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Sample records for cell inactivation rates

  1. Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells

    Science.gov (United States)

    Cucinotta, F. A.; Wilson, J. W.; Shavers, M. R.; Katz, R.

    1996-01-01

    It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

  2. Cell inactivation by heavy charged particles

    Energy Technology Data Exchange (ETDEWEB)

    Blakely, E A [Lawrence Berkeley Lab., CA (United States). Cell and Molecular Biology Div.

    1992-06-01

    The inactivation of cells resulting in lethal or aberrant effects by charged particles is of growing interest. Charged particles at extremely high LET are capable of completely eliminating cell-type and cell-line differences in repair capacity. It is still not clear however whether the repair systems are inactivated, or merely that heavy-ion lesions are less repairable. Studies correlating the particle inactivation dose of radioresistant cells with intact DNA analyzed with pulse field gel electrophoresis and other techniques may be useful, but more experiments are also needed to assess the fidelity of repair. For particle irradiations between 40-100 keV/{mu}m there is however evidence for particle-induced activation of specific genes in mammalian cells, and certain repair processes in bacteria. New data are available on the inactivation of developmental processes in several systems including seeds, and cells of the nematode C. elegans. Future experimental and theoretical modeling research emphasis should focus on exploring particle-induced inactivation of endpoints assessing functionality and not just lethality, and on analyzing molecular damage and genetic effects arising in damage but non-inactivated survivors. The discrete nature of selective types of particle damage as a function of radiation quality indicates the value of accelerated ions as probes of normal and aberrant biological processes. Information obtained from molecular analyses of damage and repair must however be integrated into the context of cellular and tissue functions of the organism. (orig.).

  3. Promotion of initiated cells by radiation-induced cell inactivation.

    Science.gov (United States)

    Heidenreich, W F; Paretzke, H G

    2008-11-01

    Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

  4. X Inactivation and Progenitor Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ruben Agrelo

    2011-04-01

    Full Text Available In mammals, silencing of one of the two X chromosomes is necessary to achieve dosage compensation. The 17 kb non-coding RNA called Xist triggers X inactivation. Gene silencing by Xist can only be achieved in certain contexts such as in cells of the early embryo and in certain hematopoietic progenitors where silencing factors are present. Moreover, these epigenetic contexts are maintained in cancer progenitors in which SATB1 has been identified as a factor related to Xist-mediated chromosome silencing.

  5. Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

    Science.gov (United States)

    Capes, Deborah L; Goldschen-Ohm, Marcel P; Arcisio-Miranda, Manoel; Bezanilla, Francisco; Chanda, Baron

    2013-08-01

    Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

  6. Inactivation of bacterial cells by cyclotron beams

    Energy Technology Data Exchange (ETDEWEB)

    Yatagai, F [Waseda Univ., Tokyo (Japan). School of Science and Engineering; Takahashi, T; Matsuyama, A

    1975-06-01

    B. subtilis spores, E. coli Bsub(s-1) and E. coli B/r were bombarded with ..cap alpha..-particles and heavy ions of carbon, nitrogen and oxygen accelerated in the IPCR Cyclotron. The RBE versus LETsub(infinity) curve for B. subtilis spores showed a maximum peak at 120 keV/..mu..m, while those for E. coli Bsub(s-1) and E. coli B/r declined without any maximum as LETsub(infinity) values increased. In the region of ..cap alpha..-particles, the effective inactivation cross section (Ssub(eff)) for these three strains increased with increasing LETsub(infinity), and the rates of increase in Ssub(eff) in the LET region from --30 to --150 keV/..mu..m were 15.0, 1.5 and 2.5 times for B. subtilis spores, E. coli Bsub(s-1) and E. coli B/r, respectively. In the case of B. subtilis spores, Ssub(eff) values for heavy ions were almost independent of their energies, but the other two strains showed a considerable dependence upon beam energy. The characteristic LET dependence of Ssub(eff) observed in this study was fairly well explained by the target theory based on microdose concept.

  7. Inactivation of bacterial cells by cyclotron beams

    International Nuclear Information System (INIS)

    Yatagai, Fumio; Takahashi, Tadashi; Matsuyama, Akira.

    1975-01-01

    B. subtilis spores, E. coli Bsub(s-1) and E. coli B/r were bombarded with α-particles and heavy ions of carbon, nitrogen and oxygen accelerated in the IPCR Cyclotron. The RBE versus LETsub(infinity) curve for B. subtilis spores showed a maximum peak at 120 keV/μm, while those for E. coli Bsub(s-1) and E. coli B/r declined without any maximum as LETsub(infinity) values increased. In the region of α-particles, the effective inactivation cross section (Ssub(eff)) for these three strains increased with increasing LETsub(infinity), and the rates of increase in Ssub(eff) in the LET region from --30 to --150 keV/μm were 15.0, 1.5 and 2.5 times for B. subtilis spores, E. coli Bsub(s-1) and E. coli B/r, respectively. In the case of B. subtilis spores, Ssub(eff) values for heavy ions were almost independent of their energies, but the other two strains showed a considerable dependence upon beam energy. The characteristic LET dependence of Ssub(eff) observed in this study was fairly well explained by the target theory based on microdose concept. (auth.)

  8. High Heating Rates Affect Greatly the Inactivation Rate of Escherichia coli

    Science.gov (United States)

    Huertas, Juan-Pablo; Aznar, Arantxa; Esnoz, Arturo; Fernández, Pablo S.; Iguaz, Asunción; Periago, Paula M.; Palop, Alfredo

    2016-01-01

    Heat resistance of microorganisms can be affected by different influencing factors. Although, the effect of heating rates has been scarcely explored by the scientific community, recent researches have unraveled its important effect on the thermal resistance of different species of vegetative bacteria. Typically heating rates described in the literature ranged from 1 to 20°C/min but the impact of much higher heating rates is unclear. The aim of this research was to explore the effect of different heating rates, such as those currently achieved in the heat exchangers used in the food industry, on the heat resistance of Escherichia coli. A pilot plant tubular heat exchanger and a thermoresistometer Mastia were used for this purpose. Results showed that fast heating rates had a deep impact on the thermal resistance of E. coli. Heating rates between 20 and 50°C/min were achieved in the heat exchanger, which were much slower than those around 20°C/s achieved in the thermoresistometer. In all cases, these high heating rates led to higher inactivation than expected: in the heat exchanger, for all the experiments performed, when the observed inactivation had reached about seven log cycles, the predictions estimated about 1 log cycle of inactivation; in the thermoresistometer these differences between observed and predicted values were even more than 10 times higher, from 4.07 log cycles observed to 0.34 predicted at a flow rate of 70 mL/min and a maximum heating rate of 14.7°C/s. A quantification of the impact of the heating rates on the level of inactivation achieved was established. These results point out the important effect that the heating rate has on the thermal resistance of E. coli, with high heating rates resulting in an additional sensitization to heat and therefore an effective food safety strategy in terms of food processing. PMID:27563300

  9. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  10. An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures.

