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Sample records for protects mammalian cells

  1. Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

    PubMed Central

    Liao, Xiaoli; Rabideau, Amy E; Pentelute, Bradley L

    2014-01-01

    Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu. PMID:25250705

  2. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    PubMed Central

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  3. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    PubMed

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  4. Triclabendazole protects yeast and mammalian cells from oxidative stress: Identification of a potential neuroprotective compound

    PubMed Central

    Lee, Yong Joo; Burlet, Elodie; Wang, Shaoxiao; Xu, Baoshan; Huang, Shile; Galiano, Floyd J.; Witt, Stephan N.

    2011-01-01

    The Prestwick and NIH chemical libraries were screened for drugs that protect baker’s yeast from sugar-induced cell death (SICD). SICD is triggered when stationary-phase yeast cells are transferred from spent rich medium into water with 2% glucose and no other nutrients. The rapid, apoptotic cell death occurs because reactive oxygen species (ROS) accumulate. We found that triclabendazole, which is used to treat liver flukes in cattle and man, partially protects against SICD. Characterization of triclabendazole revealed that it also protects yeast cells from death induced by the Parkinson’s disease-related protein alpha-synuclein (α-syn), which is known to induce the accumulation of ROS. PMID:21946065

  5. Differential chemical protection of mammalian cells from the exotoxins of Corynebacterium diphtheriae and Pseudomonas aeruginosa.

    PubMed Central

    Middlebrook, J L; Dorland, R B

    1977-01-01

    Many drugs or chemicals had markedly different effects on the cytotoxicity induced by Pseudomonas aeruginosa exotoxin A (PE) or Corynebacterium diphtheriae exotoxin (DE). The glycolytic inhibitor NaF protected cells from DE but potentiated the cytotoxicity of PE. Another energy inhibitor, salicylic acid, also protected cells from DE but had no effect with PE. Colchicine and colcemid did not affect the cytotoxicity of either toxin. Cytochalasin B exhibited a modest protection from DE but no effect with PE. Ouabain, a specific inhibitor of the Na+, K+-dependent adenosine 5'-triphosphatase (ATPase), did not affect the cytotoxicity of either toxin. Ruthenium red, a specific inhibitor of the Ca2+, Mg2+,-dependent ATPase, conferred marked protection from DE-induced cytotoxicity but did not affect PE-induced cytotoxicity. A number of local anesthetics were tested, and they too presented differential results with PE and DE. Most chemicals that affected toxin-induced cytotoxicity had little or no influence on the in vitro adenosine 5'-diphosphate-ribosylation catalyzed by either toxin. This work presents further evidence that PE and DE have different mechanisms of intoxication and suggests that these differences lie in the attachment or internalization stages of intoxication. PMID:141424

  6. Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy*

    PubMed Central

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-01-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  7. Conophylline protects cells in cellular models of neurodegenerative diseases by inducing mammalian target of rapamycin (mTOR)-independent autophagy.

    PubMed

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-03-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  8. Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro

    SciTech Connect

    Sakurai, Toshiharu; Itoh, Katsuhiko; Liu Yu; Higashitsuji, Hiroaki; Sumitomo, Yasuhiko; Sakamaki, Kazuhiro; Fujita, Jun . E-mail: jfujita@virus.kyoto-u.ac.jp

    2005-10-01

    Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 deg. C) on cells exposed to a variety of stress in vitro. We found that 32 deg. C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H{sub 2}O{sub 2}, and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 deg. C to 32 deg. C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53 {sup -/-}/Mdm2 {sup -/-} mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53 {sup -/-}/Mdm2 {sup -/-} cells exposed to thapsigargin was increased when cultured at 32 deg. C compared with 37 deg. C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.

  9. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    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)

  10. Nanoelectrochemistry of mammalian cells

    PubMed Central

    Sun, Peng; Laforge, François O.; Abeyweera, Thushara P.; Rotenberg, Susan A.; Carpino, James; Mirkin, Michael V.

    2008-01-01

    There is a significant current interest in development of new techniques for direct characterization of the intracellular redox state and high-resolution imaging of living cells. We used nanometer-sized amperometric probes in combination with the scanning electrochemical microscope (SECM) to carry out spatially resolved electrochemical experiments in cultured human breast cells. With the tip radius ≈1,000 times smaller than that of a cell, an electrochemical probe can penetrate a cell and travel inside it without apparent damage to the membrane. The data demonstrate the possibility of measuring the rate of transmembrane charge transport and membrane potential and probing redox properties at the subcellular level. The same experimental setup was used for nanoscale electrochemical imaging of the cell surface. PMID:18178616

  11. Nanoelectrochemistry of mammalian cells.

    PubMed

    Sun, Peng; Laforge, François O; Abeyweera, Thushara P; Rotenberg, Susan A; Carpino, James; Mirkin, Michael V

    2008-01-15

    There is a significant current interest in development of new techniques for direct characterization of the intracellular redox state and high-resolution imaging of living cells. We used nanometer-sized amperometric probes in combination with the scanning electrochemical microscope (SECM) to carry out spatially resolved electrochemical experiments in cultured human breast cells. With the tip radius approximately 1,000 times smaller than that of a cell, an electrochemical probe can penetrate a cell and travel inside it without apparent damage to the membrane. The data demonstrate the possibility of measuring the rate of transmembrane charge transport and membrane potential and probing redox properties at the subcellular level. The same experimental setup was used for nanoscale electrochemical imaging of the cell surface. PMID:18178616

  12. Reprogramming mammalian somatic cells.

    PubMed

    Rodriguez-Osorio, N; Urrego, R; Cibelli, J B; Eilertsen, K; Memili, E

    2012-12-01

    Somatic cell nuclear transfer (SCNT), the technique commonly known as cloning, permits transformation of a somatic cell into an undifferentiated zygote with the potential to develop into a newborn animal (i.e., a clone). In somatic cells, chromatin is programmed to repress most genes and express some, depending on the tissue. It is evident that the enucleated oocyte provides the environment in which embryonic genes in a somatic cell can be expressed. This process is controlled by a series of epigenetic modifications, generally referred to as "nuclear reprogramming," which are thought to involve the removal of reversible epigenetic changes acquired during cell differentiation. A similar process is thought to occur by overexpression of key transcription factors to generate induced pluripotent stem cells (iPSCs), bypassing the need for SCNT. Despite its obvious scientific and medical importance, and the great number of studies addressing the subject, the molecular basis of reprogramming in both reprogramming strategies is largely unknown. The present review focuses on the cellular and molecular events that occur during nuclear reprogramming in the context of SCNT and the various approaches currently being used to improve nuclear reprogramming. A better understanding of the reprogramming mechanism will have a direct impact on the efficiency of current SCNT procedures, as well as iPSC derivation. PMID:22979962

  13. Radiation protection of in vitro mammalian cells: effects of hydroxyl radical scavengers on the slopes and shoulders of survival curves

    SciTech Connect

    Ewing, D.; Walton, H.L. )

    1991-05-01

    We have tested several chemical compounds, characterized and widely used as hydroxyl radical (.OH) scavengers, for their effects on the radiation sensitivity of Chinese hamster V79 cells irradiated in air or nitrogen. Our purpose is to reexamine the proposed relationship between the level of protection and the rates at which the scavengers react with .OH. We found that the additives can have two apparently independent effects on the shape of survival curves: a reduction in sensitivity (i.e., 'protection,' a decrease in the value of k) and an increase in the size of the shoulder of the survival curve (an increase in the value of Dq). We measured intracellular scavenger concentrations, and, using these values in our analysis, we found that neither of the two effects is correlated with the rates at which the scavengers react with .OH. Although these results could mean that .OH do not cause lethal damage, the interpretation we believe most probably correct is that these scavengers protect in multiple ways. The protection would occur in addition to or instead of simple .OH removal.

  14. Myocardial ischemic protection in natural mammalian hibernation.

    PubMed

    Yan, Lin; Kudej, Raymond K; Vatner, Dorothy E; Vatner, Stephen F

    2015-03-01

    Hibernating myocardium is an important clinical syndrome protecting the heart with chronic myocardial ischemia, named for its assumed resemblance to hibernating mammals in winter. However, the effects of myocardial ischemic protection have never been studied in true mammalian hibernation, which is a unique strategy for surviving extreme winter environmental stress. The goal of this investigation was to test the hypothesis that ischemic stress may also be protected in woodchucks as they hibernate in winter. Myocardial infarction was induced by coronary occlusion followed by reperfusion in naturally hibernating woodchucks in winter with and without hibernation and in summer, when not hibernating. The ischemic area at risk was similar among groups. Myocardial infarction was significantly less in woodchucks in winter, whether hibernating or not, compared with summer, and was similar to that resulting after ischemic preconditioning. Whereas several genes were up or downregulated in both hibernating woodchuck and with ischemic preconditioning, one mechanism was unique to hibernation, i.e., activation of cAMP-response element binding protein (CREB). When CREB was upregulated in summer, it induced protection similar to that observed in the woodchuck heart in winter. The cardioprotection in hibernation was also mediated by endothelial nitric oxide synthase, rather than inducible nitric oxide synthase. Thus, the hibernating woodchuck heart is a novel model to study cardioprotection for two major reasons: (1) powerful cardioprotection occurs naturally in winter months in the absence of any preconditioning stimuli, and (2) it resembles ischemic preconditioning, but with novel mechanisms, making this model potentially useful for clinical translation. PMID:25613166

  15. Myocardial ischemic protection in natural mammalian hibernation

    PubMed Central

    Yan, Lin; Kudej, Raymond K.; Vatner, Dorothy E.

    2015-01-01

    Hibernating myocardium is an important clinical syndrome protecting the heart with chronic myocardial ischemia, named for its assumed resemblance to hibernating mammals in winter. However, the effects of myocardial ischemic protection have never been studied in true mammalian hibernation, which is a unique strategy for surviving extreme winter environmental stress. The goal of this investigation was to test the hypothesis that ischemic stress may also be protected in woodchucks as they hibernate in winter. Myocardial infarction was induced by coronary occlusion followed by reperfusion in naturally hibernating woodchucks in winter with and without hibernation and in summer, when not hibernating. The ischemic area at risk was similar among groups. Myocardial infarction was significantly less in woodchucks in winter, whether hibernating or not, compared with summer, and was similar to that resulting after ischemic preconditioning. Whereas several genes were up or downregulated in both hibernating woodchuck and with ischemic preconditioning, one mechanism was unique to hibernation, i.e., activation of cAMP-response element binding protein (CREB). When CREB was upregulated in summer, it induced protection similar to that observed in the woodchuck heart in winter. The cardioprotection in hibernation was also mediated by endothelial nitric oxide synthase, rather than inducible nitric oxide synthase. Thus, the hibernating woodchuck heart is a novel model to study cardioprotection for two major reasons: (1) powerful cardioprotection occurs naturally in winter months in the absence of any preconditioning stimuli, and (2) it resembles ischemic preconditioning, but with novel mechanisms, making this model potentially useful for clinical translation. PMID:25613166

  16. Producing Newborn Synchronous Mammalian Cells

    NASA Technical Reports Server (NTRS)

    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.

  17. Mast cells in mammalian brain.

    PubMed

    Dropp, J J

    1976-01-01

    Mast cells, which had until recently been believed to be not present in the mammalian brain, were studied in the brains of 29 mammalian species. Although there was considerable intraspecific and interspecific variation, mast cells were most numerous within the leptomeninges (especially in those overlying the cerebrum and the dorsal thalamus - most rodents, most carnivores, chimpanzees, squirrel monkeys and elephant), the cerebral cortex (most rodents, tiger, fox, chimpanzee, tarsier, and elephant) and in many nuclei of the dorsal thalamus (most rodents, tiger, lion, and fox). In some mammals, mast cells were also numerous in the stroma of the telencephalic choroid plexuses (chimpanzee, squirrel monkey), the putamen and the claustrum (chimpanzee), the subfornical organ (pack rat, tiger, chimpanzee), the olfactory peduncles (hooded rat, albino rat), the stroma of the diencephalic choroid plexus (lion, chimpanzee, squirrel monkey), the pineal organ (chimpanzee, squirrel monkey), some nuclei of the hypothalamus (tiger), the infundibulum (hooded rat, tiger, fox) the area postrema (pack rat, chinchilla, lion, spider monkey, chimpanzee, fox) and some nuclei and tracts of the metencephalon and the myelencephalon (tiger). Neither the sex of the animal nor electrolytic lesions made in the brains of some of the animals at various times prior to sacrifice appeared to effect the number and the distribution of mast cells. Age-related changes in mast cell number and distribution were detected in the albino rat. PMID:961335

  18. Mammalian cell cultivation in space

    NASA Astrophysics Data System (ADS)

    Gmünder, Felix K.; Suter, Robert N.; Kiess, M.; Urfer, R.; Nordau, C.-G.; Cogoli, A.

    Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 μl. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

  19. Cell death in mammalian development.

    PubMed

    Penaloza, C; Orlanski, S; Ye, Y; Entezari-Zaher, T; Javdan, M; Zakeri, Z

    2008-01-01

    During embryogenesis there is an exquisite orchestration of cellular division, movement, differentiation, and death. Cell death is one of the most important aspects of organization of the developing embryo, as alteration in timing, level, or pattern of cell death can lead to developmental anomalies. Cell death shapes the embryo and defines the eventual functions of the organs. Cells die using different paths; understanding which path a dying cell takes helps us define the signals that regulate the fate of the cell. Our understanding of cell death in development stems from a number of observations indicating genetic regulation of the death process. With today's increased knowledge of the pathways of cell death and the identification of the genes whose products regulate the pathways we know that, although elimination of some of these gene products has no developmental phenotype, alteration of several others has profound effects. In this review we discuss the types and distributions of cell death seen in developing mammalian embryos as well as the gene products that may regulate the process. PMID:18220829

  20. Low concentration of exogenous carbon monoxide protects mammalian cells against proliferation induced by radiation-induced bystander effect.

    PubMed

    Tong, Liping; Yu, K N; Bao, Lingzhi; Wu, Wenqing; Wang, Hongzhi; Han, Wei

    2014-01-01

    Radiation-induced bystander effect (RIBE) has been proposed to have tight relationship with the irradiation-caused secondary cancers beyond the irradiation-treated area after radiotherapy. Our previous studies demonstrated a protective effect of low concentration carbon monoxide (CO) on the genotoxicity of RIBE after α-particle irradiation. In the present work, a significant inhibitory effect of low-dose exogenous CO, generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on both RIBE-induced proliferation and chromosome aberration was observed. Further studies on the mechanism revealed that the transforming growth factor β1/nitric oxide (NO) signaling pathway, which mediated RIBE signaling transduction, could be modulated by CO involved in the protective effects. Considering the potential of exogenous CO in clinical applications and its protective effect on RIBE, the present work aims to provide a foundation for potential application of CO in radiotherapy. PMID:24333162

  1. Simplified Bioreactor For Growing Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F.

    1995-01-01

    Improved bioreactor for growing mammalian cell cultures developed. Designed to support growth of dense volumes of mammalian cells by providing ample, well-distributed flows of nutrient solution with minimal turbulence. Cells relatively delicate and, unlike bacteria, cannot withstand shear forces present in turbulent flows. Bioreactor vessel readily made in larger sizes to accommodate greater cell production quantities. Molding equipment presently used makes cylinders up to 30 centimeters long. Alternative sintered plastic techniques used to vary pore size and quantity, as necessary.

  2. Engineered Trehalose Permeable to Mammalian Cells

    PubMed Central

    Abazari, Alireza; Meimetis, Labros G.; Budin, Ghyslain; Bale, Shyam Sundhar; Weissleder, Ralph; Toner, Mehmet

    2015-01-01

    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. PMID:26115179

  3. Hacking the genetic code of mammalian cells.

    PubMed

    Schwarzer, Dirk

    2009-07-01

    A genetic shuttle: The highlighted article, which was recently published by Schultz, Geierstanger and co-workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line. PMID:19533721

  4. Isotope Labeling in Mammalian Cells

    PubMed Central

    Dutta, Arpana; Saxena, Krishna; Klein-Seetharaman, Judith

    2011-01-01

    Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. PMID:22167668

  5. Protecting effects specifically from low doses of ionizing radiation to mammalian cells challenge the concept of linearity

    SciTech Connect

    Feinendegen, L.E.; Bond, V.P.; Sondhaus, C.A.; Altman, K.I.

