the reproductive capacity, viability and protein synthesis were estimated. The results showed that a reduction of 6 log 10 cycles in the number of colonies was achieved at 6.0 kGy and after 8.0 kGy no colonies could be recovered. The viability analysis indicated that yeast cells remained viable up to 9.0 kGy. The results of protein synthesis analysis showed that the yeast cells, irradiated up to 9.0 kGy, were able to synthesize proteins. Our preliminary results indicated that for the yeast cells of S. schenckii, it is possible to find an absorbed dose in which the pathogen loses its reproductive ability, while retaining its viability, a necessary condition for the development of a radioattenuated yeast vaccine. (author)

  6. UVC-induced stress granules in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Mohamed Taha Moutaoufik

    Full Text Available Stress granules (SGs are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α. We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

  7. Gamma irradiation induced ultrastructural changes in Paracoccidioides brasiliensis yeast cells

    International Nuclear Information System (INIS)

    Demicheli, Marina C.; Andrade, Antero S.R.; Goes, Alfredo Miranda

    2007-01-01

    Paracoccidioides brasiliensis is a thermally dimorphic fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans with high prevalence in Latin America. Up to the moment no vaccine has still been reported. Ionizing radiation can be used to attenuate pathogens for vaccine development and we have successfully attenuated yeast cells of P. brasiliensis by gamma irradiation. The aim of the present study was to examine at ultrastructural level the effects of gamma irradiation attenuation on the morphology of P. brasiliensis yeast cells. P. brasiliensis (strain Pb-18) cultures were irradiated with a dose of 6.5 kGy. The irradiated cells were examined by scanning and also transmission electron microscopy. When examined two hours after the irradiation by scanning electron microscopy the 6.5 kGy irradiated cells presented deep folds or were collapsed. These lesions were reversible since examined 48 hours after irradiation the yeast have recovered the usual morphology. The transmission electron microscopy showed that the irradiated cells plasma membrane and cell wall were intact and preserved. Remarkable changes were found in the nucleus that was frequently in a very electrodense form. A extensive DNA fragmentation was produced by the gamma irradiation treatment. (author)

  8. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

    2011-01-31

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  9. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  10. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    CSIR Research Space (South Africa)

    Mthunzi, P

    2014-02-01

    Full Text Available Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including...

  11. Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

    OpenAIRE

    Chen, Yvonne Y.; Jensen, Michael C.; Smolke, Christina D.

    2010-01-01

    RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional res...

  12. Synthetic yeast based cell factories for vanillin-glucoside production

    DEFF Research Database (Denmark)

    Strucko, Tomas

    and controlled expression/overexpression of genes of interest. De novo biosynthetic pathway for vanillin-β-glucoside production was employed as a model system for several case studies in this project. In order to construct yeast cell factories fulfilling current demands of industrial biotechnology, methods......The yeast Saccharomyces cerevisiae is well a characterized microorganism and widely used as eukaryotic model organism as well as a key cell factory for bioproduction of various products. The latter comprise a large variety of scientifically and industrially relevant products such as low-value bulk...... chemicals and biofuels, food additives, high-value chemicals and recombinant proteins. Despite the recent achievements in the fields of systems biology and metabolic engineering together with availability of broad genetic engineering toolbox, the full potential of S. cerevisiae as a cell factory is not yet...

  13. Sorption of strontium by magnetically modified yeast cells

    International Nuclear Information System (INIS)

    Hu Yantao; Ji Yanqin; Tian Qing; Shao Xianzhang; Shi Jianhe; Ivo Safarik; Zhang Shengdong; Li Jinying

    2008-01-01

    Magnetically modified fodder's yeast (Kluyveromyces fragilis) cells using water based magnetic fluid, were characterized by scanning electron microscopy (SEM) and Vibrating Sample Magnetometer (VSM). The sorption-desorption properties of Sr 2+ by these yeast cells from nitrate salt of Sr 2+ were studied. The results demonstrated that the Sr 2+ sorption volume by these cells enhanced with increasing pH and reached a plateau between pH 4.0 and 7.0. A minor effect by temperature was observed. The sorption volumes are 19.5 mg/g and 53.5 mg/g from 10 ppm and 40 ppm Sr 2+ solution respectively within 20 min. The sorption of Sr 2+ in these cells can be desorbed under 0.1 mol/L HNO 3 solution. The maximum Sr 2+ sorption volume is 96.7 mg/g at 20℃. The sorption characteristic fits Langmuir model well with 140.8 mg/g calculated maximum sorption volume by these yeast cells. (authors)

  14. Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division

    NARCIS (Netherlands)

    Kumar, Sanjeev; de Boer, Rinse; van der Klei, Ida J

    2018-01-01

    Here we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and younger peroxisomes. During budding the peroxisome

  15. Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

    NARCIS (Netherlands)

    Novarina, Daniele; Mavrova, Sara N.; Janssens, Georges E.; Rempel, Irina L.; Veenhoff, Liesbeth M.; Chang, Michael

    The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast

  16. High power density yeast catalyzed microbial fuel cells

    Science.gov (United States)

    Ganguli, Rahul

    Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density

  17. Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

    Science.gov (United States)

    Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

    2017-05-26

    Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Yeast and mammalian metabolism continuous monitoring by using pressure recording as an assessment technique for xenobiotic agent effects

    Science.gov (United States)

    Milani, Marziale; Ballerini, Monica; Ferraro, Lorenzo; Marelli, E.; Mazza, Francesca; Zabeo, Matteo

    2002-06-01

    Our work is devoted to the study of Saccharomyces cerevisiae and human lymphocytes cellular metabolism in order to develop a reference model to assess biological systems responses to chemical or physical agents exposure. CO2 variations inside test-tubes are measured by differential pressure sensors; pressure values are subsequently converted in voltage. The system allows to test up to 16 samples at the same time. Sampling manages up to 100 acquisitions per second. Values are recorded by a data acquisition card connected to a computer. This procedure leads to a standard curve (pressure variation versus time), typical of the cellular line, that describe cellular metabolism. The longest time lapse used is of 170 h. Different phases appear in this curve: an initial growth up to a maximum, followed by a decrement that leads to a typical depression (pressure value inside the test-tubes is lower than the initial one) after about 35 h from the beginning of yeast cells. The curve is reproducible within an experimental error of 4%. The analysis of many samples and the low cost of the devices allow a good statistical significance of the data. In particular as a test we will compare two sterilizing agents effects: UV radiation and amuchina.

  19. Different characteristics between menadione and menadione sodium bisulfite as redox mediator in yeast cell suspension

    OpenAIRE

    Yamashoji, Shiro

    2016-01-01

    Menadione promoted the production of active oxygen species (AOS) in both yeast cell suspension and the crude enzymes from the cells, but menadione sodium bisulfite (MSB) had little effect on the production of AOS in the cell suspension. MSB kept the stable increase in the electron transfer from intact yeast cells to anode compared to menadione, but the electron transfer promoted by MSB was inhibited in permeabilized yeast cell suspension. Menadione promoted oxidation of NAD(P)H much faster th...

  20. Content of endogenous thiols and radioresistance of gemmating cells of Saccharomyces ellipsoideus and Saccharomyces cerevisiale yeasts

    International Nuclear Information System (INIS)

    Simonyan, N.V.; Avakyan, Ts.M.; Dzhanpoladyan, N.L.; Stepanyan, L.G.

    1983-01-01

    It has been shown that gemmating cells of ''wild type'' yeasts are more radioresistant and contain more endogenous thiols, than resting cells. Gemmating cells of Saccharomyces cerevisial yeasts, carrying the mutation rad 51, as to radioresistance and content of SH groups do not differ from resting cells. The results obtained testify to a connec-- tion between increased radioresistance of the yeast gemmating cells and increased content of endogenous thiols in them

  1. Modelling a tethered mammalian sperm cell undergoing hyperactivation

    KAUST Repository

    Curtis, M.P.

    2012-09-01

    The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions. © 2012 Elsevier Ltd.

  2. Modelling a tethered mammalian sperm cell undergoing hyperactivation

    KAUST Repository

    Curtis, M.P.; Kirkman-Brown, J.C.; Connolly, T.J.; Gaffney, E.A.

    2012-01-01

    The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions. © 2012 Elsevier Ltd.

  3. Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells

    International Nuclear Information System (INIS)

    Moonen, P.; Mermod, J.J.; Ernst, J.F.; Hirschi, M.; DeLamarter, J.F.

    1987-01-01

    Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein-lacking any carbohydrate- had by far the highest specific activity observed for the recombinant hGM-CSFs

  4. Yeast cells proliferation on various strong static magnetic fields and temperatures

    International Nuclear Information System (INIS)

    Otabe, E S; Kuroki, S; Nikawa, J; Matsumoto, Y; Ooba, T; Kiso, K; Hayashi, H

    2009-01-01

    The effect of strong magnetic fields on activities of yeast cells were investigated. Experimental yeast cells were cultured in 5 ml of YPD(Yeast extract Peptone Dextrose) for the number density of yeast cells of 5.0 ±0.2 x 10 6 /ml with various temperatures and magnetic fields up to 10 T. Since the yeast cells were placed in the center of the superconducting magnet, the effect of magnetic force due to the diamagnetism and magnetic gradient was negligibly small. The yeast suspension was opened to air and cultured in shaking condition. The number of yeast cells in the yeast suspension was counted by a counting plate with an optical microscope, and the time dependence of the number density of yeast cells was measured. The time dependence of the number density of yeast cells, ρ, of initial part is analyzed in terms of Malthus equation as given by ρ = ρo exp(kt), where k is the growth coefficient. It is found that, the growth coefficient under the magnetic field is suppressed compared with the control. The growth coefficient decreasing as increasing magnetic field and is saturated at about 5 T. On the other hand, it is found that the suppression of growth of yeast cells by the magnetic field is diminished at high temperatures.

  5. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    International Nuclear Information System (INIS)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    1987-01-01

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells. (author)

  6. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    Science.gov (United States)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

  7. A Comparative Study of the Cell Wall Structure of Basidiomycetous and Related Yeasts

    NARCIS (Netherlands)

    Kreger-van Rij, N.J.W.; Veenhuis, M.

    1971-01-01

    The wall of basidiomycetous and related yeasts showed a lamellar structure in sections of both budding cells and hyphae fixed with potassium permanganate. The yeasts also had a typical way of bud formation and septation. These features differ from those recorded for ascomycetous yeasts. In the

  8. Molecular basis of mammalian cell invasion by Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Nobuko Yoshida

    2006-03-01

    Full Text Available Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT and mammalian tissue culture trypomastigotes (TCT. During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM e por tripomastigotas de cultura de tecido (TCT. Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoprote

  9. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall......Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective...... with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...

  10. A study of ethanol production of yeast cells immobilized with polymer carrier produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu Zhaoxin; Fujimura, Takashi

    1993-01-01

    Polymer carriers, poly(hydroxyethyl acrylate(HEA)-methoxy polyethylene glycol methylacrylate (M-23G)) and poly(hydroxyethyl acrylate(HEA)-glycidyl methylacrylate (GMA)) used for the immobilization of yeast cells were prepared by radiation polymerization at low temperature. Yeast cells were immobilized through adhesion and multiplication of yeast cells. The ethanol productivity of immobilized yeast cells with these carriers was related to the monomer composition of polymers and the optimum monomer composition was 20%:10% in poly(HEA-M-23G) and 17%:6% in poly(HEA-GMA). In this case, the ethanol productivity of immobilized yeast cells was about 4 times that of cells in free system. The relationship between the activity of immobilized yeast cells and the water content of the polymer carrier were also discussed. (author)

  11. Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell

    International Nuclear Information System (INIS)

    Magno, A.C.G.; Oliveira, I.L.; Hauck, J.V.S.

    2016-01-01

    The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation (paper)

  12. Self-organization of yeast cells on modified polymer surfaces after dewetting: new perspectives in cellular patterning

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy); Satriano, S [Department of Chemical Sciences, University of Catania, Catania (Italy); Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2006-08-23

    In recent years, biological micro-electro-mechanical systems (commonly referred to as BioMEMS) have found widespread use, becoming increasingly prevalent in diagnostics and therapeutics. Cell-based sensors are nowadays gaining increasing attention, due to cellular built-in natural selectivity and physiologically relevant response to biologically active chemicals. On the other hand, surrogate microbial systems, including yeast models, have become a useful alternative to animal and mammalian cell systems for high-throughput screening for the identification of new pharmacological agents. A main obstacle in biosensor device fabrication is the need for localized geometric confinement of cells, without losing cell viability and sensing capability. Here we illustrate a new approach for cellular patterning using dewetting processes to control cell adhesion and spatial confinement on modified surfaces. By the control of simple system parameters, a rich variety of morphologies, ranging through hexagonal arrays, polygonal networks, bicontinuous structures, and elongated fingers, can be obtained.

  13. Algal autolysate medium to label proteins for NMR in mammalian cells.

    Science.gov (United States)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  14. Algal autolysate medium to label proteins for NMR in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia [University of Florence, Magnetic Resonance Center (CERM) (Italy); Neri, Sara [Giotto Biotech S.R.L. (Italy); Fragai, Marco, E-mail: fragai@cerm.unifi.it [University of Florence, Magnetic Resonance Center (CERM) (Italy)

    2016-04-15

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in {sup 15}N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  15. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

    Directory of Open Access Journals (Sweden)

    Ying Luo

    Full Text Available The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.

  16. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

    Science.gov (United States)

    Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

    2015-01-01

    The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.

  17. Diploid yeast cells yield homozygous spontaneous mutations

    Science.gov (United States)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  18. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    DNA synthesis is one of the landmark events in the cell cycle: G 1 cells have one copy of the genome, S phase cells are actively engaged in DNA synthesis, and G 2 cells have twice as much nuclear DNA as G 1 cells. Cellular DNA content can be measured by staining with a fluorescent dye followed by a flow-cytometric readout. This method provides a quantitative measurement of cell cycle position on a cell-by-cell basis at high speed. Using flow cytometry, tens of thousands of single-cell measurements can be generated in a few seconds. This protocol details staining of cells of the budding yeast Saccharomyces cerevisiae for flow cytometry using Sytox Green dye in a method that can be scaled widely-from one sample to many thousands and operating on inputs ranging from 1 million to more than 100 million cells. Flow cytometry is preferred over light microscopy or Coulter analyses for the analysis of the cell cycle as DNA content and cell cycle position are being directly measured. © 2017 Cold Spring Harbor Laboratory Press.

  19. Research of aquatic organism addition influence on the reproduction of yeast cells in the dough

    Directory of Open Access Journals (Sweden)

    Дмитро Павлович Крамаренко

    2016-12-01

    Full Text Available The analysis of the research results of influence of various amounts of aquatic organism additions on the reproduction of yeast cells is given. A positive impact of aquatic organism addition of animal and plant origin in investigated quantities on the reproduction of yeast cells is revealed. The influence of the chemical composition of the aquatic organism additives on the reproduction of yeast cells is proved

  20. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

    2014-03-03

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

  1. Heavy ion effects on mammalian cells: Inactivation measurements with different cell lines

    International Nuclear Information System (INIS)

    Wulf, H.; Kraft-Weyrather, W.; Miltenburger, H.G.; Kraft, G.

    1985-07-01

    In track segment experiments, the inactivation of different mammalian cells by heavy charged particles between helium and uranium in the energy range between 1 and 1000 MeV/u has been measured at the heavy ion accelerator Unilac, Darmstadt, the Tandem Van de Graaf, Heidelberg and the Bevalac, Berkeley. The inactivation cross sections calculated from the final slope of the dose effect curves are given as a function of the particle energy and the LET. (orig.)

  2. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells.

    Science.gov (United States)

    Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou

    2015-07-01

    Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. © 2015 Wiley Periodicals, Inc.

  3. Binding kinetics of magnetic nanoparticles on latex beads and yeast cells studied by magnetorelaxometry

    International Nuclear Information System (INIS)

    Eberbeck, Dietmar; Bergemann, Christian; Hartwig, Stefan; Steinhoff, Uwe; Trahms, Lutz

    2005-01-01

    The ion exchange mediated binding of magnetic nanoparticles (MNP) to modified latex spheres and yeast cells was quantified using magnetorelaxometry. By fitting subsequently recorded relaxation curves, the kinetics of the binding reactions was extracted. The signal of MNP with weak ion exchanger groups bound to latex and yeast cells scales linearly with the concentration of latex beads or yeast cells whereas that of MNP with strong ion exchanger groups is proportional to the square root of concentration. The binding of the latter leads to a much stronger aggregation of yeast cells than the former MNP

  4. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  5. Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

    Energy Technology Data Exchange (ETDEWEB)

    Jetzt, Amanda E. [Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8520 (United States); Li, Xiao-Ping; Tumer, Nilgun E. [Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8520 (United States); Cohick, Wendie S., E-mail: cohick@aesop.rutgers.edu [Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8520 (United States)

    2016-11-01

    Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. - Highlights: • Arginines 193 and 235 of RTA are critical for binding to the mammalian ribosome. • R193A/R235A has full catalytic activity on RNA but not on mammalian ribosomes. • R193A/R235A is less toxic than a mutant that targets the active site. • The toxin-ribosome interaction is a therapeutic target for ricin intoxication.