    Science.gov (United States)

    Pereira, Renata C; Silva, Andrea N M R; Souza, Marta Cristina O; Silva, Marlon V; Neves, Patrícia P C C; Silva, Andrea A M V; Matos, Denise D C S; Herrera, Miguel A O; Yamamura, Anna M Y; Freire, Marcos S; Gaspar, Luciane P; Caride, Elena

    2015-08-20

    Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with β-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 μg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Inactivation of hemopoietic stem cells by lymphocytes as related to genotype of interacting cells

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, R V; Seslavina, L S; Panteleev, E I; Egorova, O S

    1975-05-01

    Inoculation of a mixture of bone marrow cells with allogeneic lymphocytes into irradiated mice of inbred strains or into F/sub 1/ hybrids results in the depression of bone marrow cell proliferation in the spleen of the recipient: the effect of inactivation of nonsyngeneic stem cells. The inactivation of stem cells by allogeneic lymphocytes can be detected in all tested combinations of mice strains - donors of lymphocytes and bone marrow cells and mice - recipients but the degree of inactivation differs and depends on the genotype of cell donors rather than on the genotype of the recipient. Lymphocytes of some mice strains (haplotypes H-2sup(k) and H-2sup(a)) are more active killers of bone marrow cells as compared with lymphocytes of other strains (hyplotypes H-2sup(b) and H-2sup(d)). Probably, the degree of stem cells inactivation by lymphocytes depends on the differences of their histocompatibility in H-2 system.

  12. An inactivated cell-culture vaccine against yellow fever.

    Science.gov (United States)

    Monath, Thomas P; Fowler, Elizabeth; Johnson, Casey T; Balser, John; Morin, Merribeth J; Sisti, Maggie; Trent, Dennis W

    2011-04-07

    Yellow fever is a lethal viral hemorrhagic fever occurring in Africa and South America. A highly effective live vaccine (17D) is widely used for travelers to and residents of areas in which yellow fever is endemic, but the vaccine can cause serious adverse events, including viscerotropic disease, which is associated with a high rate of death. A safer, nonreplicating vaccine is needed. In a double-blind, placebo-controlled, dose-escalation, phase 1 study of 60 healthy subjects between 18 and 49 years of age, we investigated the safety and immunogenicity of XRX-001 purified whole-virus, β-propiolactone-inactivated yellow fever vaccine produced in Vero cell cultures and adsorbed to aluminum hydroxide (alum) adjuvant. On two visits 21 days apart, subjects received intramuscular injections of vaccine that contained 0.48 μg or 4.8 μg of antigen. Levels of neutralizing antibodies were measured at baseline and on days 21, 31, and 42. The vaccine induced the development of neutralizing antibodies in 100% of subjects receiving 4.8 μg of antigen in each injection and in 88% of subjects receiving 0.48 μg of antigen in each injection. Antibody levels increased by day 10 after the second injection, at which time levels were significantly higher with the 4.8-μg formulation than with the 0.48-μg formulation (geometric mean titer, 146 vs. 39; Pvaccine groups than in the placebo group: mild pain, tenderness, and (much less frequently) itching at the injection site. One case of urticaria was observed on day 3 after the second dose of 4.8 μg of vaccine. A two-dose regimen of the XRX-001 vaccine, containing inactivated yellow fever antigen with an alum adjuvant, induced neutralizing antibodies in a high percentage of subjects. XRX-001 has the potential to be a safer alternative to live attenuated 17D vaccine. (Funded by Xcellerex; ClinicalTrials.gov number, NCT00995865.).

  13. Stem-Cell Inactivation on Transplantation of Haemopoietic Cell Suspensions from Genetically Different Donors

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, R. V. [Institute of Biophysics, Ministry of Public Health of the USSR, Moscow, USSR (Russian Federation)

    1969-07-15

    The transplantation of a mixture of haemopoietic or lymphoid cells from two genetically different mice into lethally irradiated F{sub 1} recipients results in marked or total inactivation of the colony-forming units of the graft. This phenomenon is observed following transplantation of mixtures of spleen cells or bone-marrow cells from animals of different genotypes: CBA + C57BL, A + CBA, A + C57BL, C3H + C57BL, CBA + (CBA x C57BL) F{sub 1}. Maximum inactivation is observed when lymph-node cells of one genotype are transplanted with spleen or bone-marrow cells of another genotype. Use of non-syngenic kidney cells or lymphoid cells inactivated by irradiation as one component of the mixture shows that inactivation of genetically heterogeneous stem cells requires the participation of viable lymphoid cells. The inactivation phenomenon is also observed with Jerne's method. This shows that inactivation affects not only colony-forming cells but also the immunologically competent precursors of antibody-producing cells. (author)

  14. Cell extracts of propionic acid bacteria reactivate cells of Escherichia coli inactivated by ultraviolet radiation

    International Nuclear Information System (INIS)

    Vorob'eva, L.I.; Nikitenko, G.V.; Khodzhaev, E.Yu.; Ponomareva, G.M.