    1998-12-31

    This report examines the origin of tissue effects that may follow from different cellular responses to low-dose irradiation, using published data. Two principal categories of cellular responses are considered. One response category relates to the probability of radiation-induced DNA damage. The other category consists of low-dose induced changes in intracellular signaling that induce mechanisms of DNA damage control different from those operating at high levels of exposure. Modeled in this way, tissue is treated as a complex adaptive system. The interaction of the various cellular responses results in a net tissue dose-effect relation that is likely to deviate from linearity in the low-dose region. This suggests that the LNT hypothesis should be reexamined. The aim of this paper is to demonstrate that by use of microdosimetric concepts, the energy deposited in cell mass can be related to the occurrence of cellular responses, both damaging and defensive.

  6. Wnt signalling pathway parameters for mammalian cells.

    PubMed

    Tan, Chin Wee; Gardiner, Bruce S; Hirokawa, Yumiko; Layton, Meredith J; Smith, David W; Burgess, Antony W

    2012-01-01

    Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters

  7. Mammalian Cell-Based Sensor System

    NASA Astrophysics Data System (ADS)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  8. Epigenetic Regulation of Mammalian Stem Cells

    PubMed Central

    Li, Xuekun

    2008-01-01

    Two critical properties of stem cells are self-renewal and multipotency. The maintenance of their “stemness” state and commitment to differentiation are therefore tightly controlled by intricate molecular networks. Epigenetic mechanisms, including DNA methylation, chromatin remodeling and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. Recent studies have shown that epigenetic regulators are key players in stem cell biology and their dysfunction can result in human diseases such as cancer and neurodevelopmental disorders. Here, we review the recent evidences that advance our knowledge in epigenetic regulations of mammalian stem cells, with focus on embryonic stem cells and neural stem cells. PMID:18393635

  9. Cold shock response in mammalian cells.

    PubMed

    Fujita, J

    1999-11-01

    Compared to bacteria and plants, the cold shock response has attracted little attention in mammals except in some areas such as adaptive thermogenesis, cold tolerance, storage of cells and organs, and recently, treatment of brain damage and protein production. At the cellular level, some responses of mammalian cells are similar to microorganisms; cold stress changes the lipid composition of cellular membranes, and suppresses the rate of protein synthesis and cell proliferation. Although previous studies have mostly dealt with temperatures below 20 degrees C, mild hypothermia (32 degrees C) can change the cell's response to subsequent stresses as exemplified by APG-1, a member of the HSP110 family. Furthermore, 32 degrees C induces expression of CIRP (cold-inducible RNA-binding protein), the first cold shock protein identified in mammalian cells, without recovery at 37 degrees C. Remniscent of HSP, CIRP is also expressed at 37 degrees C and developmentary regulated, possibly working as an RNA chaperone. Mammalian cells are metabolically active at 32 degrees C, and cells may survive and respond to stresses with different strategies from those at 37 degrees C. Cellular and molecular biology of mammalian cells at 32 degrees C is a new area expected to have considerable implications for medical sciences and possibly biotechnology. PMID:10943555

  10. AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM

    EPA Science Inventory

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

  11. Toward predictive models of mammalian cells.

    PubMed

    Ma'ayan, Avi; Blitzer, Robert D; Iyengar, Ravi

    2005-01-01

    Progress in experimental and theoretical biology is likely to provide us with the opportunity to assemble detailed predictive models of mammalian cells. Using a functional format to describe the organization of mammalian cells, we describe current approaches for developing qualitative and quantitative models using data from a variety of experimental sources. Recent developments and applications of graph theory to biological networks are reviewed. The use of these qualitative models to identify the topology of regulatory motifs and functional modules is discussed. Cellular homeostasis and plasticity are interpreted within the framework of balance between regulatory motifs and interactions between modules. From this analysis we identify the need for detailed quantitative models on the basis of the representation of the chemistry underlying the cellular process. The use of deterministic, stochastic, and hybrid models to represent cellular processes is reviewed, and an initial integrated approach for the development of large-scale predictive models of a mammalian cell is presented. PMID:15869393

  12. Genomics in mammalian cell culture bioprocessing

    PubMed Central

    Wuest, Diane M.; Harcum, Sarah W.; Lee, Kelvin H.

    2013-01-01

    Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised. PMID:22079893

  13. Freezing mammalian cells for production of biopharmaceuticals.

    PubMed

    Seth, Gargi

    2012-03-01

    Cryopreservation techniques utilize very low temperatures to preserve the structure and function of living cells. Various strategies have been developed for freezing mammalian cells of biological and medical significance. This paper highlights the importance and application of cryopreservation for recombinant mammalian cells used in the biopharmaceutical industry to produce high-value protein therapeutics. It is a primer that aims to give insight into the basic principles of cell freezing for the benefit of biopharmaceutical researchers with limited or no prior experience in cryobiology. For the more familiar researchers, key cell banking parameters such as the cell density and hold conditions have been reviewed to possibly help optimize their specific cell freezing protocols. It is important to understand the mechanisms underlying the freezing of complex and sensitive cellular entities as we implement best practices around the techniques and strategies used for cryopreservation. PMID:22226818

  14. Isolation of genomic DNA from mammalian cells.

    PubMed

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. PMID:24011044

  15. MAMMALIAN CELL MUTAGENESIS, BANBURY CONFERENCE (JOURNAL VERSION)

    EPA Science Inventory

    A conference on mammalian cell mutagenesis was held at the Banbury Center, Cold Spring Harbor, NY, USA, March 22-25, 1987. The objective of the conference was to provide a forum for discussions concerning the genetic, biochemical, and molecular basis of induced mutations in stand...

  16. [Telomere Recombination in Normal Mammalian Cells].

    PubMed

    Zhdanova, N S; Rubtsov, N B

    2016-01-01

    Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15-20% of tumors lacking active telomerase and, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the "gold standard" of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed,and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed. PMID:27183789

  17. Iron metabolism in mammalian cells.

    PubMed

    Walker, B L; Tiong, J W; Jefferies, W A

    2001-01-01

    Most living things require iron to exist. Iron has many functions within cells but is rarely found unbound because of its propensity to catalyze the formation of toxic free radicals. Thus the regulation of iron requirements by cells and the acquisition and uptake of iron into tissues in multicellular organisms is tightly regulated. In humans, understanding iron transport and utility has recently been advanced by a "great conjunction" of molecular genetics in simple organisms, identifying genes involved in genetic diseases of metal metabolism and by the application of traditional cell physiology approaches. We are now able to approach a rudimentary understanding of the "iron cycle" within mammals. In the future, this information will be applied toward modulating the outcome of therapies designed to overcome diseases involving metals. PMID:11597005

  18. Aneuploidy in mammalian somatic cells in vivo.

    PubMed

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed. PMID:3941670

  19. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  20. Space radiation effects on plant and mammalian cells

    NASA Astrophysics Data System (ADS)

    Arena, C.; De Micco, V.; Macaeva, E.; Quintens, R.

    2014-11-01

    The study of the effects of ionizing radiation on organisms is related to different research aims. The current review emphasizes the studies on the effects of different doses of sparsely and densely ionizing radiation on living organisms, with the final purpose of highlighting specific and common effects of space radiation in mammals and plants. This topic is extremely relevant in the context of radiation protection from space environment. The response of different organisms to ionizing radiation depends on the radiation quality/dose and/or the intrinsic characteristics of the living system. Macromolecules, in particular DNA, are the critical targets of radiation, even if there is a strong difference between damages encountered by plant and mammalian cells. The differences in structure and metabolism between the two cell types are responsible for the higher resistance of the plant cell compared with its animal counterpart. In this review, we report some recent findings from studies performed in Space or on Earth, simulating space-like levels of radiation with ground-based facilities, to understand the effect of ionizing radiation on mammalian and plant cells. In particular, our attention is focused on genetic alterations and repair mechanisms in mammalian cells and on structures and mechanisms conferring radioresistance to plant cells.

  1. Fundamentals of Expression in Mammalian Cells.

    PubMed

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions. PMID:27165328

  2. Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

    PubMed

    Gutternigg, Martin; Rendić, Dubravko; Voglauer, Regina; Iskratsch, Thomas; Wilson, Iain B H

    2009-04-01

    Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterized in a recombinant form and has an important role in cellular function due to its ability to cleave beta-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterize for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability. Furthermore, a Myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localization. Transcripts of the corresponding gene are expressed in a number of murine tissues. On the basis of its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be identified from mammalian sources. PMID:19040401

  3. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 32 2014-07-01 2014-07-01 false TSCA in vitro mammalian cell gene mutation test. 799.9530 Section 799.9530 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS Health Effects...

  4. Chemical analysis of individual mammalian cells

    SciTech Connect

    Tan, W.; Yeung, E.S.

    1994-12-31

    The extremely small size of mammalian cells creates an unusual challenge for the analytical chemist, both in terms of separation and detection. Under a microscope, it is possible to confirm the injection of individual cells such as erythrocyte into capillaries with 10-{mu}m i.d. by hydrostatic pressure. The ionic contents can then be separated by capillary electrophoresis after the cell lyses. Enzymes at the zeptomole level can be monitored by on-column fluorescence enzyme assay. On-column particle-counting immunoassay can be applied to a broad range of analytes (antigens), also at the zeptomole level. The authors report here the simultaneous determination of the amounts of glucose-6-phosphate dehydrogenase (G6PDH) and their activities in individual erythrocytes by using a combination of the two detection schemes. Insights into the degradation of proteins as a function of cell age can be derived.

  5. Superoxide radical and iron modulate aconitase activity in mammalian cells.

    PubMed

    Gardner, P R; Raineri, I; Epstein, L B; White, C W

    1995-06-01

    Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-.. PMID:7768942

  6. Focusing on RISC assembly in mammalian cells

    SciTech Connect

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

    2008-04-11

    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.

  7. RNAi microarray analysis in cultured mammalian cells.

    PubMed

    Mousses, Spyro; Caplen, Natasha J; Cornelison, Robert; Weaver, Don; Basik, Mark; Hautaniemi, Sampsa; Elkahloun, Abdel G; Lotufo, Roberto A; Choudary, Ashish; Dougherty, Edward R; Suh, Ed; Kallioniemi, Olli

    2003-10-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function. PMID:14525932

  8. Cell fate regulation in early mammalian development

    NASA Astrophysics Data System (ADS)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  9. Repair of radiation damage in mammalian cells

    SciTech Connect

    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.

  10. Mammalian cells defective in DNA mismatch correction

    SciTech Connect

    Branch, P.; Aquilina, G.; Hess, P.

    1994-12-31

    Mammalian cells counteract the cytotoxicity of methylating agents, including some used in antitumor chemotherapy, by removing the methylated base, O{sup 6}-methylguanine (O{sup 6}-meG) from their DNA. This removal is normally effected by a specific DNA repair enzyme (O{sup 6}-meG-DNA methyltransferase) that is expressed constitutively. In addition, an alternative type of resistance to methylating agents can be acquired after exposure of cells to the drug. This acquired resistance is highly specific for O{sup 6}-meG and is unusual in that alkylation of DNA is normal and there is no increase in the rate of repair of O{sup 6}-meG or any other damaged base. Instead, the cell is able to tolerate the presence of the usually cytotoxic O{sup 6}-meG and to replicate its DNA normally. The ambiguity of base pairing by O{sup 6}-meG and the observation that tolerant cells are also cross-resistant to the structurally similar 6-thioguanine in DNA has led to the suggestion that the cytotoxicity of O{sup 6}-meG (and 6-thioguanine) arises from ineffective attempts at DNA mismatch correction. This model postulates that tolerance arises as a consequence of loss of this important pathway.

  11. Production of cell surface and secreted glycoproteins in mammalian cells.

    PubMed

    Seiradake, Elena; Zhao, Yuguang; Lu, Weixian; Aricescu, A Radu; Jones, E Yvonne

    2015-01-01

    Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields. PMID:25502196

  12. Genome exposure and regulation in mammalian cells.

    PubMed

    Puck, T T; Webb, P; Johnson, R

    1998-09-01

    A method of measurement of exposed DNA (i.e. hypersensitive to DNase I hydrolysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nuclei of mammalian cells is described. While cell cultures exhibit some differences in behavior from day to day, the general pattern of exposed and sequestered DNA is satisfactorily reproducible and agrees with results previously obtained by other methods. The general pattern of DNA hydrolysis exhibited by all cells tested consists of a curve which at first rises sharply with increasing DNase I, and then becomes almost horizontal, indicating that roughly about half of the nuclear DNA is highly sequestered. In 4 cases where transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with its more normal homolog (3T3, and the reverse transformed versions of CHO, HL60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid, and nerve growth factor [NGF] respectively), the transformed form displayed less genome exposure than the nontransformed form at every DNase I dose tested. When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased exposure reverting to that of the transformed forms. Therefore Ca++ appears necessary for maintenance of the DNA in the more highly exposed state characteristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested. Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increase of exposure of CHO by DBcAMP but it must be administered before or simultaneously with the latter compound. Measurements on mouse biopsies reveal large differences in exposure in different normal tissues. Thus, the exposure from adult liver cells was greater than that of adult brain, but both fetal liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human

  13. Cell lineage in mammalian craniofacial mesenchyme.

    PubMed

    Yoshida, Toshiyuki; Vivatbutsiri, Philaiporn; Morriss-Kay, Gillian; Saga, Yumiko; Iseki, Sachiko

    2008-01-01

    We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells. PMID:18617001

  14. Profiling Signaling Peptides in Single Mammalian Cells Using Mass Spectrometry

    PubMed Central

    Rubakhin, Stanislav S.; Churchill, James D.; Greenough, William T.; Sweedler, Jonathan V.

    2008-01-01

    The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and non-enzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than fifteen peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and post-source decay sequencing allows identification of fourteen of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types. PMID:17037931

  15. Hypergravity signal transduction and gene expression in cultured mammalian cells

    NASA Technical Reports Server (NTRS)

    Kumei, Y.; Whitson, P. A.

    1994-01-01

    A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

  16. MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT

    EPA Science Inventory

    Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian dells and detect mutations. s part of the International Workshop on Standardization of Genotoxicity Test Procedures, ...

  17. Some factors affecting the specific toxicity of misonidazole towards hypoxic mammalian cells.

    PubMed Central

    Stratford, I. J.; Gray, P.

    1978-01-01

    The toxic action of misonidazole towards hypoxic mammalian cells has been shown to be a function of serum concentration, with higher serum concentrations enhancing the toxic effect. Added thiols protect cells against misonidazole toxicity. In addition, the action of misonidazole on hypoxic cells labelled with 5-BUdR has been examined. Cells with incroported 5-BUdR are no more sensitive to misonidazole toxicity than are cells without label. PMID:277212

  18. Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.

    PubMed

    Ogawa, Akiko; Mizui, Shinya; Chida, Yasuhito; Shimizu, Masafumi; Terada, Satoshi; Ohura, Takeshi; Kobayashi, Kyo-Ichi; Yasukawa, Saori; Moriyama, Nobuyuki

    2014-07-01

    Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS. PMID:24485744

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

    PubMed

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

    2015-12-01

    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-Fe(III) 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-Fe(III) nanocoat, mimicking the sporulation and germination processes found in nature. PMID:26528931

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

    PubMed Central

    Paves, Heiti; Truve, Erkki

    2004-01-01

    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. PMID:15102327

  1. Amino acids in the cultivation of mammalian cells.

    PubMed

    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. PMID:26832172

  2. Selection for intrabody solubility in mammalian cells using GFP fusions.

    PubMed

    Guglielmi, Laurence; Denis, Vincent; Vezzio-Vié, Nadia; Bec, Nicole; Dariavach, Piona; Larroque, Christian; Martineau, Pierre

    2011-12-01

    Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay. PMID:21997307

  3. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  4. Antioxidation activities of pteridines in mammalian cell lines

    SciTech Connect

    Zhang, Y.; Shen, R. )

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  6. Telomere homeostasis in mammalian germ cells: a review.