  6. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  7. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    International Nuclear Information System (INIS)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

    2011-01-01

    Research highlights: → We develop a method to track a quantum dot-conjugated protein in yeast cells. → We incorporate the conjugated quantum dot proteins into yeast spheroplasts. → We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  8. AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM BY ION-EXCHANGE MEMBRANES

    Science.gov (United States)

    Metabolites such as ammonia and lactic acid formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. Cell culture conducted in the presence of such accumulated metabolites is therefore limited in pro...

  9. Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein

    OpenAIRE

    Han, Seong-Jeong; Lee, Byung Cheon; Yim, Sun Hee; Gladyshev, Vadim N.; Lee, Seung-Rock

    2014-01-01

    Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells ...

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

    Directory of Open Access Journals (Sweden)

    Mazel-Sanchez Beryl

    2010-03-01

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

  11. Possibility for simultaneous electricity generation and bioremediation by using Candida melibiosica yeast in biofuel cell

    International Nuclear Information System (INIS)

    Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

    2013-01-01

    Recently, we have proved that Candida melibiosica 2491 yeast strain possesses electrogenic properties and could be used as a biocatalyst in yeast-based biofuel cells. In this paper we demonstrate that when the yeast is cultivated under polarization conditions in a biofuel cell its phytase activity exceeds that obtained during cultivation in a conventional bioreactor. Furthermore, there is a correlation between the yeast phytase activity and the electrical characteristic of the biofuel cell during the different yeast growth phases. The obtained results reveal a possibility for application of C.melibiosica for simultaneous electricity generation and bioremediation of hardly degradable polyphosphates, especially in the regions with intensive stock-farming. Keywords: Biofuel cells, yeast, Candida melibiosica, electricity generation, bioremediation

  12. Synergistic reduction of toluylene blue induced by acetaldehyde and menadione in yeast cell suspension: Application to determination of yeast cell activity

    Directory of Open Access Journals (Sweden)

    Shiro Yamashoji

    2017-03-01

    Full Text Available Membrane permeant acetaldehyde and menadione induced the synergistic reduction of toluylene blue (TB acting as non-membrane permeant redox indicator in yeast cell suspension. NADH and acetaldehyde also induced the synergistic TB reduction in permeabilized yeast cells and phosphate buffer, but menadione had no ability to promote TB reduction. The pre-incubation of acetaldehyde inhibited the above synergistic reduction of TB in intact and permeabilized yeast cell suspension. The pre-incubation of acetaldehyde might promote NADH oxidation by alcohol dehydrogenase, because acetaldehyde decreased the intracellular NAD(PH concentration. The above facts indicate that the synergistic reduction of TB is controlled by the order of addition of menadione and acetaldehyde. The synergistic reduction of TB by menadione and acetaldehyde was proportional to viable yeast cell number from 104 to 2×106 cells/ml, and this assay was applicable to cytotoxicity test. The time required for the above assay was only 2 min.

  13. Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope

    International Nuclear Information System (INIS)

    De, Tathagata; Chettoor, Antony M.; Agarwal, Pranav; Salapaka, Murti V.; Nettikadan, Saju

    2010-01-01

    The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells (Kasas and Ikai, 1995), (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.

  14. Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties

    International Nuclear Information System (INIS)

    Busch, Wibke; Bastian, Susanne; Trahorsch, Ulrike; Iwe, Maria; Kühnel, Dana; Meißner, Tobias; Springer, Armin; Gelinsky, Michael; Richter, Volkmar; Ikonomidou, Chrysanthy; Potthoff, Annegret; Lehmann, Irina; Schirmer, Kristin

    2011-01-01

    Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells.

  15. Analytical model for macromolecular partitioning during yeast cell division

    International Nuclear Information System (INIS)

    Kinkhabwala, Ali; Khmelinskii, Anton; Knop, Michael

    2014-01-01

    Asymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell — which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles — can be achieved by both passive (e.g. diffusion, localized retention sites) and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate. Here we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports. In contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning

  16. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    Directory of Open Access Journals (Sweden)

    Ida M Smith

    Full Text Available Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications

  17. Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

    Science.gov (United States)

    Smith, Ida M.; Christensen, Jeffrey E.; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  18. Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State

    Directory of Open Access Journals (Sweden)

    Corsini Joe

    2004-01-01

    Full Text Available Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K, we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

  19. Factors affecting the spontaneous mutational spectra in somatic mammalian cells

    Directory of Open Access Journals (Sweden)

    О.А. Ковальова

    2006-04-01

    Full Text Available  In our survey of references we are discussed the influence of factors biological origin on the spontaneous mutation specters in mammalian. Seasonal and age components influence on the frequence of cytogenetic anomalies. The immune and endocrinous systems are take part in control of the alteration of the spontaneous mutation specters. Genetical difference of sensibility in animal and human at the alteration of factors enviroment as and  genetical differences of repair systems activity are may influence on individual variation of spontaneous destabilization characters of chromosomal apparatus.

  20. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

    Science.gov (United States)

    Levine, Zebulon G; Walker, Suzanne

    2016-06-02

    O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.

  1. Hydrothermal decomposition of yeast cells for production of proteins and amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Lamoolphak, Wiwat [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Goto, Motonobu [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Sasaki, Mitsuru [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Suphantharika, Manop [Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400 (Thailand); Muangnapoh, Chirakarn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Prommuag, Chattip [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Shotipruk, Artiwan [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand)]. E-mail: artiwan.s@chula.ac.th

    2006-10-11

    This study examines hydrothermal decomposition of Baker's yeast cells, used as a model for spent Brewer's yeast waste, into protein and amino acids. The reaction was carried out in a closed batch reactor at various temperatures between 100 and 250 deg. C. The reaction products were separated into water-soluble and solid residue. The results demonstrated that the amount of yeast residue decreased with increasing hydrolysis temperature. After 20 min reaction in water at 250 deg. C, 78% of yeast was decomposed. The highest amount of protein produced was also obtained at this condition and was found to be 0.16 mg/mg dry yeast. The highest amount of amino acids (0.063 mg/mg dry yeast) was found at the lowest temperature tested after 15 min. The hydrolysis product obtained at 200 deg. C was tested as a nutrient source for yeast growth. The growth of yeast cells in the culture medium containing 2 w/v% of this product was comparable to that of the cells grown in the medium containing commercial yeast extract at the same concentration. These results demonstrated the feasibility of using subcritical water to potentially decompose proteinaceous waste such as spent Brewer's yeast while recovering more useful products.

  2. Hydrothermal decomposition of yeast cells for production of proteins and amino acids

    International Nuclear Information System (INIS)

    Lamoolphak, Wiwat; Goto, Motonobu; Sasaki, Mitsuru; Suphantharika, Manop; Muangnapoh, Chirakarn; Prommuag, Chattip; Shotipruk, Artiwan

    2006-01-01

    This study examines hydrothermal decomposition of Baker's yeast cells, used as a model for spent Brewer's yeast waste, into protein and amino acids. The reaction was carried out in a closed batch reactor at various temperatures between 100 and 250 deg. C. The reaction products were separated into water-soluble and solid residue. The results demonstrated that the amount of yeast residue decreased with increasing hydrolysis temperature. After 20 min reaction in water at 250 deg. C, 78% of yeast was decomposed. The highest amount of protein produced was also obtained at this condition and was found to be 0.16 mg/mg dry yeast. The highest amount of amino acids (0.063 mg/mg dry yeast) was found at the lowest temperature tested after 15 min. The hydrolysis product obtained at 200 deg. C was tested as a nutrient source for yeast growth. The growth of yeast cells in the culture medium containing 2 w/v% of this product was comparable to that of the cells grown in the medium containing commercial yeast extract at the same concentration. These results demonstrated the feasibility of using subcritical water to potentially decompose proteinaceous waste such as spent Brewer's yeast while recovering more useful products

  3. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  4. Electroporation of Mammalian Cells by Nanosecond Electric Field Oscillations and its Inhibition by the Electric Field Reversal

    Science.gov (United States)

    2015-09-08

    Report 3. DATES COVERED (From – To) March 2013 to July 2015 4. TITLE AND SUBTITLE Electroporation of mammalian cells by nanosecond electric field...Prescribed by ANSI Std. Z39.18 1Scientific RepoRts | 5:13818 | DOi: 10.1038/srep13818 www.nature.com/scientificreports Electroporation of mammalian cells...first to demonstrate that mammalian cells can be electroporated by damped sine wave electric stimuli of nanosecond duration. By comparing the

  5. Host cell reactivation and UV-enhanced reactivation in synchronized mammalian cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Schmidt, B.J.

    1981-01-01

    Does host cell reactivation (HCR) or UV-enhanced reactivation (UVER) of UV-irradiated Herpes simplex virus (UV-HSV) vary during the host mammalian cell cycle. The answer could be useful for interpreting UVER and or the two-component nature of the UV-HSV survival curve. Procedures were developed for infection of mitotically-synchronized CV-l monkey kidney cells. All virus survival curves determined at different cell cycle stages had two components with similar D 0 's and intercepts of the second components. Thus, no single stage of the host cell cycle was responsible for the second component of the virus survival curve. When the cells were UV-irradiated immediately prior to infection, enhanced survival of UV-HSV occurred for cell irradiation and virus infection initiated during late G 1 early S phase or late S early G 2 phase but not during early G 1 phase. For infection delayed by 24 h after cell irradiation, UVER was found at all investigated times. These results indicate that: (1) HCR is similar at all stages of the host cell cycle: and (2) the ''induction'' of UVER is not as rapid for cell-irradiation in early G 1 phase. This latter observation may be one reason why normal, contact-inhibited cells do not express UVER as rapidly as faster growing, less contact-inhibited cells. (author)

  6. Mammalian cell transformation: Mechanisms of carcinogenesis and assays for carcinogens

    International Nuclear Information System (INIS)

    Barrett, J.C.; Tennant, R.W.

    1985-01-01

    This book contains nine sections, each consisting of several papers. The section titles are: Molecular Changes in Cell Transformation; Differentiation, Growth Control, and Cell Transformation; Mutagenesis and Cell Transformation; Tumor Promotion and Cell Transformation; Mechanisms of Transformation of Human Fibroblasts; Mechanisms of Transformation of Epithelial Cells; Mechanisms of C 3 H 10T12 Cell Transformation; Mechanisms of Radiation-Induced Cell Transformation; and Use of Cell Transformation Assays for Carcinogen Testing

  7. Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

    Science.gov (United States)

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-02-01

    In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  8. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K

    2002-01-01

    Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein wil...

  9. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær

    2014-01-01

    , in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors....

  10. Wnt/β-catenin signaling in adult mammalian epithelial stem cells

    NARCIS (Netherlands)

    Kretzschmar, Kai; Clevers, Hans

    2017-01-01

    Adult stem cells self-renew and replenish differentiated cells in various organs and tissues throughout a mammal's life. Over the last 25 years an ever-growing body of knowledge has unraveled the essential regulation of adult mammalian epithelia by the canonical Wnt signaling with its key

  11. Exposure of Mammalian Cells to Air-Pollutant Mixtures at the Air-Liquid Interface

    Science.gov (United States)

    It has been widely accepted that exposure of mammalian cells to air-pollutant mixtures at the air-liquid interface is a more realistic approach than exposing cell under submerged conditions. The VITROCELL systems, are commercially available systems for air-liquid interface expo...

  12. A Novel Counter Sheet-flow Sandwich Cell Culture Device for Mammalian Cell Growth in Space

    Science.gov (United States)

    Sun, Shujin; Gao, Yuxin; Shu, Nanjiang; Tang, Zemei; Tao, Zulai; Long, Mian

    2008-08-01

    Cell culture and growth in space is crucial to understand the cellular responses under microgravity. The effects of microgravity were coupled with such environment restrictions as medium perfusion, in which the underlying mechanism has been poorly understood. In the present work, a customer-made counter sheet-flow sandwich cell culture device was developed upon a biomechanical concept from fish gill breathing. The sandwich culture unit consists of two side chambers where the medium flow is counter-directional, a central chamber where the cells are cultured, and two porous polycarbonate membranes between side and central chambers. Flow dynamics analysis revealed the symmetrical velocity profile and uniform low shear rate distribution of flowing medium inside the central culture chamber, which promotes sufficient mass transport and nutrient supply for mammalian cell growth. An on-orbit experiment performed on a recovery satellite was used to validate the availability of the device.

  13. Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

    International Nuclear Information System (INIS)

    Sifontes, Ángela B.; González, Gema; Tovar, Leidy M.; Méndez, Franklin J.; Gomes, Maria E.; Cañizales, Edgar; Niño-Vega, Gustavo; Villalobos, Hector; Brito, Joaquin L.

    2013-01-01

    Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m 2 g −1 . -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N 2 adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity, respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m 2 g −1 .

  14. Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

    Energy Technology Data Exchange (ETDEWEB)

    Sifontes, Ángela B., E-mail: asifonte@ivic.gob.ve [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); González, Gema [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Tovar, Leidy M.; Méndez, Franklin J. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Gomes, Maria E. [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Cañizales, Edgar [Área de Análisis Químico Inorgánico, PDVSA, INTEVEP, Los Teques 1070-A (Venezuela, Bolivarian Republic of); Niño-Vega, Gustavo; Villalobos, Hector [Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Brito, Joaquin L. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    2013-02-15

    Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m{sup 2} g{sup −1}. -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N{sub 2} adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity, respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m{sup 2} g{sup −1}.

  15. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.

    Science.gov (United States)

    Aricescu, A Radu; Owens, Raymond J

    2013-06-01

    Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. The use of a cloned bacterial gene to study mutation in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.; Debenham, P.G.; Stretch, A.; Webb, M.B.T.

    1983-01-01

    The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. (orig./AJ)

  17. Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

    Science.gov (United States)

    Moreno-García, J; Coi, A L; Zara, G; García-Martínez, T; Mauricio, J C; Budroni, M

    2018-03-01

    Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

  18. The different shades of mammalian pluripotent stem cells

    NARCIS (Netherlands)

    Kuijk, E.W.; Lopes, S.M.; Geijsen, N.; Macklon, N.; Roelen, B.A.

    2011-01-01

    BACKGROUND: Pluripotent stem cells have been derived from a variety of sources such as from the inner cell mass of preimplantation embryos, from primordial germ cells, from teratocarcinomas and from male germ cells. The recent development of induced pluripotent stem cells demonstrates that somatic

  19. Radiation equivalence of genotoxic chemicals - Validation in cultered mammalian cell lines

    International Nuclear Information System (INIS)

    Murthy, M.S.S.

    1982-01-01

    Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated. REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human. (orig.)

  20. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  1. Novel and improved yeast cell factories for biosustainable processes

    DEFF Research Database (Denmark)

    Workman, Mhairi

    2014-01-01

    The utilization of an increasingly diverse range of cheap waste substrates will be an ongoing challenge for the bio-based economy, where the mobilization of nutrients from a variety of waste products will be necessary for realization of biosustainability on an industrial scale. Bioprocesses....... In addition to plant biomass hydrolysates, glycerol is of interest here, being available in amounts relevant for industrial scale bioprocesses due to increased production of biodiesel. The well characterised cell factory Saccharomyces cerevisiae exhibits a clear preference for glucose as a carbon source......, and is highly adapted to its utilisation. Although there have been several studies on glycerol metabolism in S. cerevisiae, many industrially used strains grow poorly on glycerol (μmax = 0.01h-1). On the other hand, several non-conventional yeast species are efficient in utilization of glycerol, some...

  2. Apple Can Act as Anti-Aging on Yeast Cells

    Directory of Open Access Journals (Sweden)

    Vanessa Palermo

    2012-01-01

    Full Text Available In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrence of cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such as ascorbic acid (vitamin C and polyphenols. The substances that hinder the presence of free radicals are also able to protect cells from aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples and their components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate that all the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all apple components.

  3. Arctigenin from Fructus Arctii is a novel suppressor of heat shock response in mammalian cells

    Science.gov (United States)

    Ishihara, Keiichi; Yamagishi, Nobuyuki; Saito, Youhei; Takasaki, Midori; Konoshima, Takao; Hatayama, Takumi

    2006-01-01

    Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoter-regulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy. PMID:16817321

  4. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S; Polizzi, Karen M

    2017-06-01

    Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  5. Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S.

    2017-01-01

    ABSTRACT Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28112405

  6. Differential effect of procaine on irradiated mammalian cells in culture

    International Nuclear Information System (INIS)

    Djordjevic, B.

    1979-01-01

    HeLa and V-79 Chinese hamster cells temporarily stored in ampoules were treated with the local anesthetic procaine. Postirradiation treatment increased lethality in HeLa cells depending on drug concentration, duration of treatment, and cell density, as measured by colony-forming ability upon plating. If present during irradiation only, procaine protected from irradiation. In V-79 cells, procaine potentiated radiation lethality only in freshly trypsinized cells. Procaine effect was thus cell type specific and most likely involved the cell membrane

  7. Electricity production from microbial fuel cell by using yeast

    International Nuclear Information System (INIS)

    Vorasingha, A.; Souvakon, C.; Boonchom, K.