    1994-01-01

    Cell extracts of three Propionibacterium shermanii strains were shown to exert a reactivating effect on cells of E. coli AB 1157 inactivated by ultraviolet radiation. The reactivating effect was revealed after both preincubation and postincubation of the irradiated cells with the extracts. The effect increased with a decrease of the survival rate within the range of 1.8-0.006%. The protective factor (or factors) is dialyzable and thermolabile; it was detected both in the fraction of soluble proteins and in the fraction of nucleoproteins and nucleic acids. The protective properties of dialyzate disappear after incubation with proteinase K and trypsin, decrease after incubation with α-amylase, deoxyribonuclease-1, or ribonuclease, and do not change under the influence of lipase. The reactivating factor is believed to be of a polypeptide nature

  15. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    International Nuclear Information System (INIS)

    Highsmith, R.F.; Gallaher, M.J.

    1986-01-01

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  16. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Highsmith, R.F.; Gallaher, M.J.

    1986-03-05

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

  17. Atmospheric-pressure air microplasma jets in aqueous media for the inactivation of Pseudomonas fluorescens cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xianhui; Yang, Si-ze [Fujian Provincial Key Laboratory of Plasma and Magnetic Resonance, School of Physics and Mechanical and Electrical Engineering, Xiamen University, Xiamen, Fujian 361005 (China); Liu, Dongping [Fujian Provincial Key Laboratory of Plasma and Magnetic Resonance, School of Physics and Mechanical and Electrical Engineering, Xiamen University, Xiamen, Fujian 361005 (China); School of Physics and Materials Engineering, Dalian Nationalities University, Dalian 116600 (China); Song, Ying [School of Physics and Materials Engineering, Dalian Nationalities University, Dalian 116600 (China); School of Physics and Optoelectronic Technology, Dalian University of Technology, Dalian 116023 (China); Sun, Yue [School of Physics, Changchun University of Science and Technology, Changchun 130022 (China)

    2013-05-15

    The hollow fiber-based cold air microplasma jet array running at atmospheric pressure has been designed to inactivate Pseudomonas fluorescens (P. fluorescens) cells in vitro in aqueous media. The influences of electrode configurations, air flow rate, and applied voltage on the discharge characteristics of the single microplasma jet operating in aqueous media are presented, and the bactericidal efficiency of the hollow fibers-based and large-volume microplasma jet array is reported. Optical emission spectroscopy is utilized to identify excited species during the antibacterial testing of plasma in solutions. These well-aligned and rather stable air microplasma jets containing a variety of short-lived species, such as OH and O radicals and charged particles, are in direct contact with aqueous media and are very effective in killing P. fluorescens cells in aqueous media. This design shows its potential application for atmospheric pressure air plasma inactivation of bacteria cells in aqueous media.

  18. Biophysical mechanism of cell inactivation by ionizing particles

    International Nuclear Information System (INIS)

    Lokajicek, M.

    1986-12-01

    In radiobiological mechanism it is possible to distinguish the sequence of three different phases which can be denoted as physical, physico-chemical and biological. Mathematical models of the individual phases and their mutual interrelations are discussed. A special accent is given to the relation between the models of two non-biological phases and that of the biological one. Some detailed characteristics concerning DSB formation and repair and inactivation mechanisms in cells are analyzed with the help of the considered model chain. (author). 39 refs, 3 figs, 3 tabs

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-07-01

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

  1. Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. I. Metabolic inactivation impairs both CD and LD antigen signals

    International Nuclear Information System (INIS)

    Kelso, A.; Boyle, W.

    1982-01-01

    The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T/sub c/) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T/sub c/ responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T/sub c/ precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T/sub c/ from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T/sub c/. It is concluded that, in addition to the traditional view that these treatments damage the ''LD'' signal to helper T lymphocytes, metabolic inactivation also impairs recognition of ''CD'' determinants by precursor T/sub c/

  2. Inactivation of Ehrlich ascites tumor cells by heavy ions

    International Nuclear Information System (INIS)

    Bertsche, U.; Iliakis, G.; Kraft, G.

    1983-01-01

    Exponentially growing and plateau-phase cultures of Ehrlich ascites tumor cells were irradiated with heavy ions (Z greater than or equal to 20) and assayed for loss of reproductive capacity either immediately or at delayed times after irradiation. The results indicated no modification of the exponential dose response due to conditions which usually favor the repair of potentially lethal damage at low ionization density. Postirradiation treatment of the cells with a DNA synthesis inhibitor known to act on PLD repair resulted in effects similar to those observed without this drug and confirmed the hypothesis that at such high values of ionization density only lethal, unmodifiable damage can be expressed. The inactivation cross-section values calculated from the slope of the measured survival curves showed no significant correlations with commonly used parameters of radiation quality. Instead, a functional dependence on the primary ion energy was indicated, being smaller by a factor of two at low energies (less than or equal to 2 MeV/amu) compared with values at energies above 4 MeV/amu, where agreement with the morphological nuclear cross section of the culture was found. This suggests that at higher specific ion energies energetic secondary electrons contribute to the induction of lethal damage, and that interaction of damaged sites between the primary track and the track ends of delta electrons may occur. The data are therefore also discussed in terms of the penumbra model which emphasizes the role of delta electrons in cell killing when radiations with very high ionization density are applied

  3. Inactivation of Ehrlich ascites tumor cells by heavy ions

    International Nuclear Information System (INIS)

    Bertsche, U.; Iliakis, G.; Kraft, G.

    1983-01-01

    Exponentially growing and plateau-phase cultures of Ehrlich ascites tumor cells were irradiated with heavy ions (Z greater than or equal to 20) and assayed for loss of reproductive capacity either immediately or at delayed times after irradiation. The results indicated no modification of the exponential dose response due to conditions which usually favor the repair of potentially lethal damage at low ionization density. Postirradiation treatment of the cells with beta-arabinofuranosyladenine, a DNA synthesis inhibitor known to act on PLD repair, resulted in effects similar to those observed without this drug and confirmed the hypothesis that at such high values of ionization density only lethal, unmodifiable damage can be expressed. The inactivation cross-section values calculated from the slope of the measured survival curves showed no significant correlations with commonly used parameters of radiation quality such as LET or z 2 /beta 2. Instead, a functional dependence on the primary ion energy was indicated, being smaller by a factor of two at low energies (less than or equal to 2 MeV/amu) compared with values at energies above 4 MeV/amu, where agreement with the morphological nuclear cross section of the culture was found. This suggests that at higher specific ion energies energetic secondary electrons contribute to the induction of lethal damage, and that interaction of damaged sites between the primary track and the track ends of delta electrons may occur. The data are therefore also discussed in terms of the ''penumbra model'' which emphasizes the role of delta electrons in cell killing when radiations with very high ionization density are applied

  4. Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine

    NARCIS (Netherlands)

    Kraan, H.; Have, Ten R.; Maas, van der L.; Kersten, G.F.A.; Amorij, J.P.