    PubMed

    Reig-Viader, Rita; Garcia-Caldés, Montserrat; Ruiz-Herrera, Aurora

    2016-06-01

    Telomeres protect against genome instability and participate in chromosomal movements during gametogenesis, especially in meiosis. Thus, maintaining telomere structure and telomeric length is essential to both cell integrity and the production of germ cells. As a result, alteration of telomere homeostasis in the germ line may result in the generation of aneuploid gametes or gametogenesis disruption, triggering fertility problems. In this work, we provide an overview on fundamental aspects of the literature regarding the organization of telomeres in mammalian germ cells, paying special attention to telomere structure and function, as well as the maintenance of telomeric length during gametogenesis. Moreover, we discuss the different roles recently described for telomerase and TERRA in maintaining telomere functionality. Finally, we review how new findings in the field of reproductive biology underscore the role of telomere homeostasis as a potential biomarker for infertility. Overall, we anticipate that the study of telomere stability and equilibrium will contribute to improve diagnoses of patients; assess the risk of infertility in the offspring; and in turn, find new treatments. PMID:26525972

  7. Mitochondrial involvement in cell death of non-mammalian eukaryotes

    PubMed Central

    Abdelwahid, Eltyeb; Rolland, Stephane; Teng, Xinchen; Conradt, Barbara; Hardwick, J. Marie; White, Kristin

    2010-01-01

    Although mitochondria are essential organelles for long-term survival of eukaryotic cells, recent discoveries in biochemistry and genetics have advanced our understanding of the requirements for mitochondria in cell death. Much of what we understand about cell death is based on the identification of conserved cell death genes in Drosophila melanogaster and Caenorhabditis elegans. However, the role of mitochondria in cell death in these models has been much less clear. Considering the active role that mitochondria play in apoptosis in mammalian cells, the mitochondrial contribution to cell death in non-mammalian systems has been an area of active investigation. In this article, we review the current research on this topic in three non-mammalian models, C. elegans, Drosophila and Saccharomyces cerevisiae. In addition, we discuss how non-mammalian models have provided important insight into the mechanisms of human disease as they relate to the mitochondrial pathway of cell death. The unique perspective derived from each of these model systems provides a more complete understanding of mitochondria in programmed cell death. PMID:20950655

  8. Mammalian cell culture capacity for biopharmaceutical manufacturing.

    PubMed

    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. PMID:23748352

  9. Cell type-specific transcriptome profiling in mammalian brains.

    PubMed

    LoVerso, Peter R; Cui, Feng

    2016-01-01

    A mammalian brain contains numerous types of cells. Advances in neuroscience in the past decade allow us to identify and isolate neural cells of interest from mammalian brains. Recent developments in high-throughput technologies, such as microarrays and next-generation sequencing (NGS), provide detailed information on gene expression in pooled cells on a genomic scale. As a result, many novel genes have been found critical in cell type-specific transcriptional regulation. These differentially expressed genes can be used as molecular signatures, unique to a particular class of neural cells. Use of this gene expression-based approach can further differentiate neural cell types into subtypes, potentially linking some of them with neurological diseases. In this article, experimental techniques used to purify neural cells are described, followed by a review on recent microarray- or NGS-based transcriptomic studies of common neural cell types. The future prospects of cell type-specific research are also discussed. PMID:27100485

  10. Cell type-specific transcriptome profiling in mammalian brains

    PubMed Central

    LoVerso, Peter R.; Cui, Feng

    2016-01-01

    A mammalian brain contains numerous types of cells. Advances in neuroscience in the past decade allow us to identify and isolate neural cells of interest from mammalian brains. Recent developments in high-throughput technologies, such as microarrays and next-generation sequencing (NGS), provide detailed information on gene expression in pooled cells on a genomic scale. As a result, many novel genes have been found critical in cell type-specific transcriptional regulation. These differentially expressed genes can be used as molecular signatures, unique to a particular class of neural cells. Use of this gene expression-based approach can further differentiate neural cell types into subtypes, potentially linking some of them with neurological diseases. In this article, experimental techniques used to purify neural cells are described, followed by a review on recent microarray- or NGS-based transcriptomic studies of common neural cell types. The future prospects of cell type-specific research are also discussed. PMID:27100485

  11. Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species

    PubMed Central

    Rosselló, Ricardo Antonio; Chen, Chun-Chun; Dai, Rui; Howard, Jason T; Hochgeschwender, Ute; Jarvis, Erich D

    2013-01-01

    Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range. DOI: http://dx.doi.org/10.7554/eLife.00036.001 PMID:24015354

  12. Survival of mammalian cells under high vacuum condition for ion bombardment.

    PubMed

    Feng, Huiyun; Wu, Lijun; Xu, An; Hu, Burong; Hei, Tom K; Yu, Zengliang

    2004-12-01

    An ion beam has been used to irradiate various organisms and its effects have been studied. Because of the poor tolerance that mammalian cells have for vacuum, such studies have not been carried out on living mammalian cells until now. However, this work is important both for elucidating the mechanism of mutation in response to low-energy ions and in exploring possible new applications of ion beam technology. The current paper describes an investigation of the survival of mammalian cells (the A(L) cell line) in a high-vacuum chamber in preparation for ion bombardment studies. The ion beam facility is described and the actual vacuum profile that the cells endured in the target chamber is reported. Cells were damaged immediately following vacuum exposure; the injury was characterized by alteration of the membrane permeability, loss of firm adhesion to the dish, and increased fragility. Three cryoprotective agents were tested (glycerol, propylene glycol, and trehalose) and of these, glycerol showed the highest potency for protecting cells against vacuum stress. This was revealed by an increase in the cell survival level from <1 to >10% with a glycerol concentration of 15 and 20%. Two glycerol-based protocols were investigated (freezing-vacuum vs. non-freezing-vacuum), but there was no significant difference (P > 0.1) in their ability to improve cell survival, the values being 10.31 +/- 4.5 and 12.7 +/- 3.37%, respectively with 20% glycerol concentration. These cells had a normal growth capability, and also retained integrity of the cell surface antigen CD59. These initial experiments indicate that mammalian cells can withstand vacuum to the degree that is needed to study the effect of the ion beam. In addition to the improvements made in this study, other factors are discussed that may increase the survival of mammalian cells exposed to a vacuum in future studies. PMID:15615610

  13. Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species.

    PubMed

    Ramakrishnan, Praveen; Maclean, Michelle; MacGregor, Scott J; Anderson, John G; Grant, M Helen

    2016-06-01

    Light at wavelength 405 nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405 nm light at 36 J/cm(2) (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54 J/cm(2)), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405 nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405 nm light treated cells. The mammalian cells were significantly protected from dying at 54 J/cm(2) in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324 J/cm(2). Results therefore suggested that the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage. PMID:26916085

  14. Postreplication repair in mammalian cells after ultraviolet irradiation: a model.

    PubMed Central

    Lavin, M F

    1978-01-01

    A model is presented for bypass of ultraviolet-induced damage in DNA during replication. The overall process is initiated by the introduction of a single-strand break into parental DNA near the point of arrest of synthesis, followed by a transient crossing-over step similar to that envisaged in genetic recombination. The mechanism proposed provides an alternative explanation to existing models and is entirely consistent with available data on postreplication repair in mammalian cells. In addition the model explains the low level of recombination repair observed in mammalian cells. PMID:687763

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

    PubMed

    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. PMID:27189205

  16. Photothermal nanoblade for large cargo delivery into mammalian cells

    PubMed Central

    Wu, Ting-Hsiang; Teslaa, Tara; Kalim, Sheraz; French, Christopher T.; Maghadam, Shahriar; Wall, Randolph; Miller, Jeffery F.; Witte, Owen N.; Teitell, Michael A.; Chiou, Pei-Yu

    2011-01-01

    It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integrating the metallic nanostructure with a micropipette, the nanoblade generates a micron-sized membrane access port for delivering highly concentrated cargo (5×108 live bacteria/ml) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads to 2 μm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult to transfer cargo into mammalian cells. PMID:21247066

  17. Aptazyme-based riboswitches and logic gates in mammalian cells.

    PubMed

    Nomura, Yoko; Yokobayashi, Yohei

    2015-01-01

    This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3' untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3' UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression. PMID:25967059

  18. Liposome mediated DNA-transfer into mammalian cells.

    PubMed

    Somlyai, G; Kondorosi, E; Karikó, K; Duda, E G

    1985-01-01

    We have investigated the interaction of mammalian cells with liposome encapsulated DNA. Tissue cultured mammalian cells were exposed to large, unilamellar phosphatidyl serine liposomes containing DNA molecules from different animal cells or prokaryotic organisms. The liposomes bind rapidly to the surface and are taken up by the cells and significant proportion of the encapsulated DNA is transported to the nuclei. Transient expression of the foreign genetic material could be detected in high percentage of the treated cells for a few days. During this period of time foreign DNA is present in both free and integrated form, however, the free form soon disappears. Stable transformant cell colonies--with continuous expression of new gene(s)--were isolated under selective pressure with a frequency of approx. 10(-5). PMID:3837979

  19. Nanoscale dielectrophoretic spectroscopy of individual immobilized mammalian blood cells.

    PubMed

    Lynch, Brian P; Hilton, Al M; Simpson, Garth J

    2006-10-01

    Dielectrophoretic force microscopy (DEPFM) and spectroscopy have been performed on individual intact surface-immobilized mammalian red blood cells. Dielectrophoretic force spectra were obtained in situ in approximately 125 ms and could be acquired over a region comparable in dimension to the effective diameter of a scanning probe microscopy tip. Good agreement was observed between the measured dielectrophoretic spectra and predictions using a single-shell cell model. In addition to allowing for highly localized dielectric characterization, DEPFM provided a simple means for noncontact imaging of mammalian blood cells under aqueous conditions. These studies demonstrate the feasibility of using DEPFM to monitor localized changes in membrane capacitance in real time with high spatial resolution on immobilized cells, complementing previous studies of mobile whole cells and cell suspensions. PMID:16798803

  20. PepGMV Rep-Protein Expression in Mammalian Cells

    PubMed Central

    Chapa-Oliver, Angela María; Mejía-Teniente, Laura; García-Gasca, Teresa; Guevara-Gonzalez, Ramon Gerardo; Torres-Pacheco, Irineo

    2012-01-01

    The Geminiviruses genome is a small, single strand DNA that replicates in the plant cell nucleus. Analogous to animal DNA viruses, Geminiviruses depend on the host replication machinery to amplify their genomes and only supply the factors required to initiate their replication. Consequently, Geminiviruses remove the cell-cycle arrest and induce the host replication machinery using an endocycle process. They encode proteins, such as the conserved replication-associated proteins (Rep) that interact with retinoblastoma-like proteins in plants and alter the cell division cycle in yeasts. Therefore, the aim of this work is to analyze the impact of Pepper Golden Mosaic Virus (PepGMV) Rep protein in mammalian cells. Results indicate that the pTracer-SV40:Rep construction obtained in this work can be used to analyze the Rep protein effect in mammalian cells in order to compare the cell cycle regulation mechanisms in plants and animals. PMID:23170183

  1. Base excision repair intermediates are mutagenic in mammalian cells

    PubMed Central

    Simonelli, Valeria; Narciso, Laura; Dogliotti, Eugenia; Fortini, Paola

    2005-01-01

    Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5′-deoxyribose-5-phosphate (5′dRp) and 3′-[2,3-didehydro-2,3-dideoxy-ribose] (3′ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the ‘A-rule’ and is also consistent with the use of the 5′ neighbouring base to instruct nucleotide incorporation (5′-rule). Preferential adenine insertion was also observed after in vivo replication of 5′dRp or 3′ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives ‘faceless’ BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations. PMID:16077026

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

    PubMed

    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 promoterregulated 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

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

    PubMed Central

    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. A mechanical model for guided motion of mammalian cells

    NASA Astrophysics Data System (ADS)

    Bitter, P.; Beck, K. L.; Lenz, P.

    2015-12-01

    We introduce a generic, purely mechanical model for environment-sensitive motion of mammalian cells that is applicable to chemotaxis, haptotaxis, and durotaxis as modes of motility. It is able to theoretically explain all relevant experimental observations, in particular, the high efficiency of motion, the behavior on inhomogeneous substrates, and the fixation of the lagging pole during motion. Furthermore, our model predicts that efficiency of motion in following a gradient depends on cell geometry (with more elongated cells being more efficient).

  5. Analysis of cell cycle position in mammalian cells.

    PubMed

    Cecchini, Matthew J; Amiri, Mehdi; Dick, Frederick A

    2012-01-01

    of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases. In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control. PMID:22297617

  6. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-11-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18428384

  7. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18265370

  8. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  9. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-12-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18429293

  10. Induced DNA repair pathway in mammalian cells

    SciTech Connect

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M 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-..beta..-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.

  11. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  12. Equipment for large-scale mammalian cell culture.

    PubMed

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed. PMID:24429549

  13. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  14. Sex determination in mammalian germ cells

    PubMed Central

    Spiller, Cassy M; Bowles, Josephine

    2015-01-01

    Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model. PMID:25791730

  15. Calcium Imaging of Sonoporation of Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Sabens, David; Aehle, Matthew; Steyer, Grant; Kourennyi, Dmitri; Deng, Cheri X.

    2006-05-01

    Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real-time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved understanding of sonoporation mechanism. Our experimental results demonstrated the feasibility of calcium imaging of sonoporation in Chinese Hamster Ovary (CHO) cells. We have measured the changes in the intracellular calcium concentration using Fura-2, a fluorescent probe, which indicate influx or flow of Calcium across the cell membrane. Analysis of data identified key aspects in the dynamic sonoporation process including the formation of pores in the cell membrane, and the relative temporal duration of the pores and their resealing. These observations are obtained through the analysis of the rate the calcium concentration changes within the cells, making it possible to visualize membrane opening and repair in real-time through such changes in the intracellular calcium concentration.

  16. Mammalian cell viability in electrospun composite nanofiber structures.

    PubMed

    Canbolat, Mehmet Fatih; Tang, Christina; Bernacki, Susan H; Pourdeyhimi, Behnam; Khan, Saad

    2011-10-10

    Incorporation of mammalian cells into nanofibers (cell electrospinning) and multilayered cell-nanofiber structures (cell layering) via electrospinning are promising techniques for tissue engineering applications. We investigate the viability of 3T3-L1 mouse fibroblasts after incorporation into poly(vinyl alcohol) nanofibers and multilayering with poly(caprolactone) nanofibers and analyze the possible factors that affect cell viability. We observe that cells do not survive cell electrospinning but survive cell layering. Assessing the factors involved in cell electrospinning, we find that dehydration and fiber stretching are the main causes of cell death. In cell layering, the choice of solvent is critical, as residual solvent in the electrospun fibers could be detrimental to the cells. PMID:21984502

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

    SciTech Connect

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

    1995-09-05

    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{sup 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{sup 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 {sup 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.

  18. The receptors and cells for mammalian taste.

    PubMed

    Chandrashekar, Jayaram; Hoon, Mark A; Ryba, Nicholas J P; Zuker, Charles S

    2006-11-16

    The emerging picture of taste coding at the periphery is one of elegant simplicity. Contrary to what was generally believed, it is now clear that distinct cell types expressing unique receptors are tuned to detect each of the five basic tastes: sweet, sour, bitter, salty and umami. Importantly, receptor cells for each taste quality function as dedicated sensors wired to elicit stereotypic responses. PMID:17108952

  19. Cryopreservation of Spin-Dried Mammalian Cells

    PubMed Central

    Chakraborty, Nilay; Menze, Michael A.; Malsam, Jason; Aksan, Alptekin; Hand, Steven C.; Toner, Mehmet

    2011-01-01

    This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN2) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification. PMID:21966385

  20. Protein diffusion in mammalian cell cytoplasm.

    PubMed

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS. PMID:21886771

  1. Regulation of mammalian brain cell volume.

    PubMed

    Law, R O

    1994-02-01

    Maintenance of brain cell volume is of crucial importance for normal central nervous system (CNS) function. This review considers volume regulation primarily in response to disturbances of body fluid osmolality. Brain cells counter the tendency to swell or shrink by appropriate adjustment of their internal osmotic potential. This is achieved by loss or uptake of inorganic ions and low molecular weight organic solutes (osmolytes). The latter comprise mainly amino acids, myoinositol, choline, and methylamines. Taurine may be of particular importance in volume control, especially in young animals. Brain cell volume regulation, however, is only one contributory factor to maintenance of constant brain volume (water content), and operates in parallel with important alterations in bulk fluid and electrolyte movement across the blood-brain barrier and between the interstitium and cerebrospinal fluid, which themselves moderate the requirement for transient alteration in cell volume during acute osmotic imbalance. Although altered cerebral content of inorganic ions and osmolytes are usually regarded as responses, respectively, to acute and chronic osmotic disturbances, osmolytes (especially taurine) may also participate in short-term cell volume regulation. PMID:8301256

  2. Effect of Carbon Nanotubes on Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Chen, Michelle; Ahmed, Asma; Black, Melanie; Kawamoto, Nicole; Lucas, Jessica; Pagala, Armie; Pham, Tram; Stankiewicz, Sara; Chen, Howard

    2010-03-01

    Carbon Nanotubes possess extraordinary electrical, mechanical, and thermal properties. Research on applying the carbon nanotubes for ultrasensitive detection, disease diagnosis, and drug delivery is rapidly developing. While the fundamental and technological findings on carbon nanotubes show great promise, it is extremely important to investigate the effect of the carbon nanotubes on human health. In our experiments, we introduce purified carbon nanotubes in suspension to ovary cells cultured from Hamsters. These cells are chosen since they show robust morphological changes associated with cytotoxicity that can easily be observed under a light microscope. We will discuss the toxicity of carbon nanotubes by characterizing the cell morphology and viability as a function of time and the concentration of carbon nanotube suspension.