    2006-01-01

    The continuous search for methods to generate electricity from renewable sources such as water, solar energy, wind, nuclear or chemicals was discussed with particular focus on attaining the full power of the microbial fuel cell (MFC). Under ideal environmental conditions, the only byproducts of a biofuel cell would be water and carbon dioxide (CO 2 ). The production of energy from renewables such as biomass is important for sustainable development and reducing global emissions of CO 2 . Hydrogen can also be an important component of an energy infrastructure that reduces CO 2 emissions if the hydrogen is produced from renewable sources and used in fuel cells. Hydrogen gas can be biologically produced at high concentration from the fermentation of high sugar substrates such as glucose and sucrose. Some of the issues of MFC design were addressed, including the use of cheap substrates to derive microbial electricity. In the MFC, yeast donates electrons to a chemical electron mediator, which in turn transfers the electrons to an electrode, producing electricity. Experimental results showed that glucose yielded the highest peak voltage, but a semi-processed sugar and molasses were similar to glucose in the electricity production pattern. It was noted that this technology is only at the research stages, and more research is needed before household microbial fuel cells can be made available for producing power for prolonged periods of time. Future research efforts will focus on increasing the efficiency, finding alternatives to hazardous electron mediators and finding new microbes. 12 refs., 6 figs

  8. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  9. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

    Science.gov (United States)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  10. Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration

    International Nuclear Information System (INIS)

    Guelden, Michael; Moerchel, Sabine; Seibert, Hasso

    2005-01-01

    Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC 50 values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC 50 values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC 50 values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity

  11. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

    Science.gov (United States)

    Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

    2011-07-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  12. Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

    Science.gov (United States)

    Bi, Xiaobao; Yin, Juan; Chen Guanbang, Ashley; Liu, Chuan-Fa

    2018-06-07

    The cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Laser flow microphotometry for rapid analysis and sorting of mammalian cells

    International Nuclear Information System (INIS)

    Mullaney, P.F.; Steinkamp, J.A.; Crissman, H.A.; Cram, L.S.; Crowell, J.M.; Salzman, G.C.; Martin, J.C.; Price, B.

    1976-01-01

    Quantitative precision measurements can be made of the optical properties of individual mammalian cells using flow microphotometry. Suspended cells pass through a special flow chamber where they are lined up for exposure to blue light from an argon-ion laser. As each cell crosses the laser beam, it produces one or more optical pulses of a duration equal to cell transit time across the beam. These pulses are detected, amplified, and analyzed using the techniques of gamma ray spectroscopy. Quantitative DNA distributions made it possible to distinguish tumor cells from normal cells as well as to assay for radiation effects on tumor cells subjected to x and gamma radiation

  14. Laser flow microphotometry for rapid analysis and sorting of mammalian cells. [X and gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Mullaney, P.F.; Steinkamp, J.A.; Crissman, H.A.; Cram, L.S.; Crowell, J.M.; Salzman, G.C.; Martin, J.C.; Price, B.

    1976-01-01

    Quantitative precision measurements can be made of the optical properties of individual mammalian cells using flow microphotometry. Suspended cells pass through a special flow chamber where they are lined up for exposure to blue light from an argon-ion laser. As each cell crosses the laser beam, it produces one or more optical pulses of a duration equal to cell transit time across the beam. These pulses are detected, amplified, and analyzed using the techniques of gamma ray spectroscopy. Quantitative DNA distributions made it possible to distinguish tumor cells from normal cells as well as to assay for radiation effects on tumor cells subjected to x and gamma radiation. (HLW)

  15. Bystander effects on mammalian cells induced by carbon ions

    International Nuclear Information System (INIS)

    Wang Jufang; Zhao Jing; Ma Qiufeng; Chinese Academy of Sciences, Beijing; Li Weijian; Zhou Guangming; Dang Bingrong; Mao Limin; Feng Yan

    2004-01-01

    Bystander effects on unirradiated V79 cells were observed by irradiated conditioned medium (ICM) method and co-cultured with carbon-ion-irradiated V79 cells. The results showed that the colony formation efficiency of unirradiated cells is obviously decreased by ICM. After co-culture with carbon-ion-irradiated cells for some time, the colony formation efficiency of co-cultured cells was lower than expected results assuming no bystander effects. The micronucleus frequency and hprt gene mutation rate was almost the same as expected results. Cytotoxic factor(s), which was effective for cell growth but not for micronucleus and mutation on unirradiated cells, might be released by irradiated cells. (authors)

  16. Lack of radiation protective effect of orgotein in normal and malignant mammalian cells

    International Nuclear Information System (INIS)

    Overgaard, J.; Nielsen, O.S.; Overgaard, M.; Steenholdt, S.; Jakobsen, A.; Sell, A.

    1979-01-01

    The potential radiation protective effect of orgotein, a metalloprotein with superoxide dismutase activity, was investigated in L 1 A 2 tumour cells in vitro, jejunal crypt cells and C 3 H mouse mammary carcinoma in vivo. No effect of orgotein, given either 2 hours before irradiation or 30 min after, was observed compared to the effect of irradiation alone. Thus, it was concluded that orgotein did not influence the primary radiation response in air in mammalian cells. (Auth.)

  17. Lack of radiation protective effect of orgotein in normal and malignant mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Overgaard, J; Nielsen, O S; Overgaard, M; Steenholdt, S; Jakobsen, A; Sell, A [Institute of Cancer Research and The Department of Radiation Therapy and Oncology, The Radium Centre, Aarhus, Denmark

    1979-01-01

    The potential radiation protective effect of orgotein, a metalloprotein with superoxide dismutase activity, was investigated in L/sub 1/A/sub 2/ tumour cells in vitro, jejunal crypt cells and C/sub 3/H mouse mammary carcinoma in vivo. No effect of orgotein, given either 2 hours before irradiation or 30 min after, was observed compared to the effect of irradiation alone. Thus, it was concluded that orgotein did not influence the primary radiation response in air in mammalian cells.

  18. Repetitious nature of repaired DNA in mammalian cells

    International Nuclear Information System (INIS)

    1978-01-01

    The report consists of three appendices, as follows: summary of preliminary studies of the comparative DNA repair in normal lymphoblastoid and Burkitt's lymphoma cell lines; nonuniform reassociation of human lymphoblastoid cell DNA repair replicated following methyl methane sulfonate treatment; and preliminary DNA single-strand breakage studies in the L5178Y cell line

  19. Binucleate cell formation correlates to loss of colony-forming ability in X-irradiated cultured mammalian cells

    International Nuclear Information System (INIS)

    Sasaki, H.; Yoshinaga, H.; Kura, S.

    1986-01-01

    The relationship between binucleate cell formation and the loss of colony-forming ability was examined in several cultured mammalian cell lines irradiated with X rays. The maximum fraction of binucleate cells after X irradiation increased dose-dependently within the range in which reproductive cell death might predominate over interphase cell death. When the logarithm of percentage survival was plotted against the percentage binucleate cells, a similar correlation was found for all cell lines tested, with the exception of mouse leukemia L5178Y cells, the most radiosensitive cells used. These observations suggest that the fraction of binucleate cells in the cell population can serve as a measure of cellular radiation damage

  20. Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

    Science.gov (United States)

    Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

    2017-09-15

    Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

  1. Effects of metal salt catalysts on yeast cell growth in ethanol conversion

    Science.gov (United States)

    Chung-Yun Hse; Yin Lin

    2009-01-01

    The effects of the addition of metal salts and metal salt-catalyzed hydrolyzates on yeast cell growth in ethanol fermentation were investigated. Four yeast strains (Saccharomyces cerevisiae WT1, Saccharomyces cerevisiae MT81, Candida sp. 1779, and Klumaromyces fragilis), four metal salts (CuCl2, FeCl3, AgNO3, and I2), two metal salt-catalyzed hydrolyzates (...

  2. Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Wloch-Salamon, D.M.; Bem, A.E.

    2013-01-01

    The occurrence of programmed cell death in unicellular organisms is a subject that arouses great interest of theoreticians and experimental scientists. Already found evolutionarily conserved genes and metabolic pathways confirmed its existence in yeast, protozoa and even bacteria. In the yeast

  3. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung, E-mail: keejung@skku.edu

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  4. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    International Nuclear Information System (INIS)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

    2015-01-01

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics

  5. A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram

    The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a new...

  6. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  7. TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

    Science.gov (United States)

    Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

    2014-01-24

    Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Recent topics on the effect of high LET radiation on microorganisms and cultured mammalian cells

    International Nuclear Information System (INIS)

    Takahashi, Tan; Nakano, Kazushiro; Yatagai, Fumio; Kaneko, Ichiro; Kosaka, Toshifumi; Kasai, Kiyomi.

    1989-01-01

    Interpretation of inactivation cross sections of E. coli K-12 JC 1553 and AB 1157 by track structure of heavy ions and recent topics on the effect of heavy ions on mammalian cells are described. Calculation of the dose around the trajectory of an ion has also been made and the radial dose distribution has been compared with a recent experiment. (author)

  9. Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins

    DEFF Research Database (Denmark)

    Rosengren, K Johan; Daly, Norelle L; Fornander, Liselotte M

    2006-01-01

    alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-C...

  10. Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

    NARCIS (Netherlands)

    Michels, AA; Kanon, B; Bensaude, O; Kampinga, HH

    1999-01-01

    Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272,

  11. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    Directory of Open Access Journals (Sweden)

    Anne Mathilde Lund

    Full Text Available A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

  12. Study on immobilized yeast cells with hydrophilic polymer carrier by radiation-induced copolymerization

    International Nuclear Information System (INIS)

    Li Zhengkui; Zhang Bosen

    1993-01-01

    Various kinds of monomers 2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl acrylate (HEA), hydroxypropyl methacrylate (HPMA) and methoxy polyethylene glycol methylacrylate (M-23G) are copolymerized by radiation technique at low temperature (-78 degree C) and several kinds of copolymer carriers were obtained. Yeast cells are immobilized through adhesion and multiplication of yeast cells themselves on these carriers. The ethanol productivity of immobilized yeast cells with these carriers was related to the monomer composition and water content of copolymer carriers and the optimum monomer composition was 20%:10% in poly (HEA-M23G). In this case, the ethanol productivity of immobilized yeast cells was 26 mg/(ml · h), which was 4 times as high as that of free cells. Effect of adding crosslinking reagent (4G) in lower monomer composition of poly(HEA-M23G) on the ethanol productivity of immobilized cells was better than that in higher one in this work

  13. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    International Nuclear Information System (INIS)

    Skelton, David; Goodyear, Abbey; Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T.; Logan, Timothy M.

    2010-01-01

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U- 13 C-glucose and 15 N-glutamate as labeled precursors. This study suggests that uniformly 15 N, 13 C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

  14. Golgi structure formation, function, and post-translational modifications in mammalian cells.

    Science.gov (United States)

    Huang, Shijiao; Wang, Yanzhuang

    2017-01-01

    The Golgi apparatus is a central membrane organelle for trafficking and post-translational modifications of proteins and lipids in cells. In mammalian cells, it is organized in the form of stacks of tightly aligned flattened cisternae, and dozens of stacks are often linked laterally into a ribbon-like structure located in the perinuclear region of the cell. Proper Golgi functionality requires an intact architecture, yet Golgi structure is dynamically regulated during the cell cycle and under disease conditions. In this review, we summarize our current understanding of the relationship between Golgi structure formation, function, and regulation, with focus on how post-translational modifications including phosphorylation and ubiquitination regulate Golgi structure and on how Golgi unstacking affects its functions, in particular, protein trafficking, glycosylation, and sorting in mammalian cells.

  15. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

    2010-10-15

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

  16. Cell thickness of UV absorption by the cell: relation to UV action spectrum shift in mammalian cells in culture

    International Nuclear Information System (INIS)

    Sakharov, V.H.; Voronkova, L.N.; Blokhin, A.V.

    1985-01-01

    By means of reconstruction of series half - thin transverse sections the three - dimensional morphometry of SPEV cells for a series of their specific states in culture is performed: for exponential growth in a monolayer, in a merged monolayer, in the mitosis phase, for giant cells and suspension cells. In the monolayer the cell thickness in its central part depended mainly on the nucleus thickness and in average changed but slightly despite a wide range of changes in volumes of nuclei and cells and their density in culture. The cell thickness has noticeably increased in mitosis. For the above states of cells UV radiation absorption spectra are determined. It is shown that a certain shift of action spectrus of death of mammalian cells as compared with that for bacterial cell can be a seguence of selfshielding and not differences in the nature of active chromophores

  17. Genomic footprinting in mammalian cells with ultraviolet light

    International Nuclear Information System (INIS)

    Becker, M.M.; Wang, Z.; Grossmann, G.; Becherer, K.A.

    1989-01-01

    A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography

  18. Toxicity of Aromatic Ketone to Yeast Cell and Improvement of the Asymmetric Reduction of Aromatic Ketone Catalyzed by Yeast Cell with the Introduction of Resin Adsorption

    Directory of Open Access Journals (Sweden)

    Zhong-Hua Yang

    2008-01-01

    Full Text Available Asymmetric reduction of the prochiral aromatic ketone catalyzed by yeast cells is one of the most promising routes to produce its corresponding enantiopure aromatic alcohol, but the space-time yield does not meet people’s expectations. Therefore, the toxicity of aromatic ketone and aromatic alcohol to the yeast cell is investigated in this work. It has been found that the aromatic compounds are poisonous to the yeast cell. The activity of yeast cell decreases steeply when the concentration of acetophenone (ACP is higher than 30.0 mmol/L. Asymmetric reduction of acetophenone to chiral S-α-phenylethyl alcohol (PEA catalyzed by the yeast cell was chosen as the model reaction to study in detail the promotion effect of the introduction of the resin adsorption on the asymmetric reduction reaction. The resin acts as the substrate reservoir and product extraction agent in situ. It has been shown that this reaction could be remarkably improved with this technique when the appropriate kind of resin is applied. The enantioselectivity and yield are acceptable even though the initial ACP concentration reaches 72.2 mmol/L.

  19. Quantification of mammalian tumor cell state plasticity with digital holographic cytometry

    Science.gov (United States)

    Hejna, Miroslav; Jorapur, Aparna; Zhang, Yuntian; Song, Jun S.; Judson, Robert L.

    2018-02-01

    Individual cells within isogenic tumor populations can exhibit distinct cellular morphologies, behaviors, and molecular profiles. Cell state plasticity refers to the propensity of a cell to transition between these different morphologies and behaviors. Elevation of cell state plasticity is thought to contribute to critical stages in tumor evolution, including metastatic dissemination and acquisition of therapeutic resistance. However, methods for quantifying general plasticity in mammalian cells remain limited. Working with a HoloMonitor M4 digital holographic cytometry platform, we have established a machine learning-based pipeline for high accuracy and label-free classification of adherent cells. We use twenty-six morphological and optical density-derived features for label-free identification of cell state in heterogeneous cultures. The system is housed completely within a mammalian cell incubator, permitting the monitoring of changes in cell state over time. Here we present an application of our approach for studying cell state plasticity. Human melanoma cell lines of known metastatic potential were monitored in standard growth conditions. The rate of feature change was quantified for each individual cell in the populations. We observed that cells of higher metastatic potential exhibited more rapid fluctuation of cell state in homeostatic conditions. The approach we demonstrate will be advantageous for further investigations into the factors that influence cell state plasticity.

  20. Plasticity within stem cell hierarchies in mammalian epithelia

    NARCIS (Netherlands)

    Tetteh, Paul W; Farin, Henner F; Clevers, Hans

    Tissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur

  1. Repair processes for photochemical damage in mammalian cells

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1974-01-01

    Repair processes for photochemical damage in cells following uv irradiation are reviewed. Cultured fibroblast cells from human patients with xeroderma pigmentosum were used as an example to illustrate aspects of repair of injuries to DNA and proteins. (250 references) (U.S.)

  2. Mechanically Gated Ion Channels in Mammalian Hair Cells

    Directory of Open Access Journals (Sweden)

    Xufeng Qiu

    2018-04-01

    Full Text Available Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS/LHFPL5, transmembrane inner ear (TMIE and transmembrane channel-like proteins 1 and 2 (TMC1/2. However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function.

  3. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  4. Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

    Science.gov (United States)

    Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

    2018-02-08

    Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

  5. How do yeast cells become tolerant to high ethanol concentrations?

    DEFF Research Database (Denmark)

    Snoek, Tim; Verstrepen, Kevin J.; Voordeckers, Karin

    2016-01-01

    The brewer’s yeast Saccharomyces cerevisiae displays a much higher ethanol tolerance compared to most other organisms, and it is therefore commonly used for the industrial production of bioethanol and alcoholic beverages. However, the genetic determinants underlying this yeast’s exceptional ethanol...... and challenges involved in obtaining superior industrial yeasts with improved ethanol tolerance....

  6. InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

    Science.gov (United States)

    Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

    2007-10-15

    Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

  7. Timing robustness in the budding and fission yeast cell cycles.

    KAUST Repository

    Mangla, Karan

    2010-02-01

    Robustness of biological models has emerged as an important principle in systems biology. Many past analyses of Boolean models update all pending changes in signals simultaneously (i.e., synchronously), making it impossible to consider robustness to variations in timing that result from noise and different environmental conditions. We checked previously published mathematical models of the cell cycles of budding and fission yeast for robustness to timing variations by constructing Boolean models and analyzing them using model-checking software for the property of speed independence. Surprisingly, the models are nearly, but not totally, speed-independent. In some cases, examination of timing problems discovered in the analysis exposes apparent inaccuracies in the model. Biologically justified revisions to the model eliminate the timing problems. Furthermore, in silico random mutations in the regulatory interactions of a speed-independent Boolean model are shown to be unlikely to preserve speed independence, even in models that are otherwise functional, providing evidence for selection pressure to maintain timing robustness. Multiple cell cycle models exhibit strong robustness to timing variation, apparently due to evolutionary pressure. Thus, timing robustness can be a basis for generating testable hypotheses and can focus attention on aspects of a model that may need refinement.