    2016-01-01

    A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick

  5. Inactivation of Cronobacter malonaticus cells and inhibition of its biofilm formation exposed to hydrogen peroxide stress.

    Science.gov (United States)

    Ye, Yingwang; Zhang, Maofeng; Jiao, Rui; Ling, Na; Zhang, Xiyan; Tong, Liaowang; Zeng, Haiyang; Zhang, Jumei; Wu, Qingping

    2018-01-01

    Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H 2 O 2 on biofilm formation in C. malonaticus were also performed. Results indicated that H 2 O 2 could completely inactivate C. malonaticus in sterile water with 0.06% H 2 O 2 for 25 min, 0.08% H 2 O 2 for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H 2 O 2 . In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H 2 O 2 treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H 2 O 2 was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H 2 O 2 on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H 2 O 2 for decontamination of C. malonaticus in dairy processing. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Pathogen inactivation of Dengue virus in red blood cells using amustaline and glutathione.

    Science.gov (United States)

    Aubry, Maite; Laughhunn, Andrew; Santa Maria, Felicia; Lanteri, Marion C; Stassinopoulos, Adonis; Musso, Didier

    2017-12-01

    Dengue virus (DENV) is an arbovirus primarily transmitted through mosquito bite; however, DENV transfusion-transmitted infections (TTIs) have been reported and asymptomatic DENV RNA-positive blood donors have been identified in endemic countries. DENV is considered a high-risk pathogen for blood safety. One of the mitigation strategies to prevent arbovirus TTIs is pathogen inactivation. In this study we demonstrate that the amustaline and glutathione (S-303/GSH) treatment previously found effective against Zika virus in red blood cells (RBCs) is also effective in inactivating DENV. Red blood cells were spiked with high levels of DENV. Viral RNA loads and infectious titers were measured in the untreated control and before and after pathogen inactivation treatment of RBC samples. DENV infectivity was also assessed over five successive cell culture passages to detect any potential residual replicative virus. The mean ± SD DENV titer in RBCs before inactivation was 6.61 ± 0.19 log 50% tissue culture infectious dose (TCID 50 )/mL and the mean viral RNA load was 8.42 log genome equivalents/mL. No replicative DENV was detected either immediately after completion of treatment using S-303/GSH or after cell culture passages. Treatment using S-303/GSH inactivated high levels of DENV in RBCs to the limit of detection. In combination with previous studies showing the effective inactivation of DENV in plasma and platelets using the licensed amotosalen/UVA system, this study demonstrates that high levels of DENV can be inactivated in all blood components. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

  7. Mutation formation and inactivation of mammalian cells by heavy ions. 3

    International Nuclear Information System (INIS)

    Obaturov, G.M.

    1984-01-01

    Using parameter values of equations for cell inactivation cross-sections, theoretical curves were calculated for cell radiosensitivity (α 1 ) in dependence on LET. There is good agreement between calculated and experimental values of α 1 . The yield of lethal mutations of 1st and 2nd types and their sum in dependence on LET was also calculated. It was shown that for low LET ions cell radiosensitivity is caused by the 2nd type of mutation, independent of LET, proportional to DNA mass and inversely proportional to repair rate. For high LET ions when lethal mutation number per one track is more than one, α 1 is caused by the 1st type of mutation, proportional to cell nuclear cross-section and inversely proportional to LET. For intermediate LET ions α 1 is a complicated function of LET. It is follows from the equations that more than 17% of double strand breaks (DSB) take part in lethal mutation formation. Others are either repaired or formed in the process of experimentally determined DSB yield. (author)

  8. Cell wall as a target for bacteria inactivation by pulsed electric fields

    Science.gov (United States)

    Pillet, Flavien; Formosa-Dague, Cécile; Baaziz, Houda; Dague, Etienne; Rols, Marie-Pierre

    2016-01-01

    The integrity and morphology of bacteria is sustained by the cell wall, the target of the main microbial inactivation processes. One promising approach to inactivation is based on the use of pulsed electric fields (PEF). The current dogma is that irreversible cell membrane electro-permeabilisation causes the death of the bacteria. However, the actual effect on the cell-wall architecture has been poorly explored. Here we combine atomic force microscopy and electron microscopy to study the cell-wall organization of living Bacillus pumilus bacteria at the nanoscale. For vegetative bacteria, exposure to PEF led to structural disorganization correlated with morphological and mechanical alterations of the cell wall. For spores, PEF exposure led to the partial destruction of coat protein nanostructures, associated with internal alterations of cortex and core. Our findings reveal for the first time that the cell wall and coat architecture are directly involved in the electro-eradication of bacteria. PMID:26830154

  9. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  10. One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Protic-Sabljic, M.; Kraemer, K.H.

    1985-01-01

    The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D 0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA

  11. Vero cell technology for rapid development of inactivated whole virus vaccines for emerging viral diseases.

    Science.gov (United States)

    Barrett, P Noel; Terpening, Sara J; Snow, Doris; Cobb, Ronald R; Kistner, Otfried

    2017-09-01

    Rapid development and production of vaccines against emerging diseases requires well established, validated, robust technologies to allow industrial scale production and accelerated licensure of products. Areas covered: A versatile Vero cell platform has been developed and utilized to deliver a wide range of candidate and licensed vaccines against emerging viral diseases. This platform builds on the 35 years' experience and safety record with inactivated whole virus vaccines such as polio vaccine. The current platform has been optimized to include a novel double inactivation procedure in order to ensure a highly robust inactivation procedure for novel emerging viruses. The utility of this platform in rapidly developing inactivated whole virus vaccines against pandemic (-like) influenza viruses and other emerging viruses such as West Nile, Chikungunya, Ross River and SARS is reviewed. The potential of the platform for development of vaccines against other emerging viruses such as Zika virus is described. Expert commentary: Use of this platform can substantially accelerate process development and facilitate licensure because of the substantial existing data set available for the cell matrix. However, programs to provide vaccines against emerging diseases must allow alternative clinical development paths to licensure, without the requirement to carry out large scale field efficacy studies.