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

    NASA Astrophysics Data System (ADS)

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

    2004-12-01

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

  4. Glycosaminoglycan receptors facilitate infection of mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A growing list of viruses has been reported to use more than one receptor for binding and internalization during infection of the host cell. Sialic acid residues or glycosaminoglycans, such as heparin sulfate, frequently function in this scenario, as a first contact, charge based, low affinity bindi...

  5. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  6. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)

    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.

  7. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

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

    1993-02-01

    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.

  8. Metabolic flux rewiring in mammalian cell cultures.

    PubMed

    Young, Jamey D

    2013-12-01

    Continuous cell lines (CCLs) engage in 'wasteful' glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells. PMID:23726154

  9. Isolation of genomic DNA from mammalian cells.

    PubMed

    Gilbert, J R; Vance, J M

    2001-05-01

    This unit describes simple, cost-effective preparation of DNA from whole blood or cultured cells that yields high-molecular-weight DNA suitable for both Southern blotting and the polymerase chain reaction. Preparation time may be shortened by substituting a high-salt precipitation procedure for the dialysis step; however, this results in a smaller average fragment size. The isolation of DNA from buccal swabs, collected from the inside of the cheek, is also described. The DNA is suitable for PCR analysis. Preparation of buffered phenol for DNA extraction is described in a support protocol. This unit describes simple, cost-effective preparation of DNA from whole blood or cultured cells that yields high-molecular-we. PMID:18428220

  10. Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

    PubMed

    Palmer, N; Kaldis, P

    2016-01-01

    The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development. PMID:27475848

  11. SNAP25 Expression in Mammalian Retinal Horizontal Cells

    PubMed Central

    Hirano, Arlene A.; Brandstätter, Johann Helmut; Morgans, Catherine W.; Brecha, Nicholas C.

    2014-01-01

    Horizontal cells mediate inhibitory feedforward and feedback lateral interactions in the outer retina at photoreceptor terminals and bipolar cell dendrites; however, the mechanisms that underlie synaptic transmission from mammalian horizontal cells are poorly understood. The localization of a vesicular γ-aminobutyric acid (GABA) transporter (VGAT) to horizontal cell processes in primate and rodent retinae suggested that mammalian horizontal cells release transmitter in a vesicular manner. Toward determining whether the molecular machinery for vesicular transmitter release is present in horizontal cells, we investigated the expression of SNAP25 (synaptosomal-associated protein of 25 kDa), a key SNARE protein, by immunocytochemistry with cell type-specific markers in the retinae of mouse, rat, rabbit, and monkey. Different commercial antibodies to SNAP25 were tested on vertical sections of retina. We report the robust expression of SNAP25 in both plexiform layers. Double labeling with SNAP25 and calbindin antibodies demonstrated that horizontal cell processes and their endings in photoreceptor triad synapses were strongly labeled for both proteins in mouse, rat, rabbit, and monkey retinae. Double labeling with parvalbumin antibodies in monkey retina verified SNAP25 immunoreactivity in all horizontal cells. Pre-embedding immunoelectron microscopy in rabbit retina confirmed expression of SNAP25 in lateral elements within photoreceptor triad synapses. The SNAP25 immunoreactivity in the plexiform layers and outer nuclear layer fell into at least three patterns depending on the antibody, suggesting a differential distribution of SNAP25 isoforms. The presence of SNAP25a and SNAP25b isoforms in mouse retina was established by reverse transcriptase-polymerase chain reaction. SNAP25 expression in mammalian horizontal cells along with other SNARE proteins is consistent with vesicular exocytosis. PMID:21280047

  12. Energy deposition in selected-mammalian cell for several-MeV single-proton beam

    NASA Astrophysics Data System (ADS)

    Ding, K.; Yu, Z.

    2007-05-01

    The phenomena resulting from interaction between ion beam and mammalian cell pose important problems for biological applications. Classic Bethe-Bloch theory utilizing attached V79 mammalian cell has been conducted in order to establish the stopping powers of the mammalian cell for several-MeV single-proton microbeam. Based on the biological structure of the mammalian cell, a physical model is proposed which presumes that the attached cell is simple MWM model. According to this model and Monte Carlo simulation, we studied the energy deposition and its ratio on the selected attached mammalian cell for MeV proton implantation.

  13. Moving beyond science to protect a mammalian migration corridor.

    PubMed

    Berger, Joel; Cain, Steven L

    2014-10-01

    As the discipline of conservation biology evolves and practitioners grow increasingly concerned about how to put results into achievable conservation, it is still unclear the extent to which science drives conservation outcomes, especially across rural landscapes. We addressed this issue by examining the role of science in the protection of a biological corridor. Our focus is on a North American endemic mammal reliant on long distance migration as an adaptive strategy, the pronghorn (Antilocapra americana) of the southern Greater Yellowstone Ecosystem. The role of science in realizing policy change, while critical as a first step, was surprisingly small relative to the role of other human dimensions. In a case study, we strategically addressed a variety of conservation needs beyond science, first by building a partnership between government and private interests and then by enhancing interest in migratory phenomena across a landscape with divergent political ideologies and economic bases. By developing awareness and even people's pride in the concept of corridor conservation, we achieved local, state, and federal acceptance for protection of a 70 km long, 2 km wide pathway for the longest terrestrial migrant in the contiguous United States. Key steps included conducting and publishing research that defined the migration corridor; fostering a variety of media coverage at local, regional, and national levels; conducting public outreach through stakeholder workshops, meetings, and presentations; and meeting with and gaining the support of elected officials. All these contributed to the eventual policy change that created the first federally protected migration corridor in the United States, which in turn stimulated additional conservation actions. On the basis of our experience, we believe conservation scientists can and should step beyond traditional research roles to assist with on-the-ground conservation by engaging in aspects of conservation that involve local

  14. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

  15. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  16. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  17. Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells*

    PubMed Central

    Badal, Sujan; Her, Yeng F.; Maher, L. James

    2015-01-01

    Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics. PMID:26205818

  18. Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells.

    PubMed

    Badal, Sujan; Her, Yeng F; Maher, L James

    2015-09-01

    Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics. PMID:26205818

  19. Direct patterning of mammalian cells in an ultrasonic heptagon stencil.

    PubMed

    Bernassau, A L; Gesellchen, F; Macpherson, P G A; Riehle, M; Cumming, D R S

    2012-06-01

    We describe the construction of a ultrasonic device suitable for micro patterning particles and cells for tissue engineering applications. The device is formed by seven transducers shaped into a heptagon cavity. By exciting two and three transducers simultaneously, lines or hexagonal shapes can be formed with beads and cells. Furthermore, phase control of the transducers allows shifting the standing waves and thus patterning at different positions on a surface in a controlled manner. The paper discusses direct patterning of mammalian cells by ultrasound "stencil". PMID:22327813

  20. Toxic effects of Karenia mikimotoi extracts on mammalian cells

    NASA Astrophysics Data System (ADS)

    Chen, Yang; Yan, Tian; Yu, Rencheng; Zhou, Mingjiang

    2011-07-01

    Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphology of four mammalian cell lines (two normal cell lines and two cancer cell lines). In addition, we carried out a preliminary investigation on the mechanism of toxicity of the alga. The results show that the polar lipid-soluble component of K. mikimotoi significantly inhibited proliferation of the four cell lines, and resulted in the cells becoming spherical, swollen and damaged. The result of Annexin V and PI double-staining confirmed that cell membranes were disrupted. The malonaldehyde (MDA) contents in the medium of the four cell lines treated with the polar-lipid extracts all increased significantly, which indicates that the polar-lipid toxins produced by K. mikimotoi could adversely affect mammalian cells by inducing lipid peroxidation. We conclude that K. mikimotoi is a potential threat to human health, and the comprehensive effect of this dinoflagellate and its mechanisms should be investigated further.

  1. Acoustophoretic sorting of viable mammalian cells in a microfluidic device.

    PubMed

    Yang, Allen H J; Soh, H Tom

    2012-12-18

    We report the first use of ultrasonic acoustophoresis for the label-free separation of viable and nonviable mammalian cells within a microfluidic device. Cells that have undergone apoptosis are physically smaller than viable cells, and our device exploits this fact to achieve efficient sorting based on the strong size dependence of acoustic radiation forces within a microchannel. As a model, we have selectively enriched viable MCF-7 breast tumor cells from heterogeneous mixtures of viable and nonviable cells. We found that this mode of separation is gentle and enables efficient, label-free isolation of viable cells from mixed samples containing 10(6) cells/mL at flow rates of up to 12 mL/h. We have extensively characterized the device, and we report the effects of piezoelectric voltage and sample flow rate on device performance and describe how these parameters can be tuned to optimize recovery, purity, or throughput. PMID:23157478

  2. NMR spectroscopy and perfusion of mammalian cells using surface microprobes.

    PubMed

    Ehrmann, Klaus; Pataky, Kristopher; Stettler, Matthieu; Wurm, Florian Maria; Brugger, Jürgen; Besse, Pierre-André; Popovic, Radivoje

    2007-03-01

    NMR spectra of mammalian cells are taken using surface microprobes that are based on microfabricated planar coils. The surface microprobe resembles a miniaturized Petri dish commonly used in biological research. The diameter of the planar coils is 1 mm. Chinese Hamster Ovaries are immobilized in a uniform layer on the microprobe surface or patterned by an ink-jet printer in the centre of the microcoil, where the rf-field of the planar microcoil is most uniform. The acquired NMR spectra show the prevalent metabolites found in mammalian cells. The volumes of the detected samples range from 25 nL to 1 nL (or 50,000 to 1800 cells). With an extended set-up that provides fluid inlets and outlets to the microprobe, the cells can be perfused within the NMR-magnet while constantly taking NMR spectra. Perfusion of the cells opens the way to increased cell viability for long acquisitions or to analysis of the cells' response to environmental change. PMID:17330170

  3. Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.

    PubMed

    Watanabe, Satoshi; Watanabe, Sachiko; Sakamoto, Naoaki; Sato, Masahiro; Akasaka, Koji

    2006-09-01

    An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells. PMID:16923122

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

    PubMed

    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. PMID:27322762

  5. Retinoic Acid Stimulates Regeneration of Mammalian Auditory Hair Cells

    NASA Astrophysics Data System (ADS)

    Lefebvre, Philippe P.; Malgrange, Brigitte; Staecker, Hinrich; Moonen, Gustave; van de Water, Thomas R.

    1993-04-01

    Sensorineural hearing loss resulting from the loss of auditory hair cells is thought to be irreversible in mammals. This study provides evidence that retinoic acid can stimulate the regeneration in vitro of mammalian auditory hair cells in ototoxic-poisoned organ of Corti explants in the rat. In contrast, treatment with retinoic acid does not stimulate the formation of extra hair cells in control cultures of Corti's organ. Retinoic acid-stimulated hair cell regeneration can be blocked by cytosine arabinoside, which suggests that a period of mitosis is required for the regeneration of auditory hair cells in this system. These results provide hope for a recovery of hearing function in mammals after auditory hair cell damage.

  6. Mammalian retinal Müller cells have circadian clock function

    PubMed Central

    Xu, Lili; Ruan, Guoxiang; Dai, Heng; Liu, Andrew C.; Penn, John

    2016-01-01

    Purpose To test whether Müller glia of the mammalian retina have circadian rhythms. Methods We used Müller glia cultures isolated from mouse lines or from humans and bioluminescent reporters of circadian clock genes to monitor molecular circadian rhythms. The clock gene dependence of the Müller cell rhythms was tested using clock gene knockout mouse lines or with siRNA for specific clock genes. Results We demonstrated that retinal Müller glia express canonical circadian clock genes, are capable of sustained circadian oscillations in isolation from other cell types, and exhibit unique features of their molecular circadian clock compared to the retina as a whole. Mouse and human Müller cells demonstrated circadian clock function; however, they exhibited species-specific differences in the gene dependence of their clocks. Conclusions Müller cells are the first mammalian retinal cell type in which sustained circadian rhythms have been demonstrated in isolation from other retinal cells. PMID:27081298

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

    PubMed

    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. PMID:26010998

  8. Stochastic mRNA synthesis in mammalian cells.

    PubMed

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  9. Carbon nanotube fibers are compatible with Mammalian cells and neurons.

    PubMed

    Dubin, R A; Callegari, G; Kohn, J; Neimark, A

    2008-03-01

    We demonstrate the biocompatibility of carbon nanotube fibers (CNFs) fabricated from single-wall carbon nanotubes. Produced by a particle-coagulation spinning process, CNFs are "hair-like" conductive microwires, which uniquely combine properties of porous nanostructured scaffolds, high-area electrodes, and permeable microfluidic conduits. We report that CNFs are nontoxic and support the attachment, spreading, and growth of mammalian cells and the extension of processes from neurons in vitro. Our findings suggest that CNF may be employed for an electrical interfacing of nerve cells and external devices. PMID:18334451

  10. Adaptor Protein Cerebral Cavernous Malformation 3 (CCM3) Mediates Phosphorylation of the Cytoskeletal Proteins Ezrin/Radixin/Moesin by Mammalian Ste20-4 to Protect Cells from Oxidative Stress*

    PubMed Central

    Fidalgo, Miguel; Guerrero, Ana; Fraile, María; Iglesias, Cristina; Pombo, Celia M.; Zalvide, Juan

    2012-01-01

    While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role. PMID:22291017

  11. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

    PubMed Central

    Somyajit, Kumar; Saxena, Sneha; Babu, Sharath; Mishra, Anup; Nagaraju, Ganesh

    2015-01-01

    Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis. PMID:26354865

  12. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  13. cDNA expression cloning in mammalian cells.

    PubMed

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  14. DNA ligase I mediates essential functions in mammalian cells.

    PubMed Central

    Petrini, J H; Xiao, Y; Weaver, D T

    1995-01-01

    DNA replication, repair, and recombination are essential processes in mammalian cells. Hence, the application of gene targeting to the study of these DNA metabolic pathways requires the creation of nonnull mutations. We have developed a method for introducing partially defective mutants in murine embryonic stem cells that circumvents the problem of cellular lethality of targeted mutations at essential loci. Using this approach, we have determined that mammalian DNA ligase I is essential for cell viability. Thus, DNA ligases II and III are not redundant with DNA ligase I for the function(s) associated with cell proliferation. Partial complementation of the lethal DNA ligase I null mutation allowed the creation of deficient embryonic stem cell lines. We found that a wild-type DNA ligase I cDNA, as well as a variant DNA ligase I cDNA, was able to rescue the lethality of the homozygous null mutation, whereas an N-terminal deletion mutant consisting of the minimal DNA ligase I catalytic domain was not. This observation demonstrates that sequences outside the DNA ligase I catalytic domain are essential for DNA ligase I function in vivo. PMID:7623824

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

    PubMed

    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. PMID:27613045

  16. Mammalian Cell Competitions, Cell-in-Cell Phenomena and Their Biomedical Implications.

    PubMed

    Huang, H; Chen, Z; Sun, Q

    2015-01-01

    Cell competition was first identified four decades ago as a mechanism to eliminate less fit cells during development in Drosophila melanogaster, and later postulated to be involved in tumorigenesis of human beings. However, evidence for a similar mechanism functional in mammals and tumors was missed until recent years. Like cell competition in fly, multiple forms of competition mechanisms were reported in mammalian system, and some of them were found participating in tumor initiation. Lately, entosis, a mechanism of cell cannibalisms responsible for the formation of cell-in-cell structures in human tumors, was identified as a novel member of ever-expanding family of mammalian cell competitions (MaCCs), and proposed to be able to promote clonal selection and tumor evolution. Thus, engulfment by neighboring cells other than the professional phagocytes, an issue still in debate in fly, was clearly demonstrated in mammals to be responsible for loser elimination. Competition mediated by cell-in-cell structures, formed by multiple cannibal mechanisms, constitutes a novel class of MaCCs. This review will summarize current research on mammalian cell competitions, followed by feature and mechanism analysis and their potential implications in the pathology and treatment of human tumors. PMID:26511708

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

    PubMed

    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. PMID:25906116

  18. Biochemistry of growth inhibition by ammonium ions in mammalian cells

    SciTech Connect

    Ryll, T.; Valley, U.; Wagner, R. . Cell Culture Techniques Dept.)

    1994-06-20

    The intracellular pool of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine has been shown to act as a central target during the inhibitory action of ammonium ions in vitro cultivated mammalian cell cultures. This pool has been demonstrated to be elevated at the end of a batch cultivation and very quickly as a response to exogenously applied ammonium chloride by using four different cell lines (hybridoma, BHK, CHO, and Ltk-929). The amount of enlarged UDP aminohexoses is correlated to the inhibitor concentration and additionally dependent on the cell line. The formation of the UDP sugars is associated with a transient reduction of the UTP pool. Moreover, the quick formation of UDP-GNAC is strictly dependent on the presence of, glucose and ammonium. Both metabolites act as biochemical precursors. Additionally, the formation of UDP-GNAc after ammonium application has been shown to increase with an elevated cultivation pH and to be independent of the inhibition of transcription and translation processes. The intracellular amount of UDP-GNAc correlates with the level of growth inhibition in mammalian cell lines.