  8. Use of non-conventional cell disruption method for extraction of proteins from black yeasts

    Directory of Open Access Journals (Sweden)

    Maja eLeitgeb

    2016-04-01

    Full Text Available The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2 by varying pressure at fixed temperature (35 °C. The black yeasts cell walls were disrupted and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV-Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2 treated cells. The advantages of the proposed method are in a simple use which is also possible for heat sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions.

  9. Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

    Science.gov (United States)

    Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

    2016-01-01

    The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

  10. Cell dualism: presence of cells with alternative membrane potentials in growing populations of bacteria and yeasts.

    Science.gov (United States)

    Ivanov, Volodymyr; Rezaeinejad, Saeid; Chu, Jian

    2013-10-01

    It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ⁻-cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ⁺-cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC⁻) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ⁻-cells was 93.6 ± 1.8 % for bacteria and 90.4 ± 4.0 % for yeasts and the content of live Δψ⁺-cells was 0.9 ± 0.3 % for bacteria and 2.4 ± 0.7 % for yeasts. Hypothetically, existence of Δψ⁺-cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K⁺, Na⁺, and Ca²⁺ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ(⁻-cells to Δψ⁺-cells can be explained by slow influx of K⁺ ions into Δψ⁻-cell to the trigger level of K⁺ concentration ("compression of potassium spring"), which is forming "alternative" Δψ⁺-cell for a short period, following with fast efflux of K⁺ ions out of Δψ⁺-cell ("release of potassium spring") returning cell to normal Δψ⁻ state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ⁻- and Δψ⁺- cells.

  11. Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival.

    LENUS (Irish Health Repository)

    Creagh, Emma M

    2009-01-01

    Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. Here, we have utilized a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal\\/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal\\/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man.

  12. UV-enhanced reactivation in mammalian cells: increase by caffeine

    International Nuclear Information System (INIS)

    Lytle, C.D.; Iacangelo, A.L.; Lin, C.H.; Goddard, J.G.

    1981-01-01

    It has been reported that caffeine decreases UV-enhanced reactivation of UV-irradiated Herpes simplex virus in CV-l monkey kidney cells. That occurred when there was no delay between cell irradiation and virus infection. In the present study, virus infection was delayed following cell irradiation to allow an 'induction' period separate from the 'expression' period which occurs during the virus infection. Thus, the effects of caffeine on 'induction' and 'expression' could be determined separately. Caffeine increased the expression of UV-enhanced reactivation, while causing a small decrease in the 'induction' of enhanced reactivation. (author)

  13. Risk Mitigation in Preventing Adventitious Agent Contamination of Mammalian Cell Cultures.

    Science.gov (United States)

    Shiratori, Masaru; Kiss, Robert

    2017-11-14

    Industrial-scale mammalian cell culture processes have been contaminated by viruses during the culturing phase. Although the historical frequency of such events has been quite low, the impact of contamination can be significant for the manufacturing company and for the supply of the product to patients. This chapter discusses sources of adventitious agent contamination risk in a cell culture process, provides a semiquantitative assessment of such risks, and describes potential process barriers that can be used to reduce contamination risk. High-temperature, short-time (HTST) heat treatment is recommended as the process barrier of choice, when compatible with the process. A case study assessing the compatibility of HTST heat treatment with a cell culture medium is presented, and lessons learned are shared from our experiences over many years of developing and implementing virus barriers in mammalian cell culture processes. Graphical Abstract.

  14. The Effects of Ionizing Radiation on Mammalian Cells.

    Science.gov (United States)

    Biaglow, John E.

    1981-01-01

    Discusses the effects of radiation on dividing cells and factors influencing these effects; also briefly reviews the radical mechanism for radiation damage. Emphasizes the importance of oxygen in radiation effects. (CS)

  15. Oxygen sensitization of mammalian cells under different irradiation conditions

    International Nuclear Information System (INIS)

    Ling, C.C.; Michaels, H.B.; Gerweck, L.E.; Epp, E.R.; Peterson, E.C.

    1981-01-01

    The oxygen dependence of the radiosensitivity of cultured CHO cells was examined in detail with particular attention paid to avoiding possible artifacts due to radiolytic oxygen depletion. Two methods of gas equilibration and irradiation were used. In the first approach, cells were irradiated with 50-kVp X rays in a thin-layer geometry which offered maximum interchange between the cells and the surrounding gas. The second technique employed 280-kVp X irradiation of cells under full-medium conditions with mechanical agitation to minimize the effect of radiochemical oxygen consumption by promoting rapid oxygen replenishment. With these techniques oxygen radiosensitization was clearly resolved at an oxygen concentration of 0.03% in the gas phase. The oxygen K curves measured by these two methods were similar in shape over a wide range of oxygen concentration

  16. In Vitro Cytotoxicity of Nanoparticles in Mammalian Germline Stem Cells

    OpenAIRE

    Braydich-Stolle, Laura; Hussain, Saber; Schlager, John J.; Hofmann, Marie-Claude

    2005-01-01

    Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyba...

  17. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells.

    Science.gov (United States)

    Braydich-Stolle, Laura; Hussain, Saber; Schlager, John J; Hofmann, Marie-Claude

    2005-12-01

    Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots have received enormous national attention in the creation of new types of analytical tools for biotechnology and the life sciences. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health and the environment. Thus, there are limited studies available on toxicity of nanoparticles for risk assessment of nanomaterials. The purpose of this study was to assess the suitability of a mouse spermatogonial stem cell line as a model to assess nanotoxicity in the male germline in vitro. The effects of different types of nanoparticles on these cells were evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. Our results demonstrate a concentration-dependent toxicity for all types of particles tested, whereas the corresponding soluble salts had no significant effect. Silver nanoparticles were the most toxic while molybdenum trioxide (MoO(3)) nanoparticles were the least toxic. Our results suggest that this cell line provides a valuable model with which to assess the cytotoxicity of nanoparticles in the germ line in vitro.

  18. In vitro ochratoxin A adsorption by commercial yeast cell walls

    Directory of Open Access Journals (Sweden)

    Carina Maricel Pereyra

    2015-03-01

    Full Text Available ABSTRACT. Pereyra C.M., Cavaglieri L.R., Keller K.M., Chiacchiera, S.M., Rosa C.A.R. & Dalcero A.M. In vitro ochratoxin A adsorption by commercial yeast cell walls. [Adsorção in vitro de ocratoxina A por paredes celulares de levedura comercial.] Revista Brasileira de Medicina Veterinária, 37(1:25-28, 2015. Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, (5800 Río Cuarto, Córdoba, Argentina. E-mail: lcavaglieri@exa.unrc.edu.ar The aim of the present study was to evaluate the ochratoxin A (OTA adsorption capacity by two kinds of commercial yeast cell walls (YCW1 and YCW2 used as dietary supplements. An in vitro test was designed to mimic the temperature (37ºC, pH (2 and passage time (30 min through the stomach of a monogastric animal. The test was performed using different concentrations of YWC (100 and 200 µg/mL and OTA (10; 80; 160 and 1000 ng/mL and extracts were quantified by HPLC. For each OTA concentration, two independent trials varying the concentration of each YCW were performed. The two YCW assayed containing different percentages of polysaccharides, were able to adsorb similar amounts of OTA. Furthermore, the relationship mannans/β-glucans does not influence the rate of adsorption of OTA. In general, it was observed that the adsorption capacity increased with decreasing OTA concentration. Results from this work related to adsorption capacity of OTA with YCW allow predicting that other factor than 3D-structure and β-glucans and mannans could be involved. Future studies could be conducted to test the in vivo binding ability to alleviate the toxic effects of OTA under field conditions. Both YCW are a promising mycotoxin adsorbent to be used in animal feed to prevent mycotoxicoses.

  19. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    Science.gov (United States)

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  20. The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

    Science.gov (United States)

    Hares, Michelle C; Hinchliffe, Stewart J; Strong, Philippa C R; Eleftherianos, Ioannis; Dowling, Andrea J; ffrench-Constant, Richard H; Waterfield, Nick

    2008-11-01

    The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further

  1. Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

    Science.gov (United States)

    Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

    2017-06-22

    Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Effect of tributyltin on mammalian endothelial cell integrity.

    Science.gov (United States)

    Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

    2015-01-01

    Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Mechanisms of radiation-induced changes in mammalian cell properties

    International Nuclear Information System (INIS)

    Elkind, M.M.; Han, A.; Ben-Hur, E.; Hill, C.K.; Myers, C.; Suzuki, F.; Utsumi, H.; Liu, C.M.; Theriot, L.D.

    1981-01-01

    The primary focus of this research is to determine the presence or absence of repair processes relative to linear or so-called single-hit dose effects. Experimental techniques and protocols are developed to test if repair processes contribute to the linear components of the induction of cell killing, mutation, and transformation and, if the slopes of such linear components are dependent upon dose rate. Principal methods are those cell culture techniques for assessing survival, altered phenotype, and transformation. Chinese hamster cells incubated in medium containing 90% D 2 O are inhibited from repairing potentially lethal x-ray and neutron damage (fisson-spectrum neutrons). The sector of damage whose repair is affected by D 2 O medium partially overlaps with that affected by anisotonic buffer. As in the instance of anisotonic buffer, enhanced cell killing due to D 2 O medium does not prevent cells from repairing sublethal damage when incubation in normal medium is resumed. Usng lt of human risk associated with nuclearing collective dose commitment will result in more attention being paid to potential releases of radionuclides at relatively short times after disposal

  4. On the origins of the universal dynamics of endogenous granules in mammalian cells.

    Science.gov (United States)

    Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

    2009-12-01

    Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

  5. Radiosensitizers and the oxygen effects in mammalian cells

    International Nuclear Information System (INIS)

    Millar, B.C.; Fielden, E.M.; Steele, J.J.

    1979-01-01

    The survival curves for Chinese Hamster cells irradiated under various oxygen tensions have been determined. The variation in OER with oxygen concentration shows two distinct components. Between 1.4 and 7.0 μM the OER is constant with a value of 1.9. Experiments with nitroaromatic radiosensitizers in combination with low concentrations of oxygen show that they can all mimic the 'low concentration' oxygen effect. Of the compounds tested only misonidazole can apparently mimic the 'high concentration' oxygen effect although the full OER cannot be obtained with the authors cell line because of toxicity by the sensitizer. (Auth.)

  6. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Science.gov (United States)

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  7. The effects of selenium on glutathione peroxidase activity and radioprotection in mammalian cells

    International Nuclear Information System (INIS)

    Diamond, A.M.; Murray, J.L.; Dale, P.; Tritz, R.; Grdina, D.J.

    1995-01-01

    The media of representative mammalian cell lines were supplemented with low levels of selenium in the form of sodium selenite in order to investigate the effects of selenium on mammalian cells. Following incubation in 30 nM sodium selenite, these cells were assayed for changes in glutathione peroxidase (GPx) activity. The cells examined included NIH 3T3 mouse fibroblasts, PC12 rat sympathetic precursor cells, SupT-1 human lymphocytes, MCF-7 adr human breast carcinoma cells and AA8 Chinese hamster ovary cells. Selenium supplementation resulted in a marginal increase in GPx activity for the NIH 3T3, MCF-7 adr and Supt-1 cells but stimulated GPx activity approximately 5-fold in PC12 and AA8 cells. AA8 cells were selected to evaluate whether selenium supplementation was radioprotective against 60 cobalt gamma irradiation. Protection against radiation-induced mutation was measured by evaluating mutation frequency at the hprt locus. In this assay, preincubation of AA8 CHO cells significantly protected these cells from exposure to 8 Gy

  8. The indentation of pressurized elastic shells: from polymeric capsules to yeast cells

    KAUST Repository

    Vella, D.; Ajdari, A.; Vaziri, A.; Boudaoud, A.

    2011-01-01

    of capsules. Our results are relevant for determining the internal pressure in bacterial, fungal or plant cells. As an illustration of this, we apply our results to recent measurements of the stiffness of baker's yeast and infer from these experiments

  9. Modeling population dynamics of mitochondria in mammalian cells

    Science.gov (United States)

    Kornick, Kellianne; Das, Moumita

    Mitochondria are organelles located inside eukaryotic cells and are essential for several key cellular processes such as energy (ATP) production, cell signaling, differentiation, and apoptosis. All organisms are believed to have low levels of variation in mitochondrial DNA (mtDNA), and alterations in mtDNA are connected to a range of human health conditions, including epilepsy, heart failure, Parkinsons disease, diabetes, and multiple sclerosis. Therefore, understanding how changes in mtDNA accumulate over time and are correlated to changes in mitochondrial function and cell properties can have a profound impact on our understanding of cell physiology and the origins of some diseases. Motivated by this, we develop and study a mathematical model to determine which cellular parameters have the largest impact on mtDNA population dynamics. The model consists of coupled ODEs to describe subpopulations of healthy and dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, and mutation. We study the time evolution and stability of each sub-population under specific selection biases and pressures by tuning specific terms in our model. Our results may provide insights into how sub-populations of mitochondria survive and evolve under different selection pressures. This work was supported by a Grant from the Moore Foundation.

  10. Sensitization of in vitro mammalian cells by nitrous oxide

    International Nuclear Information System (INIS)

    Ewing, D.

    1984-01-01

    Powers and his colleagues showed almost ten years ago that sensitization by nitrous oxide required two radiolytic products: OH radicals and hydrogen peroxide. That observation with bacterial spores has been confirmed and extended with spores and several strains of bacteria. OH must be present to form hydrogen peroxide, but, in addition, OH must also be present with the hydrogen peroxide for damage to occur. (Reagent hydrogen peroxide, except at very high concentrations, will not sensitize unless OH radicals are present.) The authors have now tested nitrous oxide with two Chinese hamster cell lines, V79 and CHO. The responses in nitrogen and nitrous oxide are the same for each. The authors have tentatively concluded that insufficient hydrogen peroxide is formed in the cells' suspending fluid for damage from nitrous oxide to occur. Several results support this conclusion: reagent hydrogen peroxide is a potent sensitizer of either cell line tested in nitrogen or nitrous oxide and an assay for radiolytic hydrogen peroxide confirms that only minimal levels are formed at the doses used in these survival curves. The authors also present results of other tests to further complement work with procaryotic cells

  11. Mammalian cells exposed to ionizing radiation: structural and biochemical aspects

    International Nuclear Information System (INIS)

    Sabanero, M.; Flores V, L. L.; Azorin V, J. C.; Vallejo, M. A.; Cordova F, T.; Sosa A, M.; Castruita D, J. P.; Barbosa S, G.

    2015-10-01

    Acute or chronic exposure to ionizing radiation is a factor that may be hazardous to health. It has been reported that exposure to low doses of radiation (less than 50 mSv / year) and subsequently exposure to high doses have greater effects in people. However, it is unknown molecular and biochemical level alteration. This study, analyzes the susceptibility of a biological system (HeLa Atcc CCL-2 human cervix cancer cell line) to ionizing radiation (6 and 60 mSv/ 90). Our evaluate multiple variables such as: total protein profile, mitochondrial metabolic activity (XTT assay), cell viability (Trypan blue exclusion assay), cytoskeleton (actin micro filaments), nuclei (D API), genomic DNA. The results indicate, that cells exposed to ionizing radiation structurally show alterations in nuclear phenotype and aneuploidy, further disruption in the tight junctions and consequently on the distribution of actin micro filaments. Similar alterations were observed in cells treated with a genotoxic agent (200μM H 2 O 2 /1 h). In conclusion, this multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems. However, it is necessary to determine stress markers for integration of the effects of ionizing radiation. (Author)

  12. Mammalian cells exposed to ionizing radiation: structural and biochemical aspects

    Energy Technology Data Exchange (ETDEWEB)

    Sabanero, M.; Flores V, L. L. [Universidad de Guanajuato, Departamento de Biologia, DCNE, Noria Alta s/n, 36250 Guanajuato, Gto. (Mexico); Azorin V, J. C.; Vallejo, M. A.; Cordova F, T.; Sosa A, M. [Universidad de Guanajuato, Departamento de Ingenieria Fisica, DCI, Loma del Bosque 103, Col. Lomas del Campestre, 37150 Leon, Guanajuato (Mexico); Castruita D, J. P. [Universidad de Guadalajara, Departamento de Ecologia, CUCBA, Las Agujas, 45100 Zapopan, Jalisco (Mexico); Barbosa S, G., E-mail: myrna.sabanero@gmail.com [Universidad de Guanajuato, Departamento de Ciencias Medicas, DCS, 20 de Enero No. 929, Col. Obregon, 37000 Leon, Guanajuato (Mexico)

    2015-10-15

    Acute or chronic exposure to ionizing radiation is a factor that may be hazardous to health. It has been reported that exposure to low doses of radiation (less than 50 mSv / year) and subsequently exposure to high doses have greater effects in people. However, it is unknown molecular and biochemical level alteration. This study, analyzes the susceptibility of a biological system (HeLa Atcc CCL-2 human cervix cancer cell line) to ionizing radiation (6 and 60 mSv/ 90). Our evaluate multiple variables such as: total protein profile, mitochondrial metabolic activity (XTT assay), cell viability (Trypan blue exclusion assay), cytoskeleton (actin micro filaments), nuclei (D API), genomic DNA. The results indicate, that cells exposed to ionizing radiation structurally show alterations in nuclear phenotype and aneuploidy, further disruption in the tight junctions and consequently on the distribution of actin micro filaments. Similar alterations were observed in cells treated with a genotoxic agent (200μM H{sub 2}O{sub 2}/1 h). In conclusion, this multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems. However, it is necessary to determine stress markers for integration of the effects of ionizing radiation. (Author)

  13. Photo-transfection of mammalian cells via femtosecond laser pulses

    CSIR Research Space (South Africa)

    Mthunzi, P

    2009-06-01

    Full Text Available on transient photo-transfection of ovary (CHO-Kl), neuroblastoma (NG-I08 & SKN-SH) and embryonic kidney (HEK-293) as well as primary non-differentiated stem cells (EI4g2a) using a tightly focused titanium sapphire laser beam (1.1 urn diameter spot size...