  12. Influence of Ouabain on cell inactivation by irradiation

    International Nuclear Information System (INIS)

    Verheye-Dua, F.A.; Boehm, L.

    1996-01-01

    Background: It has been suggested that irradiation affects the function of the Na + -K + -ATPase. Here we examine the influence of the inhibitor ouabain on the cytotoxicity or irradiation. Material and Methods: Cell colony assay, cell survival, 86 Rb-uptake, flow cytometry. Results: In V79, HeLa and A549 cell ouabain alone causes a significant growth reduction at medium concentrations of 10 -4 M, 10 -6 M and 10 -7 M, respectively. When cells were exposed to the drug for 1 h and subsequently irradiated, the SF2 values decreased from 0.55 to 0.41, from 0.42 to 0.18 and from 0.57 to 0.35 in V79, HeLa and A549 cells, respectively. These effects were manifest at drug concentrations of 10 -3 M, 10 -6 M and 10 -7 M respectively, where Na + -K + -ATPase activity as mesured by 86 Rb-uptake was reduced to 40 to 60% of the control value. Addition of the drug after irradiation and when the G2/M cell cycle block was firmly established, markedly delayed the recovery of cells for well over 6 h and G1 levels remained at 50% of the control values. Conclusion: It is concluded that ouabain is strongly dose modifying in the human cell lines HeLa and A549 at concentrations which correlate with the inhibition of the Na + -K + -ATPase. Ouabain also inhibits the recovery of cells blocked in the cell cycle by irradiation. (orig.) [de

  13. A key inactivation factor of HeLa cell viability by a plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Takehiko; Yokoyama, Mayo [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: sato@ifs.tohoku.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)

    2011-09-21

    Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H{sub 2}O{sub 2} in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H{sub 2}O{sub 2}, we assessed the differences in the effects of plasma-treated medium and H{sub 2}O{sub 2}-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H{sub 2}O{sub 2} into the cells, response to H{sub 2}O{sub 2} decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H{sub 2}O{sub 2} is one of the main factors responsible for inactivation of HeLa cell viability. (fast track communication)

  14. Real time, in situ observation of the photocatalytic inactivation of Saccharomyces cerevisiae cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jingtao [School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou 450002 (China); Environment Functional Materials Division, Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Wang, Xiaoxin [Environment Functional Materials Division, Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Li, Qi, E-mail: qili@imr.ac.cn [Environment Functional Materials Division, Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Shang, Jian Ku [Environment Functional Materials Division, Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

    2015-04-01

    An in situ microscopy technique was developed to observe in real time the photocatalytic inactivation process of Saccharomyces cerevisiae (S. cerevisiae) cells by palladium-modified nitrogen-doped titanium oxide (TiON/PdO) under visible light illumination. The technique was based on building a photocatalytic micro-reactor on the sample stage of a fluorescence/phase contrast microscopy capable of simultaneously providing the optical excitation to activate the photocatalyst in the micro-reactor and the illumination to acquire phase contrast images of the cells undergoing the photocatalytic inactivation process. Using TiON/PdO as an example, the technique revealed for the first time the vacuolar activities inside S. cerevisiae cells subjected to a visible light photocatalytic inactivation. The vacuoles responded to the photocatalytic attack by the first expansion of the vacuolar volume and then contraction, before the vacuole disappeared and the cell structure collapsed. Consistent with the aggregate behavior observed from the cell culture experiments, the transition in the vacuolar volume provided clear evidence that photocatalytic disinfection of S. cerevisiae cells started with an initiation period in which cells struggled to offset the photocatalytic damage and moved rapidly after the photocatalytic damage overwhelmed the defense mechanisms of the cells against oxidative attack. - Highlights: • Palladium-modified nitrogen-doped titanium oxidephotocatalyst (TiON/PdO) • Effective visible-light photocatalytic disinfection of yeast cells by TiON/PdO • Real time, in situ observation technique was developed for photocatalytic disinfection. • The fluorescence/phase contrast microscope with a photocatalytic micro-reactor • Yeast cell disinfection happened before the cell structure collapsed.

  15. Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1982-01-01

    Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex

  16. Comparison of protection from homologous cell-free vs cell-associated SIV challenge afforded by inactivated whole SIV vaccines.

    NARCIS (Netherlands)

    J.L. Heeney (Jonathan); P. de Vries (Petra); R. Dubbes (Rob); W. Koornstra (Willem); H. Niphuis; P. ten Haaft (Peter); J. Boes (Jolande); M.E.M. Dings (Marlinda); B. Morein (Bror); A.D.M.E. Osterhaus (Albert)

    1992-01-01

    textabstractThis study attempted to determine if SIV vaccines could protect against challenge with peripheral blood mononuclear cells (PBMCs) from an SIV infected rhesus monkey. Mature Macaca mulatta were vaccinated four times with formalin inactivated SIVmac32H administered in MDP adjuvant (n = 8)

  17. Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.

    Science.gov (United States)

    Cullen, Kirsty S; Matschinsky, Franz M; Agius, Loranne; Arden, Catherine

    2011-12-01

    The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non-β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non-β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.

  18. Psoralen/UV inactivation of HIV-1-infected cells for use in cytologic and immunologic procedures

    International Nuclear Information System (INIS)

    Watson, A.J.; Klaniecki, J.; Hanson, C.V.