  19. Enhanced Production of Docosahexaenoic Acid in Mammalian Cells

    PubMed Central

    Zhu, Guiming; Jiang, Xudong; Ou, Qin; Zhang, Tao; Wang, Mingfu; Sun, Guozhi; Wang, Zhao; Sun, Jie; Ge, Tangdong

    2014-01-01

    Docosahexaenoic acid (DHA), one of the important polyunsaturated fatty acids (PUFA) with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA). However, the acumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased prodution of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA. PMID:24788769

  20. Inorganic Polyphosphate and Energy Metabolism in Mammalian Cells*

    PubMed Central

    Pavlov, Evgeny; Aschar-Sobbi, Roozbeh; Campanella, Michelangelo; Turner, Raymond J.; Gómez-García, María R.; Abramov, Andrey Y.

    2010-01-01

    Inorganic polyphosphate (poly P) is a polymer made from as few as 10 to several hundred phosphate molecules linked by phosphoanhydride bonds similar to ATP. Poly P is ubiquitous in all mammalian organisms, where it plays multiple physiological roles. The metabolism of poly P in mammalian organisms is not well understood. We have examined the mechanism of poly P production and the role of this polymer in cell energy metabolism. Poly P levels in mitochondria and intact cells were estimated using a fluorescent molecular probe, 4′,6-diamidino-2-phenylindole. Poly P levels were dependent on the metabolic state of the mitochondria. Poly P levels were increased by substrates of respiration and in turn reduced by mitochondrial inhibitor (rotenone) or an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Oligomycin, an inhibitor of mitochondrial ATP-synthase, blocked the production of poly P. Enzymatic depletion of poly P from cells significantly altered the rate of ATP metabolism. We propose the existence of a feedback mechanism where poly P production and cell energy metabolism regulate each other. PMID:20124409

  1. Enhanced production of docosahexaenoic acid in mammalian cells.

    PubMed

    Zhu, Guiming; Jiang, Xudong; Ou, Qin; Zhang, Tao; Wang, Mingfu; Sun, Guozhi; Wang, Zhao; Sun, Jie; Ge, Tangdong

    2014-01-01

    Docosahexaenoic acid (DHA), one of the important polyunsaturated fatty acids (PUFA) with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA). However, the accumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased production of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA. PMID:24788769

  2. Transient transfection of mammalian cells using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  3. Shear stress induced stimulation of mammalian cell metabolism

    NASA Technical Reports Server (NTRS)

    Mcintire, L. V.; Frangos, J. A.; Eskin, S. G.

    1988-01-01

    A flow apparatus was developed for the study of the metabolic response of anchorage dependent cells to a wide range of steady and pulsatile shear stresses under well controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/sq cm, and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. It is demonstrated that shear stresses in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.

  4. Nuclear organization of DNA replication in primary mammalian cells.

    PubMed

    Kennedy, B K; Barbie, D A; Classon, M; Dyson, N; Harlow, E

    2000-11-15

    Using methods that conserve nuclear architecture, we have reanalyzed the spatial organization of the initiation of mammalian DNA synthesis. Contrary to the commonly held view that replication begins at hundreds of dispersed nuclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overlap with previously identified internal lamin A/C structures. These foci are established in early G(1)-phase and also contain members of the retinoblastoma protein family. Later, in S-phase, DNA replication sites distribute to regions located throughout the nucleus. As this progression occurs, association with the lamin structure and pRB family members is lost. A similar temporal progression is found in all the primary cells we have examined but not in most established cell lines, indicating that the immortalization process modifies spatial control of DNA replication. These findings indicate that in normal mammalian cells, the onset of DNA synthesis is coordinately regulated at a small number of previously unrecognized perinucleolar sites that are selected in early G(1)-phase. PMID:11090133

  5. Undetectable histone O-GlcNAcylation in mammalian cells.

    PubMed

    Gagnon, Jessica; Daou, Salima; Zamorano, Natalia; Iannantuono, Nicholas V G; Hammond-Martel, Ian; Mashtalir, Nazar; Bonneil, Eric; Wurtele, Hugo; Thibault, Pierre; Affar, El Bachir

    2015-01-01

    O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established. PMID:26075789

  6. DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP

    PubMed Central

    Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

    2009-01-01

    Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

  7. Some physiological properties of identified mammalian neuroglial cells

    PubMed Central

    Dennis, M. J.; Gerschenfeld, H. M.

    1969-01-01

    Mammalian glial cells were identified and studied in the optic nerves of anaesthetized rats. Cells with membrane potentials of 77-85 mV were located in the optic nerve with capillary micropipettes. These were shown to be neuroglia by iontophoretic injection of a fluorescent dye through the recording electrode, followed by histological verification of the location of the dye. No distinction was made between astroglia and oligodendroglia. Neuroglial cells gave no impulse activity. Their membrane potential was studied in isolated optic nerves by varying the ionic composition of the bathing fluid. The glial membrane potential depends predominantly on a transmembrane gradient of potassium ions. ImagesFig. 1Fig. 2 PMID:5821876

  8. Intracellular protein degradation in mammalian cells: recent developments.

    PubMed

    Knecht, Erwin; Aguado, Carmen; Cárcel, Jaime; Esteban, Inmaculada; Esteve, Juan Miguel; Ghislat, Ghita; Moruno, José Félix; Vidal, José Manuel; Sáez, Rosana

    2009-08-01

    In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes. PMID:19399586

  9. Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

    PubMed Central

    Ventoso, Iván; Kochetov, Alex; Montaner, David; Dopazo, Joaquín; Santoyo, Javier

    2012-01-01

    In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed. PMID

  10. Interplay between autophagy and programmed cell death in mammalian neural stem cells

    PubMed Central

    Chung, Kyung Min; Yu, Seong-Woon

    2013-01-01

    Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390] PMID:23977985

  11. Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

    PubMed

    Dizhe, E B; Ignatovich, I A; Burov, S V; Pohvoscheva, A V; Akifiev, B N; Efremov, A M; Perevozchikov, A P; Orlov, S V

    2006-12-01

    This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene. PMID:17223788

  12. CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    PubMed

    Du, Dan; Qi, Lei S

    2016-01-01

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems. PMID:26729910

  13. RNAi pathway participates in chromosome segregation in mammalian cells

    PubMed Central

    Huang, Chuan; Wang, Xiaolin; Liu, Xu; Cao, Shuhuan; Shan, Ge

    2015-01-01

    The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells.

  14. Garcinielliptone FC: antiparasitic activity without cytotoxicity to mammalian cells.

    PubMed

    Silva, Ana P; Silva, Marcos P; Oliveira, Cristiano G; Monteiro, Daniela C; Pinto, Pedro L; Mendonça, Ronaldo Z; Costa Júnior, Joaquim S; Freitas, Rivelilson M; de Moraes, Josué

    2015-06-01

    Garcinielliptone FC (GFC) is a natural prenylated benzophenone found in the seeds of Platonia insignis Mart. (Clusiaceae), a native Brazilian plant. It has been chemically characterized and it is known that GFC has several biological activities such as antioxidant and vasorelaxant properties. In this study, we report the in vitro effect of GFC against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. The anti-S. mansoni activity and cytotoxicity toward mammalian cells were determined for the compound. GFC⩾6.25 μM showed antischistosomal activity and confocal laser scanning microscopy analysis demonstrated several morphological alterations on the tegument of worms, and a correlation between viability and tegumental damage was observed. In addition, at sub-lethal concentrations of GFC (⩽3.125 μM), the number of S. mansoni eggs was reduced. More importantly, GFC exhibited no activity toward mammalian cells and, therefore, there is an appreciable selectivity of this compound against the helminths. In conclusion, these findings indicate the potential of GFC as an antiparasitic agent. PMID:25553916

  15. Cytotoxicity and genotoxicity of urban particulate matter in mammalian cells

    PubMed Central

    Dumax-Vorzet, Audrey F.; Tate, M.; Walmsley, Richard; Elder, Rhod H.; Povey, Andrew C.

    2015-01-01

    Ambient air particulate matter (PM)-associated reactive oxygen species (ROS) have been linked to a variety of altered cellular outcomes. In this study, three different PM samples from diesel exhaust particles (DEPs), urban dust standard reference material SRM1649a and air collected in Manchester have been tested for their ability to oxidise DNA in a cell-free assay, to increase intracellular ROS levels and to induce CYP1A1 gene expression in mammalian cells. In addition, the cytotoxicity and genotoxicity of PM were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline comet assay, respectively. All PM samples catalysed the Fenton reaction in a cell-free assay, but only DEP resulted in the generation of ROS as measured by dichlorodihydrofluorescein diacetate oxidation in mammalian cells. However, there was no evidence that increased ROS was a consequence of polycyclic aromatic hydrocarbon metabolism via CYP1A1 induction as urban dust, the Manchester dust samples but not DEP-induced CYP1A1 expression. Urban dust was more cytotoxic in murine embryonic fibroblasts (MEFs) than the other PM samples and also induced expression of GADD45a in the GreenScreen Human Cell assay without S9 activation suggesting the presence of a direct-acting genotoxicant. Urban dust and DEP produced comparable levels of DNA damage, as assessed by the alkaline comet assay, in MEFs at higher levels than those induced by Manchester PM. In conclusion, results from the cytotoxic and genotoxic assays are not consistent with ROS production being the sole determinant of PM-induced toxicity. This suggests that the organic component can contribute significantly to this toxicity and that further work is required to better characterise the extent to which ROS and organic components contribute to PM-induced toxicity. PMID:26113525

  16. Computing in mammalian cells with nucleic acid strand exchange.

    PubMed

    Groves, Benjamin; Chen, Yuan-Jyue; Zurla, Chiara; Pochekailov, Sergii; Kirschman, Jonathan L; Santangelo, Philip J; Seelig, Georg

    2016-03-01

    DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution. PMID:26689378

  17. Secretion of a bacterial protein by mammalian cells.

    PubMed

    Clément, J M; Jehanno, M

    1995-12-15

    The MalE protein is a periplasmic maltooligosaccharide binding protein from Escherichia coli. This protein is widely used as a model for protein export in bacteria and as a vector for the export and one-step affinity purification of foreign polypeptides. Expression of MalE was studied in various animal cell lines. The protein was exported into the culture medium, following the classical pathway of eukaryotic protein secretion. This was shown by a combination of approaches including the use of inhibitors of the Golgi complex and immunocytological methods. The signal sequence of MalE is required for secretion and a specific signal can be added to MalE that targets it to the endoplasmic reticulum. This work opens the way to the study of the secretion of a bacterial protein and to its use as a vector for protein secretion and purification from mammalian cells. PMID:8590643

  18. Methods for the Detection of Autophagy in Mammalian Cells.

    PubMed

    Zhang, Ziyan; Singh, Rajat; Aschner, Michael

    2016-01-01

    Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. © 2016 by John Wiley & Sons, Inc. PMID:27479363

  19. Computing in mammalian cells with nucleic acid strand exchange

    PubMed Central

    Pochekailov, Sergii; Kirschman, Jonathan L.; Santangelo, Philip J.; Seelig, Georg

    2015-01-01

    DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution. PMID:26689378

  20. Biology of Heme in Mammalian Erythroid Cells and Related Disorders

    PubMed Central

    Fujiwara, Tohru; Harigae, Hideo

    2015-01-01

    Heme is a prosthetic group comprising ferrous iron (Fe2+) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis. PMID:26557657

  1. Computing in mammalian cells with nucleic acid strand exchange

    NASA Astrophysics Data System (ADS)

    Groves, Benjamin; Chen, Yuan-Jyue; Zurla, Chiara; Pochekailov, Sergii; Kirschman, Jonathan L.; Santangelo, Philip J.; Seelig, Georg

    2016-03-01

    DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

  2. Engineering of ribozyme-based riboswitches for mammalian cells.

    PubMed

    Wieland, Markus; Ausländer, David; Fussenegger, Martin

    2012-03-01

    Artificial RNA riboswitches--apart from protein-based gene regulation systems, which have been known about for a long time--have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer-ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells. PMID:22305857

  3. Visualization of Intracellular Pathways of Engineered Baculovirus in Mammalian Cells

    PubMed Central

    Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

    2014-01-01

    Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy. PMID:24457070

  4. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    PubMed Central

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  5. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    PubMed Central

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-01

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  6. Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2015-01-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e. better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials. PMID:24749393

  7. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    PubMed Central

    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

  8. Radioimmunotherapy of Cryptococcus neoformans spares bystander mammalian cells

    PubMed Central

    Bryan, Ruth A; Jiang, Zewei; Morgenstern, Alfred; Bruchertseifer, Frank; Casadevall, Arturo; Dadachova, Ekaterina

    2013-01-01

    Aim Previously, we showed that radioimmunotherapy (RIT) for cryptococcal infections using radioactively labeled antibodies recognizing the cryptococcal capsule reduced fungal burden and prolonged survival of mice infected with Cryptococcus neoformans. Here, we investigate the effects of RIT on bystander mammalian cells. Materials & methods Heat-killed C. neoformans bound to anticapsular antibodies, unlabeled or labeled with the β-emitter rhenium-188 (16.9-h half-life) or the α-emitter bismuth-213 (46-min half-life), was incubated with macrophage-like J774.16 cells or epithelial-like Chinese hamster ovary cells. Lactate dehydrogenase activity, crystal violet uptake, reduction of tetrazolium dye (2,3)-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-terazolium-5-carboxanilide and nitric oxide production were measured. Results The J774.16 and Chinese hamster ovary cells maintained membrane integrity, viability and metabolic activity following exposure to radiolabeled C. neoformans. Conclusion RIT of C. neoformans is a selective therapy with minimal effects on host cells and these results are consistent with observations that RIT-treated mice with cryptococcal infection lacked RIT-related pathological changes in lungs and brain tissues. PMID:24020737

  9. Special delivery: distributing iron in the cytosol of mammalian cells

    PubMed Central

    Philpott, Caroline C.; Ryu, Moon-Suhn

    2014-01-01

    Eukaryotic cells contain hundreds of proteins that require iron cofactors for activity. These iron enzymes are located in essentially every subcellular compartment; thus, iron cofactors must travel to every compartment in the cell. Iron cofactors exist in three basic forms: Heme, iron–sulfur clusters, and simple iron ions (also called non-heme iron). Iron ions taken up by the cell initially enter a kinetically labile, exchangeable pool that is referred to as the labile iron pool. The majority of the iron in this pool is delivered to mitochondria, where it is incorporated into heme and iron–sulfur clusters, as well as non-heme iron enzymes. These cofactors must then be distributed to nascent proteins in the mitochondria, cytosol, and membrane-bound organelles. Emerging evidence suggests that specific systems exist for the distribution of iron cofactors within the cell. These systems include membrane transporters, protein chaperones, specialized carriers, and small molecules. This review focuses on the distribution of iron ions in the cytosol and will highlight differences between the iron distribution systems of simple eukaryotes and mammalian cells. PMID:25101000

  10. The effect of ascetic acid on mammalian cells

    SciTech Connect

    Mariana, Oana C; Trujillo, Antoinette; Sanders, Claire K; Burnett, Kassidy S; Freyer, James P; Mourant, Judith R

    2010-01-01

    Effects of the contrast agent, acetic acid, on mammalian cells are studied using light scattering measurements, viability and fluorescence pH assays. Results depend on whether cells are in PBS or are live and metabolizing. Acetic acid is a contrast agent used to aid the detection of cancerous and precancerous lesions of the uterine cervix. Typically 3% or 5% acetic acid is applied to the swface of the cervix and areas of the tissue that turn 'acetowhite' are considered more likely to be precancerous. The mechanism of action of acetic acid has never been understood in detail, although there are several hypotheses. One is that a decrease in pH causes cytokeratins in epithelial cells to polymerize. We will present data demonstrating that this is not the sole mechanism of acetowhitening. Another hypothesis is that a decrease in pH in the nucleus causes deacetylation of the histones which in turn results in a dense chromatin structure. Relevant to this hypothesis we have measured the internal pH of cells. Additional goals of this work are to understand what physical changes result in acetowhitening, to understand why there is variation in how cells respond to acetic acid, and to investigate how acetowhitening affects the light scatter properties measured by a fiber-optic probe we have developed for cervical cancer diagnostics.

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

    SciTech Connect

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

    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.