  14. Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

    Science.gov (United States)

    Chen, Ke-Quan; Li, Jian; Ma, Jiang-Feng; Jiang, Min; Wei, Ping; Liu, Zhong-Min; Ying, Han-Jie

    2011-01-01

    The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  15. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    International Nuclear Information System (INIS)

    Vázquez-Iglesias, Lorena; Lostalé-Seijo, Irene; Martínez-Costas, José; Benavente, Javier

    2012-01-01

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  16. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    Energy Technology Data Exchange (ETDEWEB)

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  17. A high-throughput method for quantifying metabolically active yeast cells

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander

    2015-01-01

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved....... The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e.......g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts....

  18. Stable current outputs and phytate degradation by yeast-based biofuel cell.

    Science.gov (United States)

    Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

    2014-09-01

    In this paper, we report for the first time that Candida melibiosica 2491 yeast strain expresses enhanced phytase activity when used as a biocatalyst in biofuel cells. The polarization also results in an increase of the yeast biomass. Higher steady-state electrical outputs, assigned to earlier production of an endogenous mediator, were achieved at continuous polarization under constant load. The obtained results prove that the C. melibiosica yeast-based biofuel cell could be used for simultaneous electricity generation and phytate bioremediation. In addition, the higher phytase activity obtained by interruptive polarization suggests a new method for increasing the phytase yield from microorganisms. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Chromium cytotoxic effects on mammalian cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Levis, A G; Buttignol, M; Vettorato, L

    1975-01-01

    Chromium compounds, which have several industrial uses, are reported to be carcinogenic. The author, have, therefore, undertaken the study of K/sub 2/Cr/sub 2/O/sub 7/ effects on a cell line of Hamster fibroblasts (BHK), using specific radioactive precursors and determining the acid soluble pool, the RNA, DNA, and protein contents and specific activities. The K/sub 2/Cr/sub 2/O/sub 7/ induced changes in nucleic acid and protein specific activities are related to two different, dissociable effects: (1) a sudden inhibition of macromolecular syntheses, followed by a recovery period, and (2) an immediate, drastic stimulation of nucleoside transport into the cell, whereas amino acid transport is reduced. The effects on precursor permeability are not related to non specific changes of the plasma membrane, but they seem to be due to specific simulations and inhibitions of nucleoside and amino acid transport mechanisms. A human cell line (HEp) has been also tested, which is more sensitive to K/sup 2/Cr/sub 2/O/sub 7/ action than the BHK line. DNA synthesis as well as survival in single cell plating conditions show the same difference in sensitivity to K/sub 2/Cr/sub 2/O/sub 7/. Thus, the loss of indefinite cell division ability could be due to the blockage of DNA replication. It is suggested that the main chromium cytotoxic effect lies in a multiple attack to the DNA molecule, which gradually alters the DNA tertiary structure resulting in the blockage of replication capacity. This blockage may be reversible owing to the breakage of chromium-DNA bonds and to the induced instability of phosphodiester internucleotide bonds.

  20. Chromosomal damages and mutagenesis in mammalian and human cells induced by ionizing radiations with different LET

    International Nuclear Information System (INIS)

    Govorun, R.D.

    1997-01-01

    On the basis of literature and proper data the inference was made about essential role of structural chromosomal (and gene) damages in spontaneous and radiation-induced mutagenesis of mammalian and human cells on HPRT-loci. The evidences of increasing role of these damages in the mutagenesis after the influence of ionizing radiations with high LET are adduced. The consequences of HPRT-gene damages have been examined hypothetically. The geterogeneity of mutant subclones on their cytogenetical properties were revealed experimentally. The data reflect a phenomenon of the reproductive chromosomal instability in many generations of mutant cell. The mutagenesis of mammalian cells is also accompanied by the impairment of chromosome integrity with high probability as a stage of appropriate genome reorganization because of changed vital conditions

  1. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

    Science.gov (United States)

    Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

    2018-02-13

    Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

  2. Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells

    DEFF Research Database (Denmark)

    Ramos-Molina, Bruno; López-Contreras, Andrés J; Lambertos, Ana

    2015-01-01

    Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane...... transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and V max of 17.......3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor...

  3. Chemical sporulation and germination: cytoprotective nanocoating of individual mammalian cells with a degradable tannic acid-FeIII complex

    Science.gov (United States)

    Lee, Juno; Cho, Hyeoncheol; Choi, Jinsu; Kim, Doyeon; Hong, Daewha; Park, Ji Hun; Yang, Sung Ho; Choi, Insung S.

    2015-11-01

    Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature.Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature. Electronic supplementary information (ESI) available: Experimental details, LSCM images, and SEM and TEM images. See DOI: 10.1039/c5nr05573c

  4. Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, K.; Ueda, M. [Lab. of Applied Biological Chemistry, Kyoto Univ., Yoshida, Kyoto (Japan)

    2004-07-01

    The Cd{sup 2+}-chelating abilities of yeast metallothionein (YMT) and hexa-His displayed on the yeast-cell surface were compared. Display of YMT and hexa-His by {alpha}-agglutinin-based cell-surface engineering was confirmed by immunofluorescent labeling. Surface-engineered yeast cells with YMT and hexa-His fused in tandem showed superior cell-surface adsorption and recovery of Cd{sup 2+} under EDTA treatment on the cell surface than hexa-His-displaying cells. YMT was demonstrated to be more effective than hexa-His for the adsorption of Cd{sup 2+}. Yeast cells displaying YMT and/or hexa-His exhibited a higher potential for the adsorption of Cd{sup 2+} than Escherichia coli cells displaying these molecules. In order to investigate the effect of the displayed YMT and hexa-His on sensitivity to toxic Cd{sup 2+}, growth in Cd{sup 2+}-containing liquid medium was monitored. Unlike hexa-His-displaying cells, cells displaying YMT and hexa-His fused in tandem induced resistance to Cd{sup 2+} through active and enhanced adsorption of toxic Cd{sup 2+}. These results indicate that YMT-displaying yeast cells are a unique bioadsorbent with a functional chelating ability superior to that of E. coli. (orig.)

  5. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  6. Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

    Science.gov (United States)

    Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2006-01-01

    During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994

  7. Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells.

    Science.gov (United States)

    Ramos-Molina, Bruno; López-Contreras, Andrés J; Lambertos, Ana; Dardonville, Christophe; Cremades, Asunción; Peñafiel, Rafael

    2015-05-01

    Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7 cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and Vmax of 17.3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor AMXT-1501 markedly decreased agmatine uptake. In cells transfected with any of the three ornithine decarboxylase antizymes (AZ1, AZ2 and AZ3), agmatine uptake was dramatically reduced. On the contrary, transfection with antizyme inhibitors (AZIN1 and AZIN2) markedly increased the transport of agmatine. Furthermore, whereas putrescine uptake was significantly decreased in cells transfected with ornithine decarboxylase (ODC), the accumulation of agmatine was stimulated, suggesting a trans-activating effect of intracellular putrescine on agmatine uptake. All these results indicate that ODC and its regulatory proteins (antizymes and antizyme inhibitors) may influence agmatine homeostasis in mammalian tissues.

  8. Autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity.

    Science.gov (United States)

    Xu, Tingting; Close, Dan M; Webb, James D; Ripp, Steven A; Sayler, Gary S

    2013-10-28

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

  9. Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes.

    Directory of Open Access Journals (Sweden)

    Christel Logez

    Full Text Available The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

  10. Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes.

    Science.gov (United States)

    Logez, Christel; Berger, Sylvie; Legros, Céline; Banères, Jean-Louis; Cohen, William; Delagrange, Philippe; Nosjean, Olivier; Boutin, Jean A; Ferry, Gilles; Simonin, Frédéric; Wagner, Renaud

    2014-01-01

    The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

  11. Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

    Science.gov (United States)

    Subramanian, A; Ranganathan, P; Diamond, S L

    1999-09-01

    Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

  12. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  13. Intracellular proteins produced by mammalian cells in response to environmental stress

    Science.gov (United States)

    Goochee, Charles F.; Passini, Cheryl A.

    1988-01-01

    The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

  14. Radon exposure system for mammalian cells in culture: Design, operation, and dosimetry

    International Nuclear Information System (INIS)

    Seed, T.M.; Kretz, N.D.; Schlenker, R.A.

    1991-01-01

    A novel system for Rn gas exposure of mammalian cells in culture has been designed, constructed, and used to directly assess both the magnitude and the nature of chronic, low-dose Rn/Rn daughter toxicity of exposed vital lung cells isolated from normal pulmonary tissue, propagated and exposed in vitro. Direct correlations between atmospheric Rn concentrations, alpha-particle fluences, and macro- and microdoses of absorbed radiation doses by lung cells provide for a heretofore unavailable assessment of critical doses to vital cells

  15. Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

    Science.gov (United States)

    Choi, Jinsu; Lee, Hojae; Choi, Insung S.; Yang, Sung Ho

    2017-09-01

    Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.

  16. Gold nanoparticles delivery in mammalian live cells: a critical review

    Directory of Open Access Journals (Sweden)

    Raphaël Lévy

    2010-02-01

    Full Text Available Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, ‘nanoparticle and cell’ hit. Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable. To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping. Raphaël Lévy is a BBSRC David Phillips Research Fellow at the University of Liverpool. He graduated in Physics at the University Louis Pasteur in Strasbourg (France. In 2002, after a Master in Soft Condensed Matter Physics, he obtained a PhD in Physics at the University Louis Pasteur. He then moved to

  17. Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

    Science.gov (United States)

    Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

    2014-02-01

    The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

  18. Metabolic diversification of cells during the development of yeast colonies

    Czech Academy of Sciences Publication Activity Database

    Váchová, Libuše; Kučerová, Helena; Devaux, F.; Úlehlová, M.; Palková, Z.

    2009-01-01

    Roč. 11, č. 2 (2009), s. 494-504 ISSN 1462-2912 R&D Projects: GA ČR GA204/05/0294; GA ČR GA204/08/0718; GA MŠk(CZ) LC531 Grant - others:GB(GB) Howard Hughes Medical Institute International Research Award Institutional research plan: CEZ:AV0Z50200510 Keywords : yeast * yeast colonies * saccharomyces cerevisiae Subject RIV: EE - Microbiology, Virology Impact factor: 4.909, year: 2009

  19. Interplay between autophagy and programmed cell death in mammalian neural stem cells

    Directory of Open Access Journals (Sweden)

    Kyung Min Chung

    2013-08-01

    Full Text Available Mammalian neural stem cells (NSCs are of particular interestbecause of their role in brain development and function. Recentfindings suggest the intimate involvement of programmed celldeath (PCD in the turnover of NSCs. However, the underlyingmechanisms of PCD are largely unknown. Although apoptosis isthe best-defined form of PCD, accumulating evidence hasrevealed a wide spectrum of PCD encompassing apoptosis,autophagic cell death (ACD and necrosis. This mini-reviewaims to illustrate a unique regulation of PCD in NSCs. Theresults of our recent studies on autophagic death of adulthippocampal neural stem (HCN cells are also discussed. HCNcell death following insulin withdrawal clearly provides areliable model that can be used to analyze the molecularmechanisms of ACD in the larger context of PCD. Moreresearch efforts are needed to increase our understanding of themolecular basis of NSC turnover under degenerating conditions,such as aging, stress and neurological diseases. Efforts aimed atprotecting and harnessing endogenous NSCs will offer novelopportunities for the development of new therapeutic strategiesfor neuropathologies. [BMB Reports 2013; 46(8: 383-390

  20. Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells

    OpenAIRE

    Elustondo, Pia; Zakharian, Eleonora; Pavlov, Evgeny

    2012-01-01

    Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long-chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB-accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short-chain (or complexed) cPHB (10–100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous ...

  1. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  2. NMR-based metabolomics of mammalian cell and tissue cultures

    International Nuclear Information System (INIS)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan; Niemitz, Matthias; Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D.

    2011-01-01

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  3. NMR-based metabolomics of mammalian cell and tissue cultures

    Energy Technology Data Exchange (ETDEWEB)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan [Bristol-Myers Squibb Company (United States); Niemitz, Matthias [PERCH Solutions Ltd. (Finland); Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D., E-mail: michael.reily@bms.com [Bristol-Myers Squibb Company (United States)

    2011-04-15

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  4. Fast kinetics of the oxygen effect in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Watts, M.E.; Maughan, R.L.; Michael, B.D.

    1978-01-01

    A technique using a fast gas transfer with a single pulse of electrons (the gas-explosion technique) has been used to investigate the time-dependence of the dose-modifying action of oxygen in irradiated V79 Chinese hamster cells. Oxygen did not significantly alter the shapes of the survival curves. The dose-modifying factor between the fully oxic and fully hypoxic (oxygen at 9000 ms) curve was 2.6. The dose-modifying factor for the survival curve drawn for oxygen contact at 0.3 ms after irradiation was 1.5 relative to the hypoxic curve. The duration of the post-effect (oxygen contact after irradiation) indicated that oxygen-dependent damage has a lifetime extending into the ms time-range. In the pre-effect time region (oxygen contact before irradiation) 1 to 2 ms oxygen contact was required to achieve the full sensitization. The results are discussed with reference to the diffusion time for oxygen to reach the sensitive site within the cell. (U.K.)

  5. Detection and quantitative determination by PIXE of the mutagen Sn2+ in yeast cells

    International Nuclear Information System (INIS)

    Viau, C.M.; Yoneama, M.-L.; Dias, J.F.; Pungartnik, C.; Brendel, M.; Henriques, J.A.P.

    2006-01-01

    The main goal of this work was to determine the concentration of Sn 2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl 2 . To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl 2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn 2+ induces DNA lesions and that the absorption depends on the genetic background of each strain

  6. Effect of selenium on the Hg, Zn, Fe and Co content of yeast cells

    International Nuclear Information System (INIS)

    Czauderna, M.; Peplowski, A.; Smolinski, S.

    1992-01-01

    The yeast cells, Saccharomyces cerevisiae, were exposed to Hg 2+ ions (10 -4 M) and SeO 2 (2x10 -4 -10 -2 M) or Se-methionine (2x10 -4 M). Instrumental neutron activation analysis (INAA) was used to analyze changes in the Hg, Zn,Fe and Co levels in these cells. When the yeast was incubated in a medium containing 10 -3 M and 10 -2 M Se) 2 , the Hg content of the yeast markedly increased. It was also found that the uptake of Se and Hg influenced the levels of Zn, Fe and Co found in the cells. While the presence of Se-methionine (Se-Met), SeO 2 or Hg 2+ ions caused increases in the intracellular Zn levels, the combined presence of Hg 2+ and SeO 2 and their assumed interaction, reduced the efficiency of Se for increasing the Zn content of yeast. (author) 17 refs.; 3 tabs

  7. Adhesion of yeast cells on surface of polymers produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu, Zhaoxin; Takehisa, Masaaki; Xie Zongchuan.

    1995-01-01

    The adhesion of yeast (Saccharomyces formesences) cells on polymers was studied thermodynamically. The polymers were laminally prepared by means of radiation polymerization. By measuring contact angles, we calculated dispersion component and polar component of surface free energy of the polymers and the cells, and interfacial free energy between the polymer and the cells. Then interfacial free energy change of the cell adhesion to surface of the polymer was evaluated. The adhesion behavior of yeast cells on the polymers was observed by optical microscope. From above results, we conclude that the initial adhesion of the cells is related to the surface free energy of the polymer, but the irreversible adhesion may be close to the polar component in surface free energy. The high polar component is favourable the irreversible adhesion of yeast cells. (author)

  8. Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

    Directory of Open Access Journals (Sweden)

    Valeria Zampini

    2011-04-01

    Full Text Available Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

  9. Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

    Science.gov (United States)

    Zampini, Valeria; Rüttiger, Lukas; Johnson, Stuart L; Franz, Christoph; Furness, David N; Waldhaus, Jörg; Xiong, Hao; Hackney, Carole M; Holley, Matthew C; Offenhauser, Nina; Di Fiore, Pier Paolo; Knipper, Marlies; Masetto, Sergio; Marcotti, Walter

    2011-04-01

    Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

  10. The food additive vanillic acid controls transgene expression in mammalian cells and mice.

    Science.gov (United States)

    Gitzinger, Marc; Kemmer, Christian; Fluri, David A; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

    2012-03-01

    Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

  11. Endocytosis and exocytosis of nanoparticles in mammalian cells

    Science.gov (United States)

    Oh, Nuri; Park, Ji-Ho

    2014-01-01

    Engineered nanoparticles that can be injected into the human body hold tremendous potential to detect and treat complex diseases. Understanding of the endocytosis and exocytosis mechanisms of nanoparticles is essential for safe and efficient therapeutic application. In particular, exocytosis is of significance in the removal of nanoparticles with drugs and contrast agents from the body, while endocytosis is of great importance for the targeting of nanoparticles in disease sites. Here, we review the recent research on the endocytosis and exocytosis of functionalized nanoparticles based on various sizes, shapes, and surface chemistries. We believe that this review contributes to the design of safe nanoparticles that can efficiently enter and leave human cells and tissues. PMID:24872703

  12. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    Science.gov (United States)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  13. Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells.