    1990-01-01

    A rapid procedure for the inactivation of HIV-1-infected cells using psoralen and ultraviolet (UV) light is described. Exposure of HIV-1-infected cells to 5 micrograms/ml psoralen followed by UV irradiation (320-380 nm) for 5 minutes yields cells that are noninfectious as assessed by extended infectivity assays. The psoralen/UV inactivation procedure described is effective with cells chronically or acutely infected with HIV-1 and is unaffected by cell densities up to 12 x 10(6)/ml. At 5 micrograms/ml psoralen does little damage to cellular permeability as shown by the ability of treated cells to exclude trypan blue and propidium iodide. Psoralen/UV treatment of HIV-1-infected cells does not cause a significant decrease in the reactivity of HIV-1 core and envelope antigens or cellular antigens to monoclonal antibodies. Experiments are presented demonstrating the use of these cells for flow cytometry studies and for cell surface labeling using the lactoperoxidase 125 I iodination procedure

  19. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    Science.gov (United States)

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

  20. Detailed analysis of the cell-inactivation mechanism by accelerated protons and light ions

    Czech Academy of Sciences Publication Activity Database

    Kundrát, Pavel

    2006-01-01

    Roč. 51, - (2006), s. 1185-1199 ISSN 0031-9155 R&D Projects: GA ČR GA202/05/2728 Institutional research plan: CEZ:AV0Z10100502 Keywords : biological effects of ionizing particles * cell inactivation * modelling * protons * light ions * hadron radiotherapy Subject RIV: BF - Elementary Particles and High Energy Physics Impact factor: 2.873, year: 2006

  1. Progeric effects of catalase inactivation in human cells.

    Science.gov (United States)

    Koepke, Jay I; Wood, Christopher S; Terlecky, Laura J; Walton, Paul A; Terlecky, Stanley R

    2008-10-01

    Peroxisomes generate hydrogen peroxide, a reactive oxygen species, as part of their normal metabolism. A number of pathological situations exist in which the organelle's capacity to degrade the potentially toxic oxidant is compromised. It is the peroxidase, catalase, which largely determines the functional antioxidant capacity of the organelle, and it is this enzyme that is affected in aging, in certain diseases, and in response to exposure to specific chemical agents. To more tightly control the enzymatic activity of peroxisomal catalase and carefully document the effects of its impaired action on human cells, we employed the inhibitor 3-amino-1,2,4-triazole. We show that by chronically reducing catalase activity to approximately 38% of normal, cells respond in a dramatic manner, displaying a cascade of accelerated aging reactions. Hydrogen peroxide and related reactive oxygen species are produced, protein and DNA are oxidatively damaged, import into peroxisomes and organelle biogenesis is corrupted, and matrix metalloproteinases are hyper-secreted from cells. In addition, mitochondria are functionally impaired, losing their ability to maintain a membrane potential and synthesize reactive oxygen species themselves. These latter results suggest an important redox-regulated connection between the two organelle systems, a topic of considerable interest for future study.

  2. Progeric effects of catalase inactivation in human cells

    International Nuclear Information System (INIS)

    Koepke, Jay I.; Wood, Christopher S.; Terlecky, Laura J.; Walton, Paul A.; Terlecky, Stanley R.

    2008-01-01

    Peroxisomes generate hydrogen peroxide, a reactive oxygen species, as part of their normal metabolism. A number of pathological situations exist in which the organelle's capacity to degrade the potentially toxic oxidant is compromised. It is the peroxidase, catalase, which largely determines the functional antioxidant capacity of the organelle, and it is this enzyme that is affected in aging, in certain diseases, and in response to exposure to specific chemical agents. To more tightly control the enzymatic activity of peroxisomal catalase and carefully document the effects of its impaired action on human cells, we employed the inhibitor 3-amino-1,2,4-triazole. We show that by chronically reducing catalase activity to approximately 38% of normal, cells respond in a dramatic manner, displaying a cascade of accelerated aging reactions. Hydrogen peroxide and related reactive oxygen species are produced, protein and DNA are oxidatively damaged, import into peroxisomes and organelle biogenesis is corrupted, and matrix metalloproteinases are hyper-secreted from cells. In addition, mitochondria are functionally impaired, losing their ability to maintain a membrane potential and synthesize reactive oxygen species themselves. These latter results suggest an important redox-regulated connection between the two organelle systems, a topic of considerable interest for future study

  3. Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

    Science.gov (United States)

    Cantone, Irene; Fisher, Amanda G

    2017-11-05

    X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

  4. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  5. Reactivation in UV inactivated Escherichia coli by cell-free extracts of propionic acid bacteria

    International Nuclear Information System (INIS)

    Vorob'eva, L.I.; Nikitenko, G.V.; Khodzhaev, E.Yu.; Ponomareva, G.M.

    1993-01-01

    For the first time reactivation of cell extraction of three strains of Propionibacterium shermanii in UV inactivated not filament-forming strain Escherichia colli AB 1157 is shown. Reactivation was demonstrated in prencubated and postincubated test-culture and increased as survival of E.coli decreased in a range 1,8-0,006%. The factor (factores) of defense in dialysable, thermolable and is present as in a fraction of nucleoproteins and nucleic acids so in a fraction of soluble proteins. The extracts were inactivated by incubation with proteinase K and trypsin, partly decreased activity by incubation with alpha-amylase and selected nuclease but not with lipase. Polypeltide nature of reactivative factor is supposed

  6. Risk scaling factors from inactivation to chromosome aberrations, mutations and oncogenic transformations in mammalian cells

    International Nuclear Information System (INIS)

    Alkaharam, A.S.; Watt, D.E.

    1997-01-01

    Analyses of bio-effect mechanisms of damage to mammalian cells in terms of the quality parameter 'mean free path for primary ionisation', for heavy charged particles, strongly suggests that there is a common mechanism for the biological endpoints of chromosome aberrations, mutations and oncogenic transformation. The lethal lesions are identified as unrepaired double-strand breaks in the intracellular DNA. As data for the various endpoints studied can be represented in a unified scheme, for any radiation type, it follows that radiation risk factors can be determined on the basis of simple ratios to the inactivation cross sections. There are intrinsic physical reasons why neutrons can never reach the saturation level of heavier particles for equal fluences. The probabilities of risk with respect to inactivation, for chromosome dicentrics, mutation of the HPRT gene and of oncogenic transformation are respectively 0.24, 5.8 x 10 -5 , and 4.1 x 10 -3 . (author)

  7. Solar Radiation Disinfection of Drinking Water at Temperate Latitudes: Inactivation rates for an optimized reactor configuration

    Science.gov (United States)

    Solar radiation-driven inactivation of bacteria, virus and protozoan pathogen models was quantified in simulated drinking water at a temperate latitude (34°S). The water was seeded with Enterococcus faecalis, Clostridium sporogenes spores, and P22 bacteriophage, each at ca 1 x 10...