  12. Rationally designed logic integration of regulatory signals in mammalian cells

    NASA Astrophysics Data System (ADS)

    Leisner, Madeleine; Bleris, Leonidas; Lohmueller, Jason; Xie, Zhen; Benenson, Yaakov

    2010-09-01

    Molecular-level information processing is essential for `smart' in vivo nanosystems. Natural molecular computing, such as the regulation of messenger RNA (mRNA) synthesis by special proteins called transcription factors, has inspired engineered systems that can control the levels of mRNA with certain combinations of transcription factors. Here, we show an alternative approach to achieving general-purpose control of mRNA and protein levels by logic integration of transcription factor input signals in mammalian cells. The transcription factors regulate synthetic genes coding for small regulatory RNAs (called microRNAs), which, in turn, control the mRNA of interest (the output) via an RNA interference pathway. The simplicity of these modular interactions makes it possible, in theory, to implement any arbitrary logic relation between the transcription factors and the output. We construct, test and optimize increasingly complex circuits with up to three transcription factor inputs, establishing a platform for in vivo molecular computing.

  13. Hypoxia-mediated regulation of gene expression in mammalian cells

    PubMed Central

    Shih, Shu-Ching; Claffey, Kevin P.

    1998-01-01

    The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post-transcriptional mechanisms are important to the hypoxia-mediated regulation of gene expression. In this article, the cis-acting elements and trans-acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post-transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia-responsive genes are mechanistically linked and co-operative in nature. PMID:10319016

  14. Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species

    SciTech Connect

    Vanderhaeghen, P.; Schurmans, S.; Vassart, G.; Parmentier, M.

    1997-02-01

    Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. We have previously shown that genes belonging to this family were expressed in the male germ line from both dog and human. We have subsequently demonstrated the presence of one of the corresponding olfactory receptor proteins during dog spermatogenesis and in mature sperm cells. In this study, we investigated whether the unexpected pattern of expression of olfactory receptors in the male germ line was conserved in other mammalian species. Using reverse transcription-PCR with primers specific for the olfactory receptor gene family, about 20 olfactory receptor cDNA fragments were cloned from the testis of each mammalian species tested. As a whole, they displayed no sequence specificity compared to other olfactory receptors, but highly homologous, possibly orthologous, genes were amplified from different species. Finally, their pattern of expression, as determined by RNase protection assay, revealed that many but not all of these receptors were expressed predominantly in testis. The male germ line from each mammalian species tested is thus characterized by a specific repertoire of olfactory receptors, which display a pattern of expression suggestive of their potential implication in the control of sperm maturation, migration, or fertilization. 34 refs., 4 figs., 1 tab.

  15. Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

    PubMed Central

    2015-01-01

    Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics. PMID:25360681

  16. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

    PubMed

    Belcourt, M F; Penketh, P G; Hodnick, W F; Johnson, D A; Sherman, D H; Rockwell, S; Sartorelli, A C

    1999-08-31

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  17. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    PubMed Central

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; Sherman, David H.; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  18. Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

    PubMed

    Schürmann, Matthias; Frese, Natalie; Beyer, André; Heimann, Peter; Widera, Darius; Mönkemöller, Viola; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian; Gölzhäuser, Armin

    2015-11-18

    Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm. PMID:26436577

  19. Different localization of Hsp105 family proteins in mammalian cells

    SciTech Connect

    Saito, Youhei; Yamagishi, Nobuyuki; Hatayama, Takumi

    2007-10-15

    Hsp105{alpha} and Hsp105{beta} of the HSP105 family are alternatively spliced products derived from an hsp 105 gene transcript. Hsp105{alpha} is constitutively expressed and also induced by various stress, whereas Hsp105{beta}, lacking 44 amino acids from Hsp105{alpha}, is specifically expressed during mild heat shock. Although Hsp105{alpha} is shown to localize in the cytoplasm of mammalian cells, cellular localization of Hsp105{beta} is not known. In this study, we showed that Hsp105{beta} localized in the nucleus of cells in contrast to cytoplasmic Hsp105{alpha}, suggesting that these proteins function in different cellular compartments of cells. Using deletion and substitution mutants of Hsp105{alpha} and Hsp105{beta}, we revealed that these proteins had a functional nuclear localization signal (NLS) and a nuclear export signal (NES). Furthermore, Hsp105{alpha} accumulated in the nucleus of cells when treated with leptomycin B, a specific inhibitor of NES-dependent nuclear export. siRNA for importin {beta}, an essential component for NLS-dependent nuclear transport, inhibited the nuclear localization of Hsp105{beta}. Furthermore, the 44 amino acids sequence found in Hsp105{alpha} but not in Hsp105{beta} suppressed the NLS activity. Thus, the different localization of Hsp105{alpha} and Hsp105{beta} is suggested to be due to the suppressed NLS activity in Hsp105{alpha}.

  20. The influence of bisphenol A on mammalian cell cultivation.

    PubMed

    Stiefel, Fabian; Paul, Albert Jesuran; Jacopo, Troisi; Sgueglia, Angelo; Stützle, Martina; Herold, Eva Maria; Hesse, Friedemann

    2016-01-01

    Bisphenol A (BPA) plays a substantial role in industry, as it is used for polycarbonate (PC) plastics and epoxy resins which are required for various plastic consumer products. However, BPA is known to be an endocrine disruptor, and its influence on humans, animals, and various cell lines was addressed in diverse studies. As the burden of BPA can be increased by using disposable plastic articles and single-use technologies for cultivation, it is essential to examine the consequences of BPA presence on mammalian cells, as they are a contributing factor in the production of complex pharmaceutical therapeutics. We selected three industrially relevant cell lines and analyzed systemic effects of BPA by comparing cell culture performance in BPA-free poly-ethylene terephthalate glycol (PETG) and in PC shaking flasks. We focused on the influence of BPA on cellular growth, viability, and several metabolic parameters. In addition, we determined the product concentration and aggregation behavior of the recombinant proteins expressed by these cell lines and the BPA concentration within the medium caused by leaching. Moreover, we performed EC50 studies to determine the toxic concentration of BPA. Our results indicated that leached BPA had no effect on specific growth rates and viability and toxicity appeared at about 10(4) times higher concentrations; however, it influenced the specific productivity rate and metabolic activity parameters of our Chinese hamster ovary (CHO) cell line. Consequently, one can neglect BPA from leaching in the culture as long as the selected cell line is BPA tolerant. Otherwise, BPA can be a hurdle for pharmaceutical production, as it can influence the specific productivity of recombinant proteins. PMID:26381666

  1. Gene stability in mammalian cells and protein consistency.

    PubMed

    Berthold, W

    1994-01-01

    The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well. PMID:7883100

  2. Origin of bistability underlying mammalian cell cycle entry.

    PubMed

    Yao, Guang; Tan, Cheemeng; West, Mike; Nevins, Joseph R; You, Lingchong

    2011-04-26

    Precise control of cell proliferation is fundamental to tissue homeostasis and differentiation. Mammalian cells commit to proliferation at the restriction point (R-point). It has long been recognized that the R-point is tightly regulated by the Rb-E2F signaling pathway. Our recent work has further demonstrated that this regulation is mediated by a bistable switch mechanism. Nevertheless, the essential regulatory features in the Rb-E2F pathway that create this switching property have not been defined. Here we analyzed a library of gene circuits comprising all possible link combinations in a simplified Rb-E2F network. We identified a minimal circuit that is able to generate robust, resettable bistability. This minimal circuit contains a feed-forward loop coupled with a mutual-inhibition feedback loop, which forms an AND-gate control of the E2F activation. Underscoring its importance, experimental disruption of this circuit abolishes maintenance of the activated E2F state, supporting its importance for the bistability of the Rb-E2F system. Our findings suggested basic design principles for the robust control of the bistable cell cycle entry at the R-point. PMID:21525871

  3. Ski represses BMP signaling in Xenopus and mammalian cells

    SciTech Connect

    kluo@lbl.gov

    2001-05-16

    The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-{beta} family members.

  4. Effects of Non-Thermal Plasma on Mammalian Cells

    PubMed Central

    Kalghatgi, Sameer; Kelly, Crystal M.; Cerchar, Ekaterina; Torabi, Behzad; Alekseev, Oleg; Fridman, Alexander; Friedman, Gary; Azizkhan-Clifford, Jane

    2011-01-01

    Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of intracellular reactive oxygen species (ROS). We have utilized γ-H2AX to detect DNA damage induced by non-thermal plasma and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks; however, plasma does not lead to formation of bulky adducts/thymine dimers. PMID:21283714

  5. Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells

    PubMed Central

    Burgess, Andrew; Lorca, Thierry; Castro, Anna

    2012-01-01

    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. PMID:23029203

  6. Hollow fibers - Their applications to the study of mammalian cell function

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.; Angeline, M.; Harkness, J.; Chu, M.; Grindleland, R.

    1984-01-01

    The use of hollow fiber technology in cell culture and transplantation is examined. The morphologies of encapsulated pituitary cells before and after implantation into the rat are defined. Implantation experiments using hollow fibers to study mammalian cell functions are described. Consideration is given to examining somatotroph, prolactin, prostrate, fibroblast, and retinal cell functions. These experiments demonstrate that hollow fiber technology is applicable for studying mammalian cell functions.

  7. Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN HELMUT; VILA, ALEJANDRO; BRECHA, NICHOLAS C.

    2009-01-01

    Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in

  8. Non-cell-autonomous effects of vector-expressed regulatory RNAs in mammalian heart cells.

    PubMed

    Kizana, E; Cingolani, E; Marbán, E

    2009-09-01

    In mammalian cells, small regulatory RNA molecules are able to modulate gene expression in a cell-autonomous manner. In contrast, this mechanism of gene regulation can occur systemically in plants and nematodes. The existence of similar cell-to-cell transmission in mammalian cells has been explored, but generalizibilty and mechanistic insights have remained elusive. Here, we show that small regulatory RNA molecules are capable of a non-cell-autonomous effect between primary cardiac myocytes through a gap-junction-dependent mechanism. Co-culture experiments showed that both Dicer-processed small-interfering RNAs (siRNAs) and Drosha-processed microRNAs (miRNAs) were capable of target gene knockdown and physiological effects in a non-cell-autonomous manner. Target gene siRNA molecules were detected in recipient cells, indicating transfer of the primary effector molecule. All of these effects were abrogated by dominant-negative molecular suppression of gap junction function. Our results show that both siRNAs and miRNAs are capable of a non-cell-autonomous effect between mammalian cells through gap junctions. The recognition of this biological process raises the novel therapeutic prospect of a bystander effect after gene transfer to tissues bearing gap junctions and for cell engineering with a view to creating regulatory RNA donor cells that exert their influence throughout a syncytium. PMID:19516277

  9. Genetic changes in Mammalian cells transformed by helium cells

    SciTech Connect

    Durante, M.; Grossi, G. . Dipt. di Scienze Fisiche); Yang, T.C.; Roots, R. )

    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.

  10. Control of mammalian germ cell entry into meiosis.

    PubMed

    Feng, Chun-Wei; Bowles, Josephine; Koopman, Peter

    2014-01-25

    Germ cells are unique in undergoing meiosis to generate oocytes and sperm. In mammals, meiosis onset is before birth in females, or at puberty in males, and recent studies have uncovered several regulatory steps involved in initiating meiosis in each sex. Evidence suggests that retinoic acid (RA) induces expression of the critical pre-meiosis gene Stra8 in germ cells of the fetal ovary, pubertal testis and adult testis. In the fetal testis, CYP26B1 degrades RA, while FGF9 further antagonises RA signalling to suppress meiosis. Failsafe mechanisms involving Nanos2 may further suppress meiosis in the fetal testis. Here, we draw together the growing knowledge relating to these meiotic control mechanisms, and present evidence that they are co-ordinately regulated and that additional factors remain to be identified. Understanding this regulatory network will illuminate not only how the foundations of mammalian reproduction are laid, but also how mis-regulation of these steps can result in infertility or germline tumours. PMID:24076097

  11. Genotoxic activity of caramel on Salmonella and cultured mammalian cells.

    PubMed

    Yu, Y N; Chen, X R; Ding, C; Cai, Z N; Li, Q G

    1984-04-01

    The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different. PMID:6371518

  12. [Synthetic RNA technologies to control functions of mammalian cells].

    PubMed

    Saito, Hirohide

    2015-01-01

    We recently succeeded in producing nanostructures made of RNA-protein (RNP) complexes. We show that RNA and the ribosomal protein L7Ae can form a triangular-like nanostructure that consists of three L7Ae proteins, which form the apices of the triangle, bound to one RNA scaffold. This shape is created through a 60° kink introduced into the RNA structure on L7Ae binding. By varying the size of the RNA scaffold we could in turn alter the overall size of the triangular nanostructure. Several functions can be added to this nanostructure by the introduction of effector proteins fused to L7Ae. The design and construction of functional RNP nanostructures that detect specific cancer cells are discussed herein. In parallel, we developed synthetic RNP translational switches to control production levels of particular proteins depending on certain input(s) within the intracellular environment. The RNP-binding module was successfully incorporated into mRNA to generate functional RNP switches. The designed ON/OFF translational switches detect expression of the trigger factor and repress or activate expression of a desired protein (e.g., apoptosis regulator) in target mammalian cells. Taken together, RNP-binding module could be employed for constructing designer genetic switches and functional nanostructures to regulate cellular processes. PMID:25759049

  13. Firefly luciferase gene: structure and expression in mammalian cells.

    PubMed Central

    de Wet, J R; Wood, K V; DeLuca, M; Helinski, D R; Subramani, S

    1987-01-01

    The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression. Images PMID:3821727

  14. Experimental Design to Evaluate Directed Adaptive Mutation in Mammalian Cells

    PubMed Central

    Chiaro, Christopher R; May, Tobias

    2014-01-01

    Background We describe the experimental design for a methodological approach to determine whether directed adaptive mutation occurs in mammalian cells. Identification of directed adaptive mutation would have profound practical significance for a wide variety of biomedical problems, including disease development and resistance to treatment. In adaptive mutation, the genetic or epigenetic change is not random; instead, the presence and type of selection influences the frequency and character of the mutation event. Adaptive mutation can contribute to the evolution of microbial pathogenesis, cancer, and drug resistance, and may become a focus of novel therapeutic interventions. Objective Our experimental approach was designed to distinguish between 3 types of mutation: (1) random mutations that are independent of selective pressure, (2) undirected adaptive mutations that arise when selective pressure induces a general increase in the mutation rate, and (3) directed adaptive mutations that arise when selective pressure induces targeted mutations that specifically influence the adaptive response. The purpose of this report is to introduce an experimental design and describe limited pilot experiment data (not to describe a complete set of experiments); hence, it is an early report. Methods An experimental design based on immortalization of mouse embryonic fibroblast cells is presented that links clonal cell growth to reversal of an inactivating polyadenylation site mutation. Thus, cells exhibit growth only in the presence of both the countermutation and an inducing agent (doxycycline). The type and frequency of mutation in the presence or absence of doxycycline will be evaluated. Additional experimental approaches would determine whether the cells exhibit a generalized increase in mutation rate and/or whether the cells show altered expression of error-prone DNA polymerases or of mismatch repair proteins. Results We performed the initial stages of characterizing our system

  15. Micropatterning of Proteins and Mammalian Cells on Indium Tin Oxide

    PubMed Central

    Shah, Sunny S.; Howland, Michael C.; Chen, Li-Jung; Silangcruz, Jaime; Verkhoturov, Stanislav V.; Schweikert, Emile A.; Parikh, Atul N.; Revzin, Alexander

    2010-01-01

    This paper describes a novel surface engineering approach that combines oxygen plasma treatment and electrochemical activation to create micropatterned cocultures on indium tin oxide (ITO) substrates. In this approach, photoresist was patterned onto an ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surface to oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagen regions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry (ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagen and PEG molecules on the same surface. Imaging ellipsometry and atomic force microscopy (AFM) were employed to further investigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selective attachment of mammalian cells on the ITO substrate. Importantly, after seeding the first cell type, the ITO surfaces could be activated by applying negative voltage (−1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another cell type onto the same surface and to create micropatterned cocultures. Micropatterned cocultures of primary hepatocytes and fibroblasts created by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineering strategy described here may be used to pattern multiple cell types on an optically transparent and conductive substrate and is envisioned to have applications in tissue engineering and biosensing. PMID:20356132

  16. Cellular track model of biological damage to mammalian cell cultures from galactic cosmic rays

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Katz, Robert; Wilson, John W.; Townsend, Lawrence W.; Nealy, John E.; Shinn, Judy L.