    Science.gov (United States)

    Bruce, Virginia J; McNaughton, Brian R

    2017-08-17

    Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research. Copyright © 2017. Published by Elsevier Ltd.

  14. Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

    Science.gov (United States)

    Mulchandani, A; Bassi, A S

    1996-01-01

    Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

  15. Non-homologous end joining dependency of {gamma}-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, Erich [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)]. E-mail: erich.heidenreich@meduniwien.ac.at; Eisler, Herfried [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)

    2004-11-22

    There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by {gamma}-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the {gamma}-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.

  16. Non-homologous end joining dependency of γ-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

    International Nuclear Information System (INIS)

    Heidenreich, Erich; Eisler, Herfried

    2004-01-01

    There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by γ-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the γ-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development

  17. Dose-rate effects on mammalian cells exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Mitchell, J.B.

    1978-01-01

    The effect of irradiation on the life cycle and on cell survival was studied for a range of different dose rates. Log phase, plateau phase and synchronized cultures of different mammalian cells were used. Cell cycle redistribution during the radiation exposure was found to be a very important factor in determining the overall dose-rate effect for log phase and synchronized cells. In fact, cell cycle redistribution during the exposure, in some instances, resulted in a lower dose rate being more effective in cell killing per unit dose than a higher dose rate. For plateau phase cultures, where cell cycle times are greatly lengthened, the effects of redistribution in regard to cell killing was virtually eliminated. Both fed and unfed plateau phase cultures exhibited a dose-rate effect, but it was found that below dose rates of 154 rad/h there is no further loss in effectiveness

  18. Fast Neutrons - LET Distributions and the Response of Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bewley, D. K. [Medical Research Council Cyclotron Unit, Hammersmith Hospital, London (United Kingdom)

    1968-03-15

    Distributions of stopping power (LET) are given for four beams of fast neutrons, namely, neutrons of 14.6 MeV, neutrons produced by bombarding a thick beryllium target with 15 MeV deuterons, neutrons of 3 MeV, and fast neutrons produced by bombardment of a {sup 235}U converter plate with thermal neutrons. The track average LET is correlated with mean neutron energy, but the dose average is approximately constant. However, neither of these types of average is expected to have much relevance to radiobiology. Further, specification of a ''biologically effective LET'' depends on the biological test used, and is not solely a function of the radiation quality. An attempt has been made to calculate the response of T.I. kidney cells in tissue culture to these four beams of neutrons, based on their response to charged particles using the track-segment method. The calculated RBE's of the neutron beams are lower than the observed values and the calculated values of the oxygen enhancement ratio are higher. These differences seem too great to be explained by errors in dosimetry and in the calculated LET spectra. The suggestion is made that LET is not an adequate criterion of radiation quality, and that the discrepancies may be explained by more detailed consideration of the part played by delta rays and by heavy recoil tracks of short range. (author)

  19. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    International Nuclear Information System (INIS)

    Alsteens, David; Dupres, Vincent; Evoy, Kevin Mc; Dufrene, Yves F; Wildling, Linda; Gruber, Hermann J

    2008-01-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls

  20. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    OpenAIRE

    Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H.; Michel, Jennifer Carlisle; Claxton, Derek P.; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K. Christopher; Gouaux, Eric

    2014-01-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient productio...

  1. Multiplexed expression and screening for recombinant protein production in mammalian cells

    Directory of Open Access Journals (Sweden)

    McCafferty John

    2006-12-01

    Full Text Available Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell

  2. [Mathematical modeling of synergistic interaction of sequential thermoradiation action on mammalian cells].

    Science.gov (United States)

    Belkina, S V; Semkina, M A; Kritskiĭ, R O; Petin, V G

    2010-01-01

    Data obtained by other authors for mammalian cells treated by sequential action of ionizing radiation and hyperthermia were used to estimate the dependence of synergistic enhancement ratio on the ratio of damages induced by these agents. Experimental results were described and interpreted by means of the mathematical model of synergism in accordance with which the synergism is expected to result from the additional lethal damage arising from the interaction of sublesions induced by both agents.

  3. Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells

    OpenAIRE

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conj...

  4. Synthetic biology in mammalian cells: Next generation research tools and therapeutics

    Science.gov (United States)

    Lienert, Florian; Lohmueller, Jason J; Garg, Abhishek; Silver, Pamela A

    2014-01-01

    Recent progress in DNA manipulation and gene circuit engineering has greatly improved our ability to programme and probe mammalian cell behaviour. These advances have led to a new generation of synthetic biology research tools and potential therapeutic applications. Programmable DNA-binding domains and RNA regulators are leading to unprecedented control of gene expression and elucidation of gene function. Rebuilding complex biological circuits such as T cell receptor signalling in isolation from their natural context has deepened our understanding of network motifs and signalling pathways. Synthetic biology is also leading to innovative therapeutic interventions based on cell-based therapies, protein drugs, vaccines and gene therapies. PMID:24434884

  5. Nanoscopic morphological changes in yeast cell surfaces caused by oxidative stress: an atomic force microscopic study.

    Science.gov (United States)

    Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

    2009-06-01

    Nanoscopic changes in the cell surface morphology of the yeasts Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354), due to their exposure to varying concentrations of hydrogen peroxide (oxidative stress), were investigated using an atomic force microscope (AFM). Increasing hydrogen peroxide concentration led to a decrease in cell viabilities and mean cell volumes, and an increase in the surface roughness of the yeasts. In addition, AFM studies revealed that oxidative stress caused cell compression in both S. cerevisiae and Schiz. pombe cells and an increase in the number of aged yeasts. These results confirmed the importance and usefulness of AFM in investigating the morphology of stressed microbial cells at the nanoscale. The results also provided novel information on the relative oxidative stress tolerance of S. cerevisiae and Schiz. pombe.

  6. Extraction of the number of peroxisomes in yeast cells by automated image analysis.

    Science.gov (United States)

    Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

    2006-01-01

    An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

  7. A platform for rapid prototyping of synthetic gene networks in mammalian cells

    Science.gov (United States)

    Duportet, Xavier; Wroblewska, Liliana; Guye, Patrick; Li, Yinqing; Eyquem, Justin; Rieders, Julianne; Rimchala, Tharathorn; Batt, Gregory; Weiss, Ron

    2014-01-01

    Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. PMID:25378321

  8. Mammalian Cell Culture Process for Monoclonal Antibody Production: Nonlinear Modelling and Parameter Estimation

    Directory of Open Access Journals (Sweden)

    Dan Selişteanu

    2015-01-01

    Full Text Available Monoclonal antibodies (mAbs are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies.

  9. Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

    International Nuclear Information System (INIS)

    Tosato, Valentina; Grüning, Nana-Maria; Breitenbach, Michael; Arnak, Remigiusz; Ralser, Markus; Bruschi, Carlo V.

    2013-01-01

    Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (“translocants”), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  10. WARBURG EFFECT AND TRANSLOCATION-INDUCED GENOMIC INSTABILITY: TWO YEAST MODELS FOR CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Valentina eTosato

    2013-01-01

    Full Text Available Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression i the activity of pyruvate kinase (PK, which recapitulates metabolic features of cancer cells, including the Warburg effect, and ii Bridge-Induced chromosome Translocation (BIT mimicking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect, and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, pyruvate kinase, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and posttranslational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (translocants, between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the Bridge-Induced Translocation system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  11. Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tosato, Valentina [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Grüning, Nana-Maria [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Breitenbach, Michael [Division of Genetics, Department of Cell Biology, University of Salzburg, Salzburg (Austria); Arnak, Remigiusz [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Ralser, Markus [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Bruschi, Carlo V., E-mail: bruschi@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy)

    2013-01-18

    Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (“translocants”), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  12. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Say Kong Ng

    2013-04-01

    Full Text Available From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX amplification technology or the glutamine synthetase (GS system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

  13. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  14. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Gang [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China); Liu, Naicheng; Wang, Zhenheng [Nanjing University, Department of Orthopedics, Jinling Hospital, School of Medicine (China); Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng, E-mail: jfzhang@nju.edu.cn [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China)

    2017-02-15

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  15. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    International Nuclear Information System (INIS)

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

    2014-01-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

  16. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    Science.gov (United States)

    Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

    2017-02-01

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  17. Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

    Energy Technology Data Exchange (ETDEWEB)

    Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz; Lukasik, Beata; Garner, Logan E.; Chworos, Arkadiusz

    2017-05-01

    Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.

  18. ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

    Science.gov (United States)

    Poppi, Lauren A; Tabatabaee, Hessam; Drury, Hannah R; Jobling, Phillip; Callister, Robert J; Migliaccio, Americo A; Jordan, Paivi M; Holt, Joseph C; Rabbitt, Richard D; Lim, Rebecca; Brichta, Alan M

    2018-01-01

    In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted

  19. Feathers and fins: non-mammalian models for hair cell regeneration.

    Science.gov (United States)

    Brignull, Heather R; Raible, David W; Stone, Jennifer S

    2009-06-24

    Death of mechanosensory cells in the inner ear results in two profound disabilities: hearing loss and balance disorders. Although mammals lack the capacity to regenerate hair cells, recent studies in mice and other rodents have offered valuable insight into strategies for stimulating hair cell regeneration in mammals. Investigations of model organisms that retain the ability to form new hair cells after embryogenesis, such as fish and birds, are equally important and have provided clues as to the cellular and molecular mechanisms that may block hair cell regeneration in mammals. Here, we summarize studies on hair cell regeneration in the chicken and the zebrafish, discuss specific advantages of each model, and propose future directions for the use of non-mammalian models in understanding hair cell regeneration.

  20. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    Science.gov (United States)

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates. © 2015 American Institute of Chemical Engineers.

  1. Effect of an aminothiol (WR-1065) on radiation-induced mutagenesis and cytotoxicity in two repair-deficient mammalian cell lines

    International Nuclear Information System (INIS)

    Grdina, D.J.; Nagy, B.; Meechan, P.J.

    1991-01-01

    WR-2721 and its free thiol WR-1065 have been found to effectively protect against radiation- and/or chemotherapy-induced mutagenesis, transformation and carcinogenesis. With respect to the antimutagenic effect, WR-1065 significantly reduced the frequency of HGPRT mutants even when it was administered up to three hours following exposure of cells to radiation. The mechanisms of action most often attributed to these agents include their ability to scavenge free radicals, enter into chemical repair processes through the donation of hydrogen atoms, and induce intracellular hypoxia by means of auto-oxidative processes. Although evidence exists for each of these processes, none is sufficiently satisfactory to account for the post-irradiation protection of WR-1065 against mutation induction in mammalian cells. The most elegant work describing the role of aminothiols on cellular enzymatic repair processes has focused on well-characterized repair-proficient and -deficient bacterial and yeast cell systems. Protection against radiation-induced cytotoxicity by the aminothiol cysteamine was absent in E. coli cell lines that were characterized as having genetically defective repair systems. Until recently, such studies could not be effectively performed with mammalian cells. However, with the isolation and characterization of rodent cell lines deficient in their ability to repair DNA damage, it is now possible to investigate the role of cell-mediated repair systems on aminothiol radioprotection. Specifically, the authors have investigated the effects of WR-1065 on radiation-induced mutagenesis and cytotoxicity in cell lines EM9 and xrs-5, which are defective in DNA single-strand break (SSB) and double-strand break (DSB) rejoining, respectively. Corresponding parental repair-proficient cell lines, AA8 and K1, were also studied for comparative purposes. 26 refs., 5 figs., 2 tabs

  2. Estimation of relative biological effectiveness for low energy protons using cytogenetic end points in mammalian cells

    International Nuclear Information System (INIS)

    Bhat, N.N.; Nairy, Rajesh; Chaurasia, Rajesh; Desai, Utkarsha; Shirsath, K.B.; Anjaria, K.B.; Sreedevi, B.

    2013-01-01

    A facility has been designed and developed to facilitate irradiation of biological samples to proton beam using folded tandem ion accelerator (FOTIA) at BARC. The primary proton beam from the accelerator was diffused using gold foil and channelled through a drift tube. Scattered beam was monitored and calibrated. Uniformity and dosimetry studies were conducted to calibrate the setup for precise irradiation of mammalian cells. Irradiation conditions and geometry were optimized for mammalian cells and other biological samples in thin layer. The irradiation facility is housed in a clean air laminar flow to help exposure of samples in aseptic conditions. The set up has been used for studying various radiobiological endpoints in many biological model systems. CHO, MCF-7, A-549 and INT-407 cell lines were studied in the present investigation using micronucleus (MN) induction as an indicator of radiation damage. The mammalian cells grown on petri plates to about 40 % confluence (log phase) were exposed to proton beam of known doses in the range of 0.1 to 2 Gy. The dose estimation was done based on specific ionization in cell medium. Studies were also conducted using 60 Co gamma radiation to compare the results. Linear quadratic response was observed for all the cell lines when exposed to 60 Co gamma radiation. In contrast, linear response was observed for proton beam. In addition, very significant increase in the MN yield was observed for proton beam compared to 60 Co gamma radiation. Estimated α and β values for CHO cells is found to be 0.02±0.003 Gy-1 and 0.042±0.006 Gy-2 respectively for 60 Co gamma radiation. For proton beam, estimated α for linear fit is found to be 0.37±0.011 Gy-1. Estimated RBE was found to be in the range of 4-8 for all the cell lines and dose ranges studied. In conclusion, the proton irradiation facility developed for mammalian cells has helped to study various radiobiological endpoints. In this presentation, facility description, MN as

  3. Thicker three-dimensional tissue from a "symbiotic recycling system" combining mammalian cells and algae.

    Science.gov (United States)

    Haraguchi, Yuji; Kagawa, Yuki; Sakaguchi, Katsuhisa; Matsuura, Katsuhisa; Shimizu, Tatsuya; Okano, Teruo

    2017-01-31

    In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac cell-layered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 μm-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro "symbiotic recycling system" composed of mammalian cells and algae.

  4. Empty Turnip yellow mosaic virus capsids as delivery vehicles to mammalian cells.

    Science.gov (United States)

    Kim, Doyeong; Lee, Younghee; Dreher, Theo W; Cho, Tae-Ju

    2018-05-03

    Turnip yellow mosaic virus (TYMV) was able to enter animal cells when the spherical plant virus was conjugated with Tat, a cell penetrating peptide (CPP). Tat was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. Baby hamster kidney (BHK) cells were incubated with either unmodified or Tat-conjugated TYMV and examined by flow cytometry and confocal microscopic analyses. Tat conjugation was shown to be more efficient than Lipofectamine in allowing TYMV to enter the mammalian cells. Tat-assisted-transfection was also associated with less loss of cell viability than lipofection. Among the CPPs tested (Tat, R8, Pep-1 and Pen), it was observed that R8 and Pen were also effective while Pep-1 was not. We also examined if the internal space of TYMV can be used to load fluorescein dye as a model cargo. When TYMV is treated by freezing and thawing, the virus is known to convert into a structure with a 6-8 nm hole and release viral RNA. When the resultant pot-like particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection. Our studies demonstrate the potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Genetic control of yeast cell radiosensitivity modification by oxygen and hypoxic sensitizers

    International Nuclear Information System (INIS)

    Zhuranovskaya, G.P.; Petin, V.G.

    1984-01-01

    Diploid yeast cells Saccharomyces cerevisiae ''of the wild type'', individual mutants, homozygous in rad 2 and rad 54 and double mutants, containing both these loci in homozygous state are considered to prove genetic determination of radiosensitivity modification of hypoxic cells by oxygen and electron acceptor compounds previously demonstrated on yeast cells of other genotypes. It is shown that both ''oxygen effect'' and the effect of hypoxic sensitizers depend on the activity of repair systems. The possible mechanism of participation of post-radiation restoration processes in the modification of cell radiosensitivity, is discussed

  6. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis

    Science.gov (United States)

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas

    2016-01-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

  7. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

    Science.gov (United States)

    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

    Science.gov (United States)

    Chen, Xianzhong

    2017-03-04

    The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

  9. The influence of high intensity terahertz radiation on mammalian cell adhesion, proliferation and differentiation.

    Science.gov (United States)

    Williams, Rachel; Schofield, Amy; Holder, Gareth; Downes, Joan; Edgar, David; Harrison, Paul; Siggel-King, Michele; Surman, Mark; Dunning, David; Hill, Stephen; Holder, David; Jackson, Frank; Jones, James; McKenzie, Julian; Saveliev, Yuri; Thomsen, Neil; Williams, Peter; Weightman, Peter

    2013-01-21

    Understanding the influence of exposure of biological systems to THz radiation is becoming increasingly important. There is some evidence to suggest that THz radiation can influence important activities within mammalian cells. This study evaluated the influence of the high peak power, low average power THz radiation produced by the ALICE (Daresbury Laboratory, UK) synchrotron source on human epithelial and embryonic stem cells. The cells were maintained under standard tissue culture conditions, during which the THz radiation was delivered directly into the incubator for various exposure times. The influence of the THz radiation on cell morphology, attachment, proliferation and differentiation was evaluated. The study demonstrated that there was no difference in any of these parameters between irradiated and control cell cultures. It is suggested that under these conditions the cells are capable of compensating for any effects caused by exposure to THz radiation with the peak powers levels employed in these studies.