  8. Inactivation of nuclear factor κB by MIP-based drug combinations augments cell death of breast cancer cells

    Directory of Open Access Journals (Sweden)

    Subramaniam M

    2018-05-01

    Full Text Available Menaga Subramaniam,1 Su Ki Liew,1 Lionel LA In,2 Khalijah Awang,3,4 Niyaz Ahmed,5 Noor Hasima Nagoor1,6 1Institute of Biological Science (Genetics & Molecular Biology, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Biotechnology, Faculty of Applied Sciences, UCSI University, Kuala Lumpur, Malaysia; 3Centre for Natural Product Research and Drug Discovery (CENAR, University of Malaya, Kuala Lumpur, Malaysia; 4Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; 5Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, India; 6Centre for Research in Biotechnology for Agriculture (CEBAR, University of Malaya, Kuala Lumpur, Malaysia Background: Drug combination therapy to treat cancer is a strategic approach to increase successful treatment rate. Optimizing combination regimens is vital to increase therapeutic efficacy with minimal side effects. Materials and methods: In the present study, we evaluated the in vitro cytotoxicity of double and triple combinations consisting of 1'S-1'-acetoxychavicol acetate (ACA, Mycobacterium indicus pranii (MIP and cisplatin (CDDP against 14 various human cancer cell lines to address the need for more effective therapy. Our data show synergistic effects in MCF-7 cells treated with MIP:ACA, MIP:CDDP and MIP:ACA:CDDP combinations. The type of interaction between MIP, ACA and CDDP was evaluated based on combination index being <0.8 for synergistic effect. Identifying the mechanism of cell death based on previous studies involved intrinsic apoptosis and nuclear factor kappa B (NF-κB and tested in Western blot analysis. Inactivation of NF-κB was confirmed by p65 and IκBα, while intrinsic apoptosis pathway activation was confirmed by caspase-9 and Apaf-1 expression Results: All combinations confirmed intrinsic apoptosis activation and NF-κB inactivation. Conclusion: Double and triple

  9. Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

    Directory of Open Access Journals (Sweden)

    Bassermann Florian

    2010-05-01

    Full Text Available Abstract Background Inactivation of the Fanconi anemia (FA pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI tumors has not yet been systematically explored. Results A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type. Conclusions As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could

  10. Application of alpha microdosimetry in possible differentiation of inactivation effect from malignant cell transformation

    International Nuclear Information System (INIS)

    Sedlak, A.

    1984-01-01

    The model is described with a certain threshold value of specific energy z 0 independent of the linear transfer of energy (LTE) whose exceedance would result in the inactivation of the cell. The survival curves may be fitted to distribution function F(z 0 ,D). The final relationship is described by the linear dependence of quotient L 3 /D 37 on L 2 . The survival curves in low and high LTE are given. It may be assumed that at high LTE there exists a certain probability of survival even after passage of a densely ionizing particle through the cell nucleus. (E.S.)

  11. Mean inactivation dose: a useful concept for intercomparison of human cell survival curves

    International Nuclear Information System (INIS)

    Fertil, B.; Dertinger, H.; Courdi, A.; Malaise, E.P.

    1984-01-01

    The mean inactivation dose (anti D) is calculated for published in vitro survival curves obtained from cell lines of both normal and neoplastic human tissues. Cells belonging to different histological categories (melanomas, carcinomas, etc.) are shown to be characterized by distinct values of anti D which are related to the clinical radiosensitivity of tumors from these categories. Compared to other ways of representing in vitro radiosensitivity, e.g., by the multitarget parameters D 8 and n, the parameter anti D has several specific advantages

  12. Recovery of prostacyclin synthesis in vascular smooth muscle cells following self-inactivation and requirement for growth factors

    International Nuclear Information System (INIS)

    Bailey, J.M.; Hla, T.T.; Pash, J.M.

    1986-01-01

    The cyclooxygenase enzyme system is a prime example of a metabolic pathway that is regulated by self inactivation. This is believed to occur in part via the irreversible reaction of the endoperoxide intermediate species with the cyclooxygenase enzyme. This inactivation and recovery of activity is similar to the inactivation observed with aspirin which irreversibly acetylates the enzyme. Self inactivation was studied in cultured rat and bovine aorta smooth muscle cells. The production of the prostanoid PGI2 was demonstrated by incubation of a monolayer of cells with 12 μM C-14 labeled arachidonic acid. Products were analyzed by thin layer chromatography and identified by their comigration with authentic standards and confirmed by gas chromatography/mass spectrometry. Preincubation of the cells for 10 minutes with arachidonic acid at concentrations as low as 1 μg/mL inactivated the cells to a second challenge with radiolabeled arachidonic acid. Recovery from self inactivation took place over a three hour time period and was similar to the recovery observed with aspirin pretreatment. Recovery was inhibited by addition of 10 μg/mL cycloheximide to the medium indicating that it involves synthesis of cyclooxygenase protein. Epidermal growth factor was identified as a serum factor responsible for the rapid recovery of cyclooxygenase activity in rat and bovine aorta smooth muscle cells

  13. Rhodosporidium BANNO: Inactivation of yeast phase cells by ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Boettcher, F.; Samsonova, I.A.