    1991-01-01

    The assessment of biological damage from the galactic cosmic rays (GCR) is a current interest for exploratory class space missions where the highly ionizing, high-energy, high-charge ions (HZE) particles are the major concern. The relative biological effectiveness (RBE) values determined by ground-based experiments with HZE particles are well described by a parametric track theory of cell inactivation. Using the track model and a deterministic GCR transport code, the biological damage to mammalian cell cultures is considered for 1 year in free space at solar minimum for typical spacecraft shielding. Included are the effects of projectile and target fragmentation. The RBE values for the GCR spectrum which are fluence-dependent in the track model are found to be more severe than the quality factors identified by the International Commission on Radiological Protection publication 26 and seem to obey a simple scaling law with the duration period in free space.

  17. Limitations of allotopic expression of mitochondrial genes in mammalian cells.

    PubMed Central

    Oca-Cossio, Jose; Kenyon, Lesley; Hao, Huiling; Moraes, Carlos T

    2003-01-01

    The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria. PMID:14573482

  18. Rapid purification of protein complexes from mammalian cells

    PubMed Central

    Medina, Dan; Moskowitz, Neal; Khan, Subarna; Christopher, Scott; Germino, Joseph

    2000-01-01

    The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin. PMID:10871384

  19. Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells.

    PubMed

    Rayner, Benjamin S; Love, Dominic T; Hawkins, Clare L

    2014-06-01

    Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions. PMID:24632382

  20. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    PubMed

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins. PMID:25283838

  1. Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

    PubMed Central

    Tomaro-Duchesneau, Catherine; Saha, Shyamali; Malhotra, Meenakshi; Kahouli, Imen; Prakash, Satya

    2013-01-01

    Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed. PMID:26555963

  2. Protease inhibitors suppress the survival increase mediated by uncouplers in X-irradiated mammalian cells.

    PubMed

    Michel, S; Laval, F

    1982-01-01

    When mammalian cells are incubated with an uncoupler of oxidative phosphorylation prior to and during X-irradiation, the survival and the mutation frequency are markedly increased. This process requires protein synthesis and is inhibited when the cells are plated in the presence of a protease inhibitor (antipain or leupeptin). These results suggest the existence of an error-prone DNA repair process in X-irradiated mammalian cells. PMID:6814524

  3. Freezing-induced uptake of trehalose into mammalian cells facilitates cryopreservation.

    PubMed

    Zhang, Miao; Oldenhof, Harriëtte; Sieme, Harald; Wolkers, Willem F

    2016-06-01

    The aim of this study was to investigate if membrane-impermeable molecules are taken up by fibroblasts when exposing the cells to membrane phase transitions and/or freezing-induced osmotic forces. The membrane-impermeable fluorescent dye lucifer yellow (LY) was used to visualize and quantify uptake during endocytosis, and after freezing-thawing. In addition, trehalose uptake after freezing and thawing was studied. Fourier transform infrared spectroscopic studies showed that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas cells display strong highly cooperative fluid-to-gel membrane phase transitions during freezing, both in the absence and presence of protectants. Cells do not show uptake of LY upon passing the suprazero membrane phase transition at 30-10°C, whereas after freezing and thawing cells show intracellular LY equally distributed within the cell. Both, LY and trehalose are taken up by fibroblasts after freezing and thawing with loading efficiencies approaching 50%. When using 250 mM extracellular trehalose during cryopreservation, intracellular concentrations greater than 100 mM were determined after thawing. A plot of cryosurvival versus the cooling rate showed a narrow inverted-'U'-shaped curve with an optimal cooling rate of 40°C min(-1). Diluting cells cryopreserved with trehalose in isotonic cell culture medium resulted in a loss of cell viability, which was attributed to intracellular trehalose causing an osmotic imbalance. Taken together, mammalian cells can be loaded with membrane-impermeable compounds, including the protective agent trehalose, by subjecting the cells to freezing-induced osmotic stress. PMID:27003129

  4. Vitamin H-regulated transgene expression in mammalian cells.

    PubMed

    Weber, Wilfried; Bacchus, William; Daoud-El Baba, Marie; Fussenegger, Martin

    2007-01-01

    Although adjustable transgene expression systems are considered essential for future therapeutic and biopharmaceutical manufacturing applications, the currently available transcription control modalities all require side-effect-prone inducers such as immunosupressants, hormones and antibiotics for fine-tuning. We have designed a novel mammalian transcription-control system, which is reversibly fine-tuned by non-toxic vitamin H (also referred to as biotin). Ligation of vitamin H, by engineered Escherichia coli biotin ligase (BirA), to a synthetic biotinylation signal fused to the tetracycline-dependent transactivator (tTA), enables heterodimerization of tTA to a streptavidin-linked transrepressor domain (KRAB), thereby abolishing tTA-mediated transactivation of specific target promoters. As heterodimerization of tTA to KRAB is ultimately conditional upon the presence of vitamin H, the system is vitamin H responsive. Transgenic Chinese hamster ovary cells, engineered for vitamin H-responsive gene expression, showed high-level, adjustable and reversible production of a human model glycoprotein in bench-scale culture systems, bioreactor-based biopharmaceutical manufacturing scenarios, and after implantation into mice. The vitamin H-responsive expression systems showed unique band pass filter-like regulation features characterized by high-level expression at low (0-2 nM biotin), maximum repression at intermediate (100-1000 nM biotin), and high-level expression at increased (>100 000 nM biotin) biotin concentrations. Sequential ON-to-OFF-to-ON, ON-to-OFF and OFF-to-ON expression profiles with graded expression transitions can all be achieved by simply increasing the level of a single inducer molecule without exchanging the culture medium. These novel expression characteristics mediated by an FDA-licensed inducer may foster advances in therapeutic cell engineering and manufacturing of difficult-to-produce protein therapeutics. PMID:17827215

  5. Engineered mammalian RNAi can elicit antiviral protection that negates the requirement for the interferon response

    PubMed Central

    Bouhaddou, Mehdi; Sachs, David; tenOever, Benjamin Robert

    2015-01-01

    SUMMARY While the intrinsic antiviral cell defenses of many kingdoms utilize pathogen-specific small RNAs, the antiviral response of chordates is primarily protein-based and not uniquely tailored to the incoming microbe. In an effort to explain this evolutionary bifurcation, we determined whether antiviral RNA interference (RNAi) was sufficient to replace the protein-based type I interferon (IFN-I) system of mammals. To this end, we recreated an RNAi-like response in mammals and determined its effectiveness to combat influenza A virus in vivo in the presence and absence of the canonical IFN-I system. Mammalian antiviral RNAi, elicited by either host- or virus-derived small RNAs, effectively attenuated virus and prevented disease independently of the innate immune response. These data find that chordates could have utilized RNAi as their primary antiviral cell defense and suggest that the IFN-I system emerged as a result of natural selection imposed by ancient pathogens. PMID:26549455

  6. Initiation of DNA Interstrand Cross-link Repair in Mammalian Cells

    PubMed Central

    Hlavin, Erica M.; Smeaton, Michael B.; Miller, Paul S.

    2010-01-01

    Interstrand cross-links (ICLs) are among the most cytotoxic DNA lesions to cells because they prevent the two DNA strands from separating, thereby precluding replication and transcription. Even though chemotherapeutic cross-linking agents are well established in clinical use, and numerous repair proteins have been implicated in the initial events of mammalian ICL repair, the precise mechanistic details of these events remain to be elucidated. This review will summarize our current understanding of how ICL repair is initiated with an emphasis on the context (replicating, transcribed or quiescent DNA) in which the ICL is recognized, and how the chemical and physical properties of ICLs influence repair. Although most studies have focused on replication-dependent repair because of the relation to highly replicative tumor cells, replication-independent ICL repair is likely to be important in the circumvention of cross-link cytotoxicity in non-dividing, terminally differentiated cells that may be challenged with exogenous or endogenous sources of ICLs. Consequently, the ICL repair pathway that should be considered ‘dominant’ appears to depend on the cell type and the DNA context in which the ICL is encountered. The ability to define and inhibit distinct pathways of ICL repair in different cell cycle phases may help in developing methods that increase cytotoxicity to cancer cells while reducing side-effects in non-dividing normal cells. This may also lead to a better understanding of pathways that protect against malignancy and aging. PMID:20658650

  7. Comparative Mammalian Cell Toxicity of N-DBPs and C-DBPs

    EPA Science Inventory

    In order to generate a quantitative, direct comparison amongst classes of drinking water disinfection by-products (DBPs), we developed and calibrated in vitro mammalian cell cytotoxicity and genotoxicity assays to integrate the analytical biology with the analytical chemistry of ...

  8. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  9. Gold nanoparticles electroporation enhanced polyplex delivery to mammalian cells.

    PubMed

    Huang, Shuyan; Deshmukh, Harshavardhan; Rajagopalan, Kartik Kumar; Wang, Shengnian

    2014-07-01

    Nonviral methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. In this paper, we combined physical and chemical methods to improve the performance of polyplex delivery of DNA and small interfering RNA. Specifically, gold nanoparticles (AuNPs) were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes as usual while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids or small interfering RNA molecules to form AuNPs-polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation. The delivery efficiency was evaluated with both model anchor cells (i.e., NIH/3T3) and suspension cells (i.e., K562), together with their impact on cell viability. We found that AuNP-polyplex showed 1.5∼2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications. PMID:24777715

  10. Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells.

    PubMed

    Cohen, Sarit; Arbely, Eyal

    2016-06-01

    We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents. PMID:27120490

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

    PubMed

    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. PMID:27106902

  12. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

    PubMed Central

    Zhuang, Hanyi; Matsunami, Hiroaki

    2009-01-01

    A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigate odorant-OR relationship in mammals has been an inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein (RTP) family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput “deorphanization” of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently-expressed mammalian odorant receptors in HEK293T cells. The stages of odorant receptor cell-surface expression include cell culture preparation, transfer of cells, transfection, and immunocytochemistry/flow cytometry, odorant stimulation, and luciferase assay. This protocol can be completed in a period of 3 days from transfer of cells to cell-surface expression detection and/or measurement of functional activation. PMID:18772867

  13. The monitoring possibility of some mammalian cells for zinc concentrations on metallic materials.

    PubMed

    Ogawa, Akiko; Okuda, Naoaki; Hio, Katsuya; Kanematsu, Hideyuki; Tamauchi, Hidekazu

    2012-05-01

    Zinc plating is widely used to protect steels against corrosion. However, the possibility of a high environmental risk for zinc has been recently discussed among advanced countries and more environmentally-friendly substitutes are required urgently. Therefore, monitoring zinc concentration changes on metallic materials such as steel is very important. We chose to measure zinc concentration changes in some mammalian cells and confirmed that V79 cells were highly sensitive to changes in zinc concentrations. In this study, the following process was applied to the proprietary production for tin-zinc alloy films on steel using V79 cells. Specimens were immersed in PBS to produce extracts. Zinc concentrations in the extracts almost corresponded to zinc concentrations on steel surfaces. When extracts were added to a V79 cell culture, colony formation was inhibited, and inhibition increased with increases in zinc concentrations. Changes in zinc concentrations on steel surfaces with heat treatment could be monitored relatively well by V79 cells, even though the results were still semi-quantitative. PMID:22331507

  14. Mammalian cell nano structures visualized by cryo Hilbert differential contrast transmission electron microscopy.

    PubMed

    Setou, Mitsutoshi; Radostin, Danev; Atsuzawa, Kimie; Yao, Ikuko; Fukuda, Yoshiyuki; Usuda, Nobuteru; Nagayama, Kuniaki

    2006-12-01

    We applied the Hilbert differential contrast phase electron microscopy technique for the first time to mammalian cells, Ptk2 cells. Intracellular architectures such as the cytoskeletal network, membranous organelles, and mitochondria were observed without prior removal of cell membranes or extraction of soluble proteins. The attachment of mitochondria and membrane organelles with microtubules were observed. Microtubules were depolymerized by nocodazole treatment as expected. Thus, Hilbert differential contrast phase electron microscopy of vitrified cells is a nano-scale molecular imaging technique that opens up new vistas for exploring the supramolecular organization of the mammalian cell. PMID:17187178

  15. Anaplasma phagocytophilum surface protein AipA mediates invasion of mammalian host cells

    PubMed Central

    Seidman, David; Ojogun, Nore; Walker, Naomi J.; Mastronunzio, Juliana; Kahlon, Amandeep; Hebert, Kathryn S.; Karandashova, Sophia; Miller, Daniel P.; Tegels, Brittney K.; Marconi, Richard T.; Fikrig, Erol; Borjesson, Dori L.; Carlyon, Jason A.

    2014-01-01

    Summary Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA1–87) were found to be essential for host cell invasion. Recombinant AipA1–87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1–87, but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1–87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis. PMID:24612118

  16. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  17. Fundamental aspects of the freezing of cells, with emphasis on mammalian ova and embryos

    SciTech Connect

    Mazur, P.

    1980-01-01

    The problem in cryobiology is how to cool cells to -196/sup 0/C and return them to normal temperatures without killing them. One important factor is the presence of a protective additive like glycerol or dimethyl sulfoxide. Mammalian cells rarely survive freezing to below -40/sup 0/C in its absence. In the presence of an additive, survival is critically dependent on the cooling rate. Supraoptimal rates and suboptimal rates are both damaging. Death at supraoptimal rates is the result of the formation of intracellular ice and its recrystallization during warming. Death at suboptimal rates is a consequence of the major alterations in aqueous solutions produced by ice formation. The chief effects are a major reduction in the fraction of the solution remaining unfrozen at a given temperature and a major increase in the solute concentration of that fraction. The introduction of molar concentrations of additive greatly reduces both the fraction frozen and the concentration of electrolytes in the unfrozen channels and in the cell interior. Usually, freezing either kills cells outright or it results in survivors that retain full capacity to function. But there is the possibility that in some cases survivors may in fact be impaired genetically or physiologically. All evidence indicates that genetic damage does not occur. But there are clear examples in which freezing does induce nonlethal physiological damage. (ERB)

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

    EPA Science Inventory

    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...

  19. Coenzyme Q10 protects hair cells against aminoglycoside.

    PubMed

    Sugahara, Kazuma; Hirose, Yoshinobu; Mikuriya, Takefumi; Hashimoto, Makoto; Kanagawa, Eiju; Hara, Hirotaka; Shimogori, Hiroaki; Yamashita, Hiroshi

    2014-01-01

    It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10) is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group). In the neomycin group, utricles were cultured with neomycin (1 mM) to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30-0.3 µM). Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear. PMID:25265538

  20. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

    PubMed Central

    Thompson, David B.; Cronican, James J.; Liu, David R.

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

  1. a Study of Biophysical Mechanisms of Damage by Ionizing Radiation to Mammalian Cells in Vitro.

    NASA Astrophysics Data System (ADS)

    Chen, Chun-Zhang

    Available from UMI in association with The British Library. An extensive survey made of published survival data of damage by ionizing radiation to mammalian cells in vitro has led to the new conclusion that the damage is determined by the specific ionization or the mean free path between ionizing events along the charged particle tracks. The optimum damage is observed when the mean free path is equivalent to the DNA double strand spacing of 1.8 nm. Therefore, the biological mechanism of ionizing radiation to mammalian cells in vitro is intra track dominant. A 100 keV electron accelerator has been constructed and commissioned to produce a broad beam irradiation field of greater than 1 cm diameter. The fluence rate may be adjusted from 10^8cm^ {-2}sec^{-1} downwards to enable further development as a chronic irradiation facility. Another new feature of the accelerator is that it incorporates a differential vacuum system which permits irradiation of the monolayer cell cultures to be carried out in normal pressure. Experiments of irradiation to Chinese hamster cells, by ^{241}Am alpha particles at low fluence rate, have supplied satisfactory data for testing a new DNA-rupture model which is under development. For V79 cells irradiated at a low fluence rate of 10^5cm^{ -2}min^{-1}, when survival data were fitted into the model, new biophysical parameters were extracted and a proposal was made that the repair phenomenon of cellular survival at very low doses is determined by three time factors: the irradiation time, the damage fixation time and the repair time. The values obtained were 3-4 hours for the mean repair time, and more than 10 hours for the damage to be considered permanent. Details of the monolayer cell culture technique developed and used in the present experiments are described. Consideration has been given to the significance of the results obtained from the study in radiation protection and in radiotherapy. In future studies it is recommended that more

  2. Phototransfection of mammalian cells using femtosecond laser pulses: optimization and applicability to stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; Dholakia, Kishan; Gunn-Moore, Frank

    2010-07-01

    Recently, femtosecond laser pulses have been utilized for the targeted introduction of genetic matter into mammalian cells. This rapidly expanding and developing novel optical technique using a tightly focused laser light beam is called phototransfection. Extending previous studies [Stevenson et al., Opt. Express 14, 7125-7133 (2006)], we show that femtosecond lasers can be used to phototransfect a range of different cell lines, and specifically that this novel technology can also transfect mouse embryonic stem cell colonies with ~25% efficiency. Notably, we show the ability of differentiating these cells into the extraembryonic endoderm using phototransfection. Furthermore, we present two new findings aimed at optimizing the phototransfection method and improving applicability: first, the influence of the cell passage number on the transfection efficiency is explored and, second, the ability to enhance the transfection efficiency via whole culture treatments. Our results should encourage wider uptake of this methodology.