  10. Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells.

    Science.gov (United States)

    Angelici, Bartolomeo; Mailand, Erik; Haefliger, Benjamin; Benenson, Yaakov

    2016-08-30

    One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    Directory of Open Access Journals (Sweden)

    Roberto Franco Teixeira Corrêa

    Full Text Available Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ, resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7 when tested at 20 µg/mL.

  12. The ciliary margin zone of the mammalian retina generates retinal ganglion cells

    Science.gov (United States)

    Marcucci, Florencia; Murcia-Belmonte, Veronica; Coca, Yaiza; Ferreiro-Galve, Susana; Wang, Qing; Kuwajima, Takaaki; Khalid, Sania; Ross, M. Elizabeth; Herrera, Eloisa; Mason, Carol

    2016-01-01

    Summary The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live-imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. As Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2−/− mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity. PMID:28009286

  13. Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species.

    Directory of Open Access Journals (Sweden)

    Smanla Tundup

    2017-03-01

    Full Text Available The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections.

  14. Repair capability of mammalian cell fractions demonstrated using infectivity of bacteriophage DNA

    International Nuclear Information System (INIS)

    Lai, S.P.; Lytle, C.D.; Benane, S.G.

    1976-01-01

    Extracts of Potoroo kidney cells (PtK2) were examined for ability to provide a repair function in vitro. The biological activity (infectivity) of uv-irradiated replicative form (RF) DNA of bacteriophage phiX174 was restored during incubation of the DNA with a nuclear extract but not with a cytoplasmic extract. The infectivity of the RF-DNA was determined in spheroplasts of E. coli C/sub s/, which is HCR - . This system for biological assay of uv-irradiated DNA repaired in vitro may be used to complement biochemical and biophysical investigations of molecular repair mechanisms in mammalian cells

  15. Assessing mRNA nuclear export in mammalian cells by microinjection.

    Science.gov (United States)

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

    Science.gov (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y.

    2016-01-01

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  17. Copper Biosorption on Magnetically Modified Yeast Cells Under Magnetic Field

    Czech Academy of Sciences Publication Activity Database

    Uzun, L.; Saglam, N.; Šafaříková, Miroslava; Šafařík, Ivo; Denizli, A.

    2011-01-01

    Roč. 46, č. 6 (2011), s. 1045-1051 ISSN 0149-6395 Institutional research plan: CEZ:AV0Z60870520 Keywords : copper removal * heavy metal removal * magnetic biosorbents * yeast Subject RIV: CE - Biochemistry Impact factor: 1.088, year: 2011

  18. Biosorption of mercury on magnetically modified yeast cells

    Czech Academy of Sciences Publication Activity Database

    Yavuz, H.; Denizli, A.; Gungunes, H.; Šafaříková, Miroslava; Šafařík, Ivo

    2006-01-01

    Roč. 52, - (2006), s. 253-260 ISSN 1383-5866 R&D Projects: GA MŠk(CZ) OC 108 Institutional research plan: CEZ:AV0Z60870520 Keywords : mercury removal * magnetic biosorbents * yeast Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.497, year: 2006

  19. Aged yeast mother cells show markers of apoptosis

    Czech Academy of Sciences Publication Activity Database

    Laun, P.; Pichová, Alena; Madeo, F.; Heeren, G.; Kohlwein, S. D.; Fröhlich, K. W.; Dawes, I.; Breitenbach, M.

    2001-01-01

    Roč. 18, S1 (2001), s. S160 ISSN 0749-503X. [International Conference on Yeast Genetics and Molecular Biology /20./. 26.08.2001-31.08.2001, Prague] Institutional research plan: CEZ:AV0Z5020903 Subject RIV: EB - Genetics ; Molecular Biology

  20. DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Inoue, Masao; Takebe, Hiraku.

    1978-01-01

    Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

  1. Immobilised Sarawak Malaysia yeast cells for production of bioethanol.

    Science.gov (United States)

    Zain, Masniroszaime Mohd; Kofli, Noorhisham Tan; Rozaimah, Siti; Abdullah, Sheikh

    2011-05-01

    Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.

  2. Information system for selection of conditions and equipment for the cultivation of mammalian cells

    Directory of Open Access Journals (Sweden)

    D. R. Batyrgazieva

    2017-01-01

    Full Text Available The use of mammals cells and their products wide application, so the actual problem is a creation of an information system in the field of their cultivation for the organizing and structuring of information on process experimental data. This work is devoted the analysis of mammalian cell cultivation. The main technologies of cell cultivation, necessary equipment and matrices are considered. The main stages of database design and information system is described. The justification of software products are provided and the results of the database and information system implementation are done. The detailed description of all modules of the system, as well as a comparative analysis of the results of the search are in the system to verify correct operation of the system. The scientific and practical significance of the work lies in the fact that the effective tool for presenting knowledge and data for search by specific parameters is required. The convenience of the system is that it is not necessary to address in various data sources to get and conditions of cultivation of mammalian cells, it has already been collected and structured according to parameters. With help of the system, it is possible to select conditions for the cultivation of mammalian cells at the stage of scientific researches that will significantly reduce the time and cost of work, also to rank of recommended technological and hardware solutions. The system has a functional completeness, i.e. in a specific subject area, it ensures the fulfillment of user's requirements, and allows to accumulate and process information.

  3. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Directory of Open Access Journals (Sweden)

    Elizabeth Muir

    Full Text Available There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed

  4. Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

    Science.gov (United States)

    Smith, I M; Baker, A; Arneborg, N; Jespersen, L

    2015-11-01

    The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast

  5. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    International Nuclear Information System (INIS)

    Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

    2014-01-01

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies

  6. Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media.

    Science.gov (United States)

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Fann, John C H

    2013-01-01

    The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed-batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top-down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers.

  7. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Tak, Hyosun, E-mail: chuberry@naver.com [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Rho, Mina, E-mail: minarho@hanyang.ac.kr [Department of Computer Science, Hanyang University, Seoul 133-791 (Korea, Republic of); Chang, Hae Ryung, E-mail: heyhae@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Yon Hui, E-mail: yhkim@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Kyung Tae, E-mail: bioktkim@ncc.re.kr [Molecular Epidemiology Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Balch, Curt, E-mail: curt.balch@gmail.com [Medical Sciences Program, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN 47405 (United States); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Nam, Seungyoon, E-mail: seungyoon.nam@ncc.re.kr [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of)

    2014-03-28

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

  8. Repair of traumatized mammalian hair cells via sea anemone repair proteins.

    Science.gov (United States)

    Tang, Pei-Ciao; Smith, Karen Müller; Watson, Glen M

    2016-08-01

    Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea. © 2016. Published by The Company of Biologists Ltd.

  9. Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

    International Nuclear Information System (INIS)

    Wan Jin; Zheng Hua; Xiao Honglei; She Zhenjue; Zhou Guomin

    2007-01-01

    Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results provide evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons

  10. Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Wan; Hua, Zheng; Honglei, Xiao; Zhenjue, She [Department of Anatomy, Histology and Embryology, Shanghai Medical School, Fudan University, 200032 Shanghai (China); Zhou Guomin [Department of Anatomy, Histology and Embryology, Shanghai Medical School, Fudan University, 200032 Shanghai (China)], E-mail: gmzhou185@yahoo.com.cn

    2007-11-16

    Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results provide evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

  11. Yeast Killer Toxin K28: Biology and Unique Strategy of Host Cell Intoxication and Killing

    Directory of Open Access Journals (Sweden)

    Björn Becker

    2017-10-01

    Full Text Available The initial discovery of killer toxin-secreting brewery strains of Saccharomyces cerevisiae (S. cerevisiae in the mid-sixties of the last century marked the beginning of intensive research in the yeast virology field. So far, four different S. cerevisiae killer toxins (K28, K1, K2, and Klus, encoded by cytoplasmic inherited double-stranded RNA viruses (dsRNA of the Totiviridae family, have been identified. Among these, K28 represents the unique example of a yeast viral killer toxin that enters a sensitive cell by receptor-mediated endocytosis to reach its intracellular target(s. This review summarizes and discusses the most recent advances and current knowledge on yeast killer toxin K28, with special emphasis on its endocytosis and intracellular trafficking, pointing towards future directions and open questions in this still timely and fascinating field of killer yeast research.

  12. Drying enhances immunoactivity of spent brewer's yeast cell wall β-D-glucans.

    Science.gov (United States)

    Liepins, Janis; Kovačova, Elena; Shvirksts, Karlis; Grube, Mara; Rapoport, Alexander; Kogan, Grigorij

    2015-07-20

    Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast β-D-glucans as BRM, and drying as an efficient pretreatment to increase β-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified β-D-glucan. However, drying increased purified β-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted β-D-glucan. This is corroborated with FT-IR analyses of the β-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality β-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast β-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Averbeck, D.; Boucher, D.

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-07-01

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

  15. Radiation induced reproductive death as a function of mammalian cell ploidy

    International Nuclear Information System (INIS)

    Philbrick, D.A.

    1976-09-01

    Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37 0 C, 4 0 C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated 3 HTdR and 125 IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37 0 C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death

  16. Radiation induced reproductive death as a function of mammalian cell ploidy

    Energy Technology Data Exchange (ETDEWEB)

    Philbrick, D.A.

    1976-09-01

    Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37/sup 0/C, 4/sup 0/C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated /sup 3/HTdR and /sup 125/IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37/sup 0/C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death.

  17. Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses

    Science.gov (United States)

    Zhang, Jun; Hartmann, Bastian; Siegel, Julian; Marchi, Gabriele; Clausen-Schaumann, Hauke; Sudhop, Stefanie; Huber, Heinz P.

    2018-01-01

    Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation. PMID:29718923

  18. The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast.

    Science.gov (United States)

    Leitao, Ricardo M; Kellogg, Douglas R

    2017-11-06

    The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast. © 2017 Leitao and Kellogg.

  19. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  20. An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

    Science.gov (United States)

    Murata, Akihiko; Yoshino, Miya; Hikosaka, Mari; Okuyama, Kazuki; Zhou, Lan; Sakano, Seiji; Yagita, Hideo; Hayashi, Shin-Ichi

    2014-01-01

    Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion. PMID:25255288

  1. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

    DEFF Research Database (Denmark)

    Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

    2016-01-01

    Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level p...

  2. Expression of death receptor 4 induces caspase-independent cell death in MMS-treated yeast.

    Science.gov (United States)

    Kang, Mi-Sun; Lee, Sung-Keun; Park, Chang-Shin; Kang, Ju-Hee; Bae, Sung-Ho; Yu, Sung-Lim

    2008-11-14

    DR4, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, is a key element in the extrinsic pathway of TRAIL/TRAIL receptor-related apoptosis that exerts a preferential toxic effect against tumor cells. However, TRAIL and DR4 are expressed in various normal cells, and recent studies indicate that DR4 has a number of non-apoptotic functions. In this study, we evaluated the effects of human DR4 expression in yeast to determine the function of DR4 in normal cells. The expression of DR4 in yeast caused G1 arrest, which resulted in transient growth inhibition. Moreover, treatment of DR4-expressing yeast with a DNA damaging agent, MMS, elicited drastic, and sustained cell growth inhibition accompanied with massive apoptotic cell death. Further analysis revealed that cell death in the presence of DNA damage and DR4 expression was not dependent on the yeast caspase, YCA1. Taken together, these results indicate that DR4 triggers caspase-independent programmed cell death during the response of normal cells to DNA damage.

  3. The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast

    Directory of Open Access Journals (Sweden)

    Ana Belén Sanz

    2017-12-01

    Full Text Available Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies.

  4. Viral Cre-LoxP tools aid genome engineering in mammalian cells.

    Science.gov (United States)

    Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

    2017-01-01

    Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

  5. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-01-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  6. Evaluation of yeast cell wall on the performance of broiles fed diets with or without mycotoxins

    Directory of Open Access Journals (Sweden)

    E Santin

    2006-12-01

    Full Text Available This experiment aimed at evaluating the effects of the interactions between aflatoxin (500 or 250 ppb and ochratoxin (500 or 250 ppb, and the possible benefits of adding yeast cell wall to prevent the effects of these mycotoxins in broiler chickens. Relative organ weight gain and live performance were evaluated at 21 and 42 days of age. Results indicated that at the levels of mycotoxins included in the experimental diets, ochratoxin reduced feed intake and body weight gain, and aflatoxin only affect feed intake of 21-day-old birds. No interaction was observed between aflatoxin and ochratoxin at the levels used in experimental study. Yeast cell wall did not significantly reduced the deleterious effects of ochratoxins. No significant differences were observed in relative organ weight gain. Yeast cell wall improved feed conversion ratio when birds were fed either contaminated or non-contaminated feeds.

  7. Damage and repair in mammalian cells after exposure to non-ionizing radiations. 1

    International Nuclear Information System (INIS)

    Harm, H.

    1978-01-01

    Cornea cells of the rat kangaroo or 'potoroo' (Potorous tridactylus) were exposed to far-UV (254 or 302 nm) radiation, with or without subsequent illumination by near-UV or visible light. The DNA of these cells was extracted and tested for the presence of photoproducts binding yeast photoreactivating enzyme (PRE). The effects on repair kinetics of the transforming DNA indicate that in UV-irradiated potoroo cornea cells up to approximately 90% of photorepairable DNA damage can be photorepaired within 15 min. However, the extent of cellular photorepair depends appreciably on experimental parameters during photoreactivating treatment, including the spectral composition of photoreactivating light. Apparently superposition of damage by the photoreactivating treatment itself is the critical factor. This may explain experimental discrepancies existing in different laboratories studying photorepair in UV-irradiated cells of placental mammals. (Auth.)

  8. The self-orientation of mammalian cells in optical tweezers—the importance of the nucleus

    International Nuclear Information System (INIS)

    Perney, Nicolas M B; Horak, Peter; Melvin, Tracy; Hanley, Neil A

    2012-01-01

    Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations. (communication)

  9. Non-lethal effects of low- and high-LET radiation on cultured mammalian cells

    International Nuclear Information System (INIS)

    Walker, J.T.

    1982-01-01

    In analyzing post-irradiation growth kinetics of cultured mammalian cells, specifically T1-E human cells, this investigation shows that the shift in post-irradiation clone-size distributions toward small colonies is due to both radiation-induced division delay and increased generation times of the irradiated population. Evidence also indicates that the final shape of the final clone-size distribution is influenced by the age density distribution of the parent cells at the time of plating. From computer-generated delay time distributions it was determined that a large percentage of the parent population was found to be in the plateau phase at early growth times and evidence indicates that these cells may contribute heavily to the total population response to radiation

  10. Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Ronen, A.; Gingerich, J.D.; Duncan, A.M.V.; Heddle, J.A.

    1984-01-01

    Diptheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. Resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [ 3 H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [ 3 H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium. 23 references, 6 figures

  11. Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

    Science.gov (United States)

    Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

    2018-04-02

    During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Ethanol production potential from fermented rice noodle wastewater treatment using entrapped yeast cell sequencing batch reactor

    Science.gov (United States)

    Siripattanakul-Ratpukdi, Sumana

    2012-03-01

    Fermented rice noodle production generates a large volume of starch-based wastewater. This study investigated the treatment of the fermented rice noodle wastewater using entrapped cell sequencing batch reactor (ECSBR) compared to traditional sequencing batch reactor (SBR). The yeast cells were applied because of their potential to convert reducing sugar in the wastewater to ethanol. In present study, preliminary treatment by acid hydrolysis was performed. A yeast culture, Saccharomyces cerevisiae, with calcium alginate cell entrapment was used. Optimum yeast cell loading in batch experiment and fermented rice noodle treatment performances using ECSBR and SBR systems were examined. In the first part, it was found that the cell loadings (0.6-2.7 × 108 cells/mL) did not play an important role in this study. Treatment reactions followed the second-order kinetics with the treatment efficiencies of 92-95%. In the second part, the result showed that ECSBR performed better than SBR in both treatment efficiency and system stability perspectives. ECSBR maintained glucose removal of 82.5 ± 10% for 5-cycle treatment while glucose removal by SBR declined from 96 to 40% within the 5-cycle treatment. Scanning electron microscopic images supported the treatment results. A number of yeast cells entrapped and attached onto the matrix grew in the entrapment matrix.

  13. Synchronized mammalian cell culture: part I--a physical strategy for synchronized cultivation under physiological conditions.

    Science.gov (United States)

    Barradas, Oscar Platas; Jandt, Uwe; Becker, Max; Bahnemann, Janina; Pörtner, Ralf; Zeng, An-Ping

    2015-01-01

    Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large-scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole-culture methods. Conventional nonsynchronizing methods with subsequent cell-specific, for example, flow cytometric analysis, can only resolve cell-limited effects of the cell cycle. In this work, we demonstrate countercurrent-flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO-K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2/M phase, with enrichment factors (Ysync) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This

  14. Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

    Science.gov (United States)

    Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

    2017-08-01

    The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.

  15. The effects of UV irradiation and gas plasma treatment on living mammalian cells and bacteria: a comparative approach

    NARCIS (Netherlands)

    Sosnin, E.A.; Stoffels - Adamowicz, E.; Erofeev, M.V.; Kieft, I.E.; Kunts, S.E.

    2004-01-01

    Living mammalian cells and bacteria were exposed to irradiation from narrow-band UV lamps and treated with a nonthermal gas plasma (plasma needle). The model systems were: Chinese Hamster Ovary (CHO-K1) cells (fibroblasts) and Escherichia Coli bacteria. UV irradiation can lead to cell death

  16. RNAi screening for characterisation of ER-associated degradation pathways in mammalian cells

    DEFF Research Database (Denmark)

    Månsson, Mats David Joakim

    in a process termed ER-associated degradation (ERAD). This mechanism proceeds through four steps involving recognition, dislocation, ubiquitination and proteasomal degradation. This report describes a high-throughput screening method for identification of hitherto unknown pathways for degradation. We present...... fluorescence-based RNAi screens in mammalian cells on TCR-α-GFP and HANSκLC, for identification of ERAD pathways. By validating the obtained screening hits we concluded that UBE2J2 is involved in TCR-α-GFP degradation, possibly by ubiquitination of C-terminal serine residues in TCR-α-GFP. Additionally, we also...

  17. Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

    OpenAIRE

    Song, Yuanda; Dilger, Emily; Bell, Jessica; Barton, William A; Fang, Xianjun

    2010-01-01

    We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29–915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated fro...

  18. Immobilization of yeast cells on hydrogel carriers obtained by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Luzhao Xin; Carenza, M.; Kaetsu, Isao; Kumakura, Minoru; Yoshida, Masaru; Fujimura, Takashi

    1992-01-01

    Polymer hydrogels were obtained by radiation-induced copolymerization at -78 o C of aqueous solutions of acrylic and methacrylic esters. The matrices were characterized by equilibrium water content measurements, by optical microscopy observations and by scanning electron microscopy analysis. Yeast cells were immobilized on these hydrogels and the ethanol productivity by batch fermentation was determined. Matrix hydrophilicity and porosity were found to deeply influence the adhesion of yeast cells and, hence, the ethanol productivity. The latter as well as other physico-chemical properties were also affected by the presence of a crosslinking agent added in small amounts to the polymerizating mixture. (author)

  19. Soft x-ray-controlled dose deposition in yeast cells: techniques, model, and biological assessment

    Science.gov (United States)

    Milani, Marziale; Batani, Dimitri; Conti, Aldo; Masini, Alessandra; Costato, Michele; Pozzi, Achille; Turcu, I. C. Edmond

    1996-12-01

    A procedure is presented to release soft x-rays onto yeast cell membrane allegedly damaging the resident enzymatic processes connected with fermentation. The damage is expected to be restricted to regulating fermentation processes without interference with respiration. By this technique fermentation is followed leading to CO2 production, and respiration resulting in global pressure measurements. A solid state pressure sensor system has been developed linked to a data acquisition system. Yeast cells cultures have been investigated at different concentrations and with different nutrients. A non-monotone response in CO2 production as a function of the delivered x-ray dose is observed.

  20. A multi-landing pad DNA integration platform for mammalian cell engineering

    Science.gov (United States)

    Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

    2018-01-01

    Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

  1. Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.

    Science.gov (United States)

    Erickson, Andrea K; Jesudhasan, Palmy R; Mayer, Melinda J; Narbad, Arjan; Winter, Sebastian E; Pfeiffer, Julie K

    2018-01-10

    RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Some important advances in DNA repair study on the mammalian cells

    International Nuclear Information System (INIS)

    Xia Shouxuan.

    1991-01-01

    In the recent years the study of DNA damage and repair in the mammalian cells has gone deeply at gene level and got the following advances: (1) For a long time DNA has been considered to be an uniform unit in case of damage and repair. Now this concept should be replaced by the non-random distribution of damage and heterogenous repair in the genome. These would allow us to study cellular mutagenesis, carcinogenesis, aging and dying processes in great detail, and would be beneficial to the elucidation of mechanisms of radiation sickness and chemical toxicology. (2) The advent of new techniques in molecular biology has made it possible to isolate and clone the human DNA repair genes. Up to now more than ten human DNA repair genes have been cloned and these works would have an important impact on the theoretical and practical study in this field. Because DNA repair system is very complicate, voluminous work should be done in the future. (3) The technique of gene transfer has been efficiently used in the study of DNA repair in mammalian cells and has made great contribution in the cellular engineering. It could modify the genetic behavior of the gene-accepting cells, and enhance the DNA repair ability to physical and chemical damages. Human gene therapy for DNA deficient diseases is now on the day

  3. Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

    Science.gov (United States)

    Kreuzer, Helen W.; Quaroni, Luca; Podlesak, David W.; Zlateva, Theodora; Bollinger, Nikki; McAllister, Aaron; Lott, Michael J.; Hegg, Eric L.

    2012-01-01

    Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue. PMID:22848359

  4. Structural and functional organization of ribosomal genes within the mammalian cell nucleolus.

    Science.gov (United States)

    Derenzini, Massimo; Pasquinelli, Gianandrea; O'Donohue, Marie-Françoise; Ploton, Dominique; Thiry, Marc

    2006-02-01

    Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.

  5. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  6. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  7. Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

    Science.gov (United States)

    Moussa, Hassan I.; Logan, Megan; Siow, Geoffrey C.; Phann, Darron L.; Rao, Zheng; Aucoin, Marc G.; Tsui, Ting Y.

    2017-12-01

    Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only 22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

  8. Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Mohamed Ali Abol Hassan

    2011-12-01

    Full Text Available The extracellular expression of monoclonal antibodies (mAbs in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi, kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam

  9. Tuning the Density of Poly(ethylene glycol Chains to Control Mammalian Cell and Bacterial Attachment

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Ani

    2017-08-01

    Full Text Available Surface modification of biomaterials with polymer chains has attracted great attention because of their ability to control biointerfacial interactions such as protein adsorption, cell attachment and bacterial biofilm formation. The aim of this study was to control the immobilisation of biomolecules on silicon wafers using poly(ethylene glycol(PEG chains by a “grafting to” technique. In particular, to control the polymer chain graft density in order to capture proteins and preserve their activity in cell culture as well as find the optimal density that would totally prevent bacterial attachment. The PEG graft density was varied by changing the polymer solubility using an increasing salt concentration. The silicon substrates were initially modified with aminopropyl-triethoxysilane (APTES, where the surface density of amine groups was optimised using different concentrations. The results showed under specific conditions, the PEG density was highest with grafting under “cloud point” conditions. The modified surfaces were characterised with X-ray photoelectron spectroscopy (XPS, ellipsometry, atomic force microscopy (AFM and water contact angle measurements. In addition, all modified surfaces were tested with protein solutions and in cell (mesenchymal stem cells and MG63 osteoblast-like cells and bacterial (Pseudomonas aeruginosa attachment assays. Overall, the lowest protein adsorption was observed on the highest polymer graft density, bacterial adhesion was very low on all modified surfaces, and it can be seen that the attachment of mammalian cells gradually increased as the PEG grafting density decreased, reaching the maximum attachment at medium PEG densities. The results demonstrate that, at certain PEG surface coverages, mammalian cell attachment can be tuned with the potential to optimise their behaviour with controlled serum protein adsorption.

  10. Intercellular contact: its influence on the Dsub(q) of mammalian cell survival curves

    International Nuclear Information System (INIS)

    Durand, R.E.; Sutherland, R.M.

    1975-01-01

    Cell survival in tissues exposed to a given dose of ionizing radiation is usually greater than that of similar cells grown individually in vitro, despite the fact that the radiosensitivities (D 0 ) are virtually identical under the two conditions. An analogous increase in cell survival is observed when Chinese hamster V79-171 cells are grown in suspension culture and irradiated as multicell spheroids. Unfortunately, the information gained from the survival curves so obtained is limited by the inhomogeneity of the cell population with respect to both degree of contact and cell cycle position. The latter can be studied using synchronized small spheroids. The ratio of Dsub(q) of spheroid cells to Dsub(q) of single cells increased as the cells progressed through the cell cycle, from a minimum of 1.3 for G 1 phase cells to a maximum of 2.2 for late S-phase cells. The enhanced survival, or 'contact effect', developed slowly as the spheroids grew, after an initial latent period of about one generation cycle of the cells. A second effect of intercellular contact on mammalian cell survival has also been observed. When cells are assayed under conditions in which intercellular contact is maintained, the net cellular survival is increased further. This effect is different from the usual repair of potentially lethal damage, in that it occurs much more slowly and results in modification of the survival-curve shoulder. Not all cell types tested have shown enhanced survival when grown as spheroids. Several MNNG-induced mutants of the Chinese hamster V79-171 line have been isolated and sublines which do and do not show the contact effect are now available. These may permit study of the mechanism(s) of contact effects. (author)

  11. Singlet Oxygen Sensor Green: Photochemical Behavior in Solution and in a Mammalian Cell

    DEFF Research Database (Denmark)

    Gollmer, Anita; Arnbjerg, Jacob; Blaikie, Frances Helen

    2011-01-01

    The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green® (SOSG), a commercially available fluorescent probe...... for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product...... of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields...

  12. Bacterial mutagenicity and mammalian cell DNA damage by several substituted anilines.

    Science.gov (United States)

    Zimmer, D; Mazurek, J; Petzold, G; Bhuyan, B K

    1980-04-01

    Several substituted alkyl- and haloanilines were tested for their ability to mutate Salmonella typhimurium and to damage the DNA of mammalian (V79) cells. These results were correlated with their reported carcinogenicity. Of 9 suspected carcinogens, 4 were bacterial mutagens and 4 (out of 7 tested) damaged DNA of V79 cells. The following compounds were weakly mutagenic (less than 150 revertants/mumole): 4-fluoroaniline, 2,3-, 2,4-, 2,5- and 3,4-dimethylaniline, and 2-methyl-4-fluoroaniline. The following compounds were strong mutagens: 2,4,5-trimethylaniline, 2-methyl-4-chloro-, and 2-methyl-4-bromo-, 4-methyl-2-chloro-, 4-methyl-2-bromo- and 2-ethyl-4-chloroaniline. The compounds which damaged DNA in V79 cells were: 2 methyl-4-chloroaniline, 2-methyl-4-bromoaniline, 2,4,5- and 2,4,6-trimethylaniline.

  13. A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors.

    Science.gov (United States)

    Xing, Zizhuo; Lewis, Amanda M; Borys, Michael C; Li, Zheng Jian

    2017-06-01

    Control of carbon dioxide within the optimum range is important in mammalian bioprocesses at the manufacturing scale in order to ensure robust cell growth, high protein yields, and consistent quality attributes. The majority of bioprocess development work is done in laboratory bioreactors, in which carbon dioxide levels are more easily controlled. Some challenges in carbon dioxide control can present themselves when cell culture processes are scaled up, because carbon dioxide accumulation is a common feature due to longer gas-residence time of mammalian cell culture in large scale bioreactors. A carbon dioxide stripping model can be used to better understand and optimize parameters that are critical to cell culture processes at the manufacturing scale. The prevailing carbon dioxide stripping models in literature depend on mass transfer coefficients and were applicable to cell culture processes with low cell density or at stationary/cell death phase. However, it was reported that gas bubbles are saturated with carbon dioxide before leaving the culture, which makes carbon dioxide stripping no longer depend on a mass transfer coefficient in the new generation cell culture processes characterized by longer exponential growth phase, higher peak viable cell densities, and higher specific production rate. Here, we present a new carbon dioxide stripping model for manufacturing scale bioreactors, which is independent of carbon dioxide mass transfer coefficient, but takes into account the gas-residence time and gas CO 2 saturation time. The model was verified by CHO cell culture processes with different peak viable cell densities (7 to 12 × 10 6  cells mL -1 ) for two products in 5,000-L and 25,000-L bioreactors. The model was also applied to a next generation cell culture process to optimize cell culture conditions and reduce carbon dioxide levels at manufacturing scale. The model provides a useful tool to understand and better control cell culture carbon dioxide

  14. Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

    Science.gov (United States)

    Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S; Ellisman, Mark H; Ting, Alice Y

    2018-01-01

    Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). PMID:28796234

  15. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer; Campbell, Keith H.S.

    2005-01-01

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  16. Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

    Science.gov (United States)

    Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

    2017-11-06

    Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  17. Immobilization of yeast cells with ionic hydrogel produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu Zhaoxin; Fujimura, T.

    1990-01-01

    The mixture of an ionic monomer of 2-acrylamido 2-methylpropane-sulfonic acid and a series of polyethylene glycol dimethacrylate monomer were polymerized at-78 deg C with 60 Co γ-rays and were used for immobilization of yeast cells. The immobilized yeast cells with these carriers had higher ethanol productivity than that without any carriers. The yield of ethanol with poly TBAS-14G carrier was the highest, and increased by 3.5 times compared with the free yeast cells. It was found that the ethanol yield increased with the increase of the glycol number in polyethylene glycol dimethacrylate. The state of the immobilized cells was observed with microscope and it was found that the difference in the ethanol productivity was mainly due to the difference in the internal structure and the properties of polymer carrier. It was considered that the polymer carrier had a proper hydrophilicity, swelling ability, cation in the surface and porousity in the internal structure for immobilizing yeast cells

  18. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    Directory of Open Access Journals (Sweden)

    Elham Aslankoohi

    Full Text Available Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.

  19. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    Science.gov (United States)

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.

  20. Methods for Synchronization and Analysis of the Budding Yeast Cell Cycle.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    Like other eukaryotes, budding yeast temporally separate cell growth and division. DNA synthesis is distinct from chromosome segregation. Storage carbohydrates are accumulated slowly and then rapidly liquidated once per cycle. Cyclin-dependent kinase associates with multiple different transcriptionally and posttranslationally regulated cyclins to drive the cell cycle. These and other crucial events of cellular growth and division are limited to narrow windows of the cell cycle. Many experiments in the yeast laboratory treat a culture of cells as a homogeneous mixture. Measurements of asynchronous cultures are, however, confounded by the presence of cells in various cell cycle stages; measuring a population average in unsynchronized cells provides at best a decreased signal and at worst an artifactual result. A number of experimentally tractable methods have been developed to generate populations of yeast cells that are synchronized with respect to cell cycle phase. Robust methods for determining cell cycle position have also been developed. These methods are introduced here. © 2017 Cold Spring Harbor Laboratory Press.

  1. Radiosensitization of mammalian cells by misonidazole and oxygen: DNA damage exposed by Micrococcus luteus enzymes

    International Nuclear Information System (INIS)

    Skov, K.A.; Palcic, B.; Skarsgard, L.D.

    1979-01-01

    When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37 0 C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract

  2. Quality by control: Towards model predictive control of mammalian cell culture bioprocesses.

    Science.gov (United States)

    Sommeregger, Wolfgang; Sissolak, Bernhard; Kandra, Kulwant; von Stosch, Moritz; Mayer, Martin; Striedner, Gerald

    2017-07-01

    The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach. The application of advanced sensor systems in combination with mathematical modelling techniques offers enhanced process understanding, allows on-line prediction of critical quality attributes and subsequently real-time product quality control. In this review opportunities and unsolved issues on the road to a successful quality by design and dynamic control implementation are discussed. A major focus is directed on the preconditions for the application of model predictive control for mammalian cell culture bioprocesses. Design of experiments providing information about the process dynamics upon parameter change, dynamic process models, on-line process state predictions and powerful software environments seem to be a prerequisite for quality by control realization. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Role of H1 linker histones in mammalian development and stem cell differentiation.

    Science.gov (United States)

    Pan, Chenyi; Fan, Yuhong

    2016-03-01

    H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Control of radiation sensitivity of mammalian cells. Regulation of expression of DNA repair genes

    International Nuclear Information System (INIS)

    Yoshida, Kayo; Morita, Takashi

    2003-01-01

    This review describes authors' investigations concerning regulation of expression of DNA repair genes for the purpose of control of radiosensitivity of mammalian cells for cancer radiotherapy. One of their experiments concerns the enhancement of sensitivity to radiation and anti-tumor agents by suppressing the expression of mammalian Rad51 gene which playing a central role in recombination repair against DNA double-strand break, by RNA interference (RNAi). Described are the mode of action of RNAi, mechanism of suppression of Rad51 gene expression by it, enhancing effect in radiosensitivity, stable suppression and enhancement by hairpin RNA and its possible usefulness in cancer therapy. The other concerns the histone H2AX gene, which delivering the repair signal post phosphorylation in chromatin against the double-strand break. Experimental results of suppression of the histone H2AX gene by tet-off system, enhancement of radiosensitivity by the suppression and functional recovery by the gene transfer are described, and the radiosensitivity can be thus artificially controlled by tetracycline in authors' F9 2AX (tet/tet) cells. (N.I.)

  5. Incorporation of functionalized gold nanoparticles into nanofibers for enhanced attachment and differentiation of mammalian cells

    Directory of Open Access Journals (Sweden)

    Jung Dongju

    2012-06-01

    Full Text Available Abstract Background Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata. Results Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells. Conclusions PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations.

  6. Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

    Directory of Open Access Journals (Sweden)

    Weeks Sara K

    2009-01-01

    Full Text Available Abstract Background The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars. Results A plasmid transfection approach was used to examine the function of the product of CT223 and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that CT223, and, to a lesser extent, adjacent inc genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells. Conclusion These studies suggest that certain Inc proteins block cytokinesis in C. trachomatis-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.

  7. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07