  3. A dual molecular analogue tuner for dissecting protein function in mammalian cells.

    PubMed

    Brosh, Ran; Hrynyk, Iryna; Shen, Jessalyn; Waghray, Avinash; Zheng, Ning; Lemischka, Ihor R

    2016-01-01

    Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes. PMID:27230261

  4. A dual molecular analogue tuner for dissecting protein function in mammalian cells

    PubMed Central

    Brosh, Ran; Hrynyk, Iryna; Shen, Jessalyn; Waghray, Avinash; Zheng, Ning; Lemischka, Ihor R.

    2016-01-01

    Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes. PMID:27230261

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

    PubMed

    Levine, Zebulon G; Walker, Suzanne

    2016-06-01

    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. PMID:27294441

  6. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells

    PubMed Central

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi

    2015-01-01

    ABSTRACT RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic

  7. ssiRNA Induced Gene Silencing is Transmitted Between Cells From the Mammalian Central Nervous System

    PubMed Central

    Zhao, Tian-Yong; Zou, Shi-Ping; Alimova, Yelena V.; Wang, Guoying; Hauser, Kurt F.; Ghandour, M. Said; Knapp, Pamela E.

    2014-01-01

    Although siRNA induced gene silencing can be transmitted between cells in plants and in C. elegans, this phenomenon has been barely studied in mammalian cells. Both immortalized oligodendrocytes and SNB-19 glioblastoma cells were transfected with siRNA constructs for PTEN (phosphatase and tensin homolog deleted on chromosome 10) or Akt (Akt/protein kinase B). Co-cultures were established between silenced cells and non-silenced cells which were hygromycin resistant and/or expressed green fluorescent protein (GFP). After fluorescence sorting or hygromycin selection to remove the silenced cells, the expression of PTEN or Akt genes in the originally unsilenced cells was in all cases significantly decreased. Importantly, silencing did not occur in transwell culture studies, suggesting that transmission of the silencing signal requires a close association between cells. These results provide the first direct demonstration that an siRNA induced silencing signal can be transmitted between mammalian central nervous system (CNS) cells. PMID:16923165

  8. Studies on the mechanisms of mammalian cell killing by a freeze-thaw cycle: conditions that prevent cell killing using nucleated freezing

    SciTech Connect

    Shier, W.T.

    1988-04-01

    Normally a freeze-thaw cycle is a very efficient method of killing mammalian cells. However, this report describes conditions that prevent killing of cultured mammalian cells by nucleated freezing at -24 degrees C. Optimal protection from cell killing at -24 degrees C was obtained in isotonic solutions containing an organic cryoprotectant such as dimethyl sulfoxide (DMSO; 10%, v/v), a saccharide such as sucrose over a broad concentration range from 50 to 150 mM, and glucose. Glycerol was also an effective cryoprotectant but other organic solvents were ineffective, although in some cases they appeared to protect cell membranes, while not protecting other vital components. A wide variety of saccharide structures were effective at protecting cells from freeze-thaw killing, with trehalose being particularly effective. The degree of resistance to killing by a freeze-thaw cycle under these conditions varied widely among different cell lines. If toxicity of DMSO was responsible for this variability of cryoprotection, it must have been due to short-term, not longer term, toxicity of DMSO. Studies on the mechanism by which cells are protected from killing under these conditions indicated that neither vitrification of the medium nor the concentrating of components during freezing were involved. One model not eliminated by the mechanistic studies proposes that the organic solvent cryoprotectant component acts by fluidizing membranes under the thawing conditions, so that any holes produced by ice crystals propagating through membranes can reseal during the thawing process. In this model one of the mechanisms by which the saccharide component could act is by entering the cells and stabilizing vital intracellular components. Consistent with this, a freeze-thaw cycle promoted the uptake of labeled sucrose into cultured cells.

  9. Auxin induces cell proliferation in an experimental model of mammalian renal tubular epithelial cells.

    PubMed

    Cernaro, Valeria; Medici, Maria Antonietta; Leonello, Giuseppa; Buemi, Antoine; Kohnke, Franz Heinrich; Villari, Antonino; Santoro, Domenico; Buemi, Michele

    2015-06-01

    Indole-3-acetic acid is the main auxin produced by plants and plays a key role in the plant growth and development. This hormone is also present in humans where it is considered as a uremic toxin deriving from tryptophan metabolism. However, beyond this peculiar aspect, the involvement of auxin in human pathophysiology has not been further investigated. Since it is a growth hormone, we evaluated its proliferative properties in an in vitro model of mammalian renal tubular epithelial cells. We employed an experimental model of renal tubular epithelial cells belonging to the LLC-PK1 cell line that is derived from the kidney of healthy male pig. Growth effects of auxin against LLC-PK1 cell lines were determined by a rapid colorimetric assay. Increasing concentrations of auxin (to give a final concentration from 1 to 1000 ng/mL) were added and microplates were incubated for 72 h. Each auxin concentration was assayed in four wells and repeated four times. Cell proliferation significantly increased, compared to control cells, 72 h after addition of auxin to cultured LLC-PK1 cells. Statistically significant values were observed when 100 ng/mL (p < 0.01) and 1000 ng/mL (p < 0.05) were used. In conclusion, auxin influences cell growth not only in plants, where its role is well documented, but also in mammalian cell lines. This observation opens new scenarios in the field of tissue regeneration and may stimulate a novel line of research aiming at investigating whether this hormone really influences human physiology and pathophysiology and in particular, kidney regeneration. PMID:25707523

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

    EPA Science Inventory

    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...

  11. Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Ogura, Makoto; Sato, Shunichi; Wakisaka, Hitoshi; Ashida, Hiroshi; Uenoyama, Maki; Masaki, Yoshinori; Obara, Minoru

    2004-06-01

    Plasmid DNA has been successfully delivered to mammalian cells by applying a nanosecond pulsed laser-induced stress wave (LISW). Cells exposed to a LISW were selectively transfected with plasmids coding for green fluorescent protein. It was also shown that transient, mild cellular heating (~43 °C) was effective in improving the transfection efficiency.

  12. Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

    PubMed

    Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

    2010-05-01

    We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. PMID:20381456

  13. Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells

    PubMed Central

    Kanekura, Kohsuke; Ishigaki, Shinsuke; Merksamer, Philip I.; Papa, Feroz R.; Urano, Fumihiko

    2014-01-01

    The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER stressed cells. Here we report the establishment of a system for monitoring ER redox state in mammalian cells. The new ER redox sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes ER redox state. The mammalian ER redox sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration and Wolfram syndrome. PMID:24042438

  14. A covalent approach for site-specific RNA labeling in Mammalian cells.

    PubMed

    Li, Fahui; Dong, Jianshu; Hu, Xiaosong; Gong, Weimin; Li, Jiasong; Shen, Jing; Tian, Huifang; Wang, Jiangyun

    2015-04-01

    Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA. PMID:25694369

  15. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

    PubMed Central

    Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

    2015-01-01

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

  16. Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit.

    PubMed

    Barbie, David A; Kudlow, Brian A; Frock, Richard; Zhao, Jiyong; Johnson, Brett R; Dyson, Nicholas; Harlow, Ed; Kennedy, Brian K

    2004-01-01

    In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit. PMID:14701733

  17. Some process control/design considerations in the development of a microgravity mammalian cell bioreactor

    NASA Technical Reports Server (NTRS)

    Goochee, Charles F.

    1987-01-01

    The purpose is to review some of the physical/metabolic factors which must be considered in the development of an operating strategy for a mammalian cell bioreactor. Emphasis is placed on the dissolved oxygen and carbon dioxide requirements of growing mammalian epithelial cells. Literature reviews concerning oxygen and carbon dioxide requirements are discussed. A preliminary, dynamic model which encompasses the current features of the NASA bioreactor is presented. The implications of the literature survey and modeling effort on the design and operation of the NASA bioreactor are discussed.

  18. Genetically Encoded Cyclopropene Directs Rapid, Photoclick Chemistry-Mediated Protein Labeling in Mammalian Cells

    PubMed Central

    Yu, Zhipeng; Pan, Yanchao; Wang, Zhiyong; Wang, Jiangyun; Lin, Qing

    2012-01-01

    Genetic incorporation of a cyclopropene amino acid, Nε-(1-methylcycloprop-2-enecarboxamido)-lysine (CpK), into sperm whale myoglobin site-specifically in E. coli as well as enhanced green fluorescent protein in mammalian cells was achieved through amber codon suppression employing an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair. Because of its high ring strain, cyclopropene exhibited fast reaction kinetics (up to 58 ± 16 M−1 s−1) in the photoclick reaction and allowed rapid (~ 2 min) bioorthogonal labeling of proteins in mammalian cells. PMID:22997015

  19. Methylated DNA-binding protein is present in various mammalian cell types

    SciTech Connect

    Supakar, P.C.; Weist, D.; Zhang, D.; Inamdar, N.; Zhang, Xianyang; Khan, R.; Ehrlich, M. ); Ehrlich, K.C. )

    1988-08-25

    A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m{sup 5}C) residues at specific positions. The authors found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

  20. Effect of uncouplers on radiosensitivity and mutagenicity in x-irradiated mammalian cells.

    PubMed Central

    Laval, F

    1980-01-01

    The number of x-irradiated mammalian cells surviving is markedly increased when the cells are incubated with an uncoupler of oxidative phosphorylation prior to or immediately after irradiation. This increase is greater in plateau-phase cells than in exponentially growing cells. The increase in survival is related to the potency of the uncouplers, which do not modify the effective x-ray dose. The influence of uncouplers on survival is related to an increase of repair and semiconservative DNA synthesis. The mutation frequency (8-azaguanine-resistant mutants) is significantly higher in irradiated cells treated with uncouplers than in untreated cells. These results suggest the existence of an error-prone repair process in mammalian cells. PMID:6930660

  1. Effect of uncouplers on radiosensitivity and mutagenicity in x-irradiated mammalian cells

    SciTech Connect

    Laval, F.

    1980-05-01

    The number of x-irradiated mammalian cells surviving is markedly increased when the cells are incubated with an uncoupler of oxidative phosphorylation prior to or immediately after irradiation. This increase is greater in plateau-phase cells than in exponentially growing cells. The increase in survival is related to the potency of the uncouplers, which do not modify the effective x-ray dose. The influence of uncouplers on survival is related to an increase of repair and semiconservative DNA synthesis. The mutation frequency (8-azaguanine-resistant mutants) is significantly higher in irradiated cells treated with uncouplers than in untreated cells. These results suggest the existence of an error-prone repair process in mammalian cells.

  2. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo.

    PubMed

    Fischer, Ulrike; Backes, Christina; Raslan, Abdulrahman; Keller, Andreas; Meier, Carola; Meese, Eckart

    2015-03-30

    In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells. PMID:25760141

  3. Study of radiation effects on mammalian cells in vitro

    NASA Technical Reports Server (NTRS)

    Sinclair, W. K.

    1968-01-01

    Radiation effect on single cells and cell populations of Chinese hamster lung tissue is studied in vitro. The rate and position as the cell progresses through the generation cycle shows division delay, changes in some biochemical processes in the cell, chromosomal changes, colony size changes, and loss of reproductive capacity.

  4. A general design strategy for protein-responsive riboswitches in mammalian cells.

    PubMed

    Ausländer, Simon; Stücheli, Pascal; Rehm, Charlotte; Ausländer, David; Hartig, Jörg S; Fussenegger, Martin

    2014-11-01

    RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits. PMID:25282610

  5. MAMMALIAN CELL CULTURE ASSAY TO QUANTITATE CHEMICALLY INDUCED ANEUPLOIDY: USE OF A MONOCHROMOSOMAL HUMAN/MOUSE CELL HYBRID

    EPA Science Inventory

    A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome seg...

  6. Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

    PubMed Central

    Stanley, Pamela; Sundaram, Subha

    2014-01-01

    Glycosylation engineering is used to generate glycoproteins, glycolipids or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans, with truncated glycans missing the sugar transferred by that glycosyltransferase, and also missing those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also give rise to spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes

  7. Direct observation of mammalian cell growth and size regulation

    PubMed Central

    Son, Sungmin; Tzur, Amit; Weng, Yaochung; Jorgensen, Paul; Kim, Jisoo; Kirschner, Marc W.; Manalis, Scott R.

    2012-01-01

    We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis. PMID:22863882

  8. Factors affecting the attachment of Treponema pallidum to mammalian cells in vitro.

    PubMed

    Wong, G H; Steiner, B; Faine, S; Graves, S

    1983-02-01

    Attachment of Treponema pallidum (Nichols) to mammalian cells is probably the first step in the pathogenesis of syphilis. It may also be important for the multiplication of T pallidum in vitro. When factors affecting the attachment of T pallidum to mammalian cells in vitro were studied significantly greater numbers of treponemes were found to attach to baby rabbit genital organ (BRGO) cells than to five other mammalian cell lines. When attached to BRGO cells T pallidum survived longer in vitro than unattached treponemes. Eagle's minimal essential medium was superior to three other culture media in increasing attachment and maintaining the survival of treponemes. Dithiothreitol (0.25-1.0 mmol/l) had no effect on the attachment of T pallidum to BRGO cells. Anaerobic conditions were superior to microaerophilic conditions, and the latter were superior to aerobic conditions for the attachment and survival of T pallidum to BRGO cells. Within the range of concentrations tested the number of treponemes attached to the BRGO cells was directly dependent on the concentrations of viable treponemes in the inoculum. Greater numbers of treponemes attached to actively metabolising BRGO cells than to quiescent or slowly growing cells. PMID:6337680

  9. Role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells

    PubMed Central

    Hu, Ningjie; Yu, Richard; Shikuma, Cecilia; Shiramizu, Bruce; Ostrwoski, Mario A.; Yu, Qigui

    2011-01-01

    Poxviruses have been extensively used as a promising vehicle to efficiently deliver a variety of antigens in mammalian hosts to induce immune responses against infectious diseases and cancer. Using recombinant vaccinia virus (VV) and canarypox virus (ALVAC) expressing enhanced green fluorescent protein (EGFP) or multiple HIV-1 gene products, we studied the role of four cellular signaling pathways, the phosphoinositide-3-OH kinase (PI3K), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK), in poxvirus-mediated foreign gene expression in mammalian cells. In nonpermissive infection (human monocytes), activation of PI3K, ERK, p38 MAPK, and JNK was observed both VV and ALVAC and blocking PI3K, p38 MAKP, and JNK pathways with their specific inhibitors significantly reduced viral and vaccine antigen gene expression. Whereas, blocking the ERK pathway had no significant effect. Among these cellular signaling pathways studied, PI3K was the most critical pathway involved in gene expression by VV- or ALVAC-infected monocytes. The important role of PI3K in poxvirus-mediated gene expression was further confirmed in mouse epidermal cells stably transfected with dominant-negative PI3K mutant, as poxvirus-mediated targeted gene expression was significantly decreased in these cells when compared with their parental cells. Signaling pathway activation was influenced gene expression at the mRNA level rather than virus binding. In permissive mammalian cells, however, VV DNA copies were also significantly decreased in the absence of normal function of PI3K pathway. Poxvirus-triggered activation of PI3K pathway could be completely abolished by atazanavir, a new generation of antiretroviral protease inhibitors (PIs). As a consequence, ALVAC-mediated EGFP or HIV-1 gag gene expression in infected primary human monocytes was significantly reduced in the presence of atazanavir. These findings implicate that

  10. Expanding roles of programmed cell death in mammalian neurodevelopment.

    PubMed

    De Zio, Daniela; Giunta, Luigi; Corvaro, Marco; Ferraro, Elisabetta; Cecconi, Francesco

    2005-04-01

    Programmed cell death is an orchestrated form of cell death in which cells are actively involved in their own demise. During neural development in mammals, many progenitor cells, immature cells or differentiated cells undergo the most clearly characterized type of cell death, apoptosis. Several pathways of apoptosis have been linked to neural development, but according to the numerous and striking phenotypes observed when apoptotic genes are inactivated, the mitochondrial death-route is the most important pathway in this context. Here, we discuss the relative importance of pro-growth/pro-death factors in the control of neural tissue development. We also discuss the impact of studying programmed cell death in development in order to better understand the basis of several human diseases and embryonic defects of the nervous system. PMID:15797838