    1977-01-01

    The inactivation of stationary phase cells by ultraviolet light (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in eight wild strains of Rhodotorula, six of which are the sporidial yeast phase of Rhodosporidium, a basidiomycetous fungus. It thas been found that (1) the UV-resistance of Rhodosporidium and Rhodotorula yeasts is higher and the MNNG-resistance lower than the resistance of Candida and Hansenula yeasts, (2) the shape of the survival curves is sigmoid in the case of UV and two-phase exponential in the case of MNNG, (3) the mutagen sensitivities but not the inactivation kinetics of the strains are different, (4) the UV- and MNNG-sensitivities for each of the strains are correlated, (5) the relatively high resistance to UV cannot be due to the carotenoid pigments of the cells, (6) mutations to UV-sensitivity can be induced with a high rate, (7) the sigmoidal character of the UV survival curves were reduced or transformed to an exponential shape by the UVS-mutations. (author)

  14. An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

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    Green David E

    2010-02-01

    Full Text Available Abstract Background Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC, shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs such as Shiga-like Toxin 1 (SLT-1 represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP derived from the cytotoxic A subunit of SLT-1 (SLT-1A, harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM allowing the toxin variant to selectively target and kill human melanoma cells. Results SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. Conclusions These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

  15. Induction of DNA double-strand breaks by ionizing radiation of different quality and their relevance for cell inactivation

    International Nuclear Information System (INIS)

    Kampf, G.

    1988-01-01

    By investigation of the production of DNA strand breaks and of DNA release from the nuclear membrane complex in Chinese hamster cells using different radiation qualities from 1 to 360 keV/μm, partly also under hypoxic conditions, and by relating the results to the induction of chromosome aberrations and to cell inactivation it has become possible to find connections between the induction of molecular lesions and the expression of this damage on the cellular level. From the studies follows that DNA pieces are cut off from the nuclear membrane complex by DNA double-strand breaks (DSB). The share and size of the released pieces depends on radiation dose and quality as well as on the oxygen conditions. The lesions can partly be repaired. In connection with the DSB rates the results of the DNA release studies led to the conclusion that the DNA in the cells must be organized in superstructure units (MASSUs) with a DNA mass of about 2 x 10 9 g/mol, which are associated to the nuclear membrane in attachment points. The numerical relations show that for a 37% survival probability about 90 DSB per genome are required with sparsely ionizing radiation; this number declines to about 40 by use of more densely ionizing radiation up to 150 keV/μm, and increases again with further rise of the ionization density. Hence, for cell inactivation not simply a certain number of DSB per cell is required but rather seems their cooperation within a small structure section of the DNA to be relevant. These critical structures are with high probability the MASSUs. An irrepairable release of DNA from such a structure unit can bring about a chromosome break detectable in the metaphase and finally lead to cell inactivation. DSB turned out to be the essential lethal events in bacteria as well. The relatively small differences to the eukaryotic cells in the position of the maximum of radiation sensitivity on the LET scale and in the lesion sensitivity towards DSB let suggest that a common critical

  16. Effect of using heat-inactivated serum with the Abbott human T-cell lymphotropic virus type III antibody test.

    OpenAIRE

    Jungkind, D L; DiRenzo, S A; Young, S J

    1986-01-01

    The Abbott enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.) for human T-cell lymphotropic virus type III (HTLV-III) antibody was evaluated to determine the effect of using heat-inactivated (56 degrees C for 30 min) serum as the sample. Each of 58 nonreactive serum samples gave a higher A492 value when tested after heat inactivation. Ten of the samples became reactive after heating. Heat-inactivated serum should not be used in the current Abbott HTLV-III antibody test, because thi...

  17. Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types.

    Science.gov (United States)

    Schleh, Marc; Romanowski, Peter; Bhebe, Prince; Zhang, Li; Chinniah, Shivanthi; Lawrence, Bill; Bashiri, Houman; Gaduh, Asri; Rajurs, Viveka; Rasmussen, Brian; Chuck, Alice; Dehghani, Houman

    2009-01-01

    Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus-spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. 2009 American Institute of Chemical Engineers

  18. Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

    Directory of Open Access Journals (Sweden)

    Ayako Nakano

    Full Text Available Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2, KG-1 (ABCB1 and HepG2 cells (ABCB1 and ABCG2. Interestingly, although side population (SP cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

  19. A tritherapy combination of inactivated allogeneic leukocytes infusion and cell vaccine with cyclophosphamide in a sequential regimen enhances antitumor immunity

    OpenAIRE

    Yishu Tang; Wenbo Ma; Chunxia Zhou; Dongmei Wang; Shuren Zhang

    2018-01-01

    Background: Tumor-induced immunosuppression can impede tumor-specific immune responses and limit the effects of cancer immunotherapy. The aim of this study was to investigate the possible effects of sequential chemoimmunotherapeutic strategies to enhance antitumor immune responses. Methods: Using the E7-expressing tumor TC-1 as the tumor model, the treatment groups were divided into the following groups: (1) inactivated allogeneic leukocyte infusion (ALI), (2) ALI + MMC-inactivated TC-1 cell ...

  20. Differential mechanism of Escherichia coli Inactivation by (+)-limonene as a function of cell physiological state and drug's concentration.

    Science.gov (United States)

    Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

    2014-01-01

    (+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about

  1. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    International Nuclear Information System (INIS)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo; Koya, Daisuke

    2012-01-01

    Highlights: ► SIRT1 inactivation decreases autophagy in THP-1 cell. ► Inhibition of autophagy induces inflammation. ► SIRT1 inactivation induces inflammation through NF-κB activation. ► The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-κB activation. ► SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD + -dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 through nuclear factor (NF)-κB signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-κB activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-κB activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and is implicated in decreased 5′-AMP activated kinase (AMPK) activation, leading to the impairment of autophagy. The mTOR inhibitor, rapamycin, abolishes

  2. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

  3. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  4. A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

    Science.gov (United States)

    Srivastava, A K; Putnak, J R; Lee, S H; Hong, S P; Moon, S B; Barvir, D A; Zhao, B; Olson, R A; Kim, S O; Yoo, W D; Towle, A C; Vaughn, D W; Innis, B L; Eckels, K H

    2001-08-14

    A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

  5. Differential analysis of the inactivation of yeast cells induced by irradiation with various ionization densities

    International Nuclear Information System (INIS)

    Grundler, W.

    1979-03-01

    A quantitative investigation is presented on the radiation-induced inactivation of yeast cells in the first generations as a function of dose, repair, and various ionization densities. The study has been made to solve two main questions, i.e.: How do these cells reproduce, and how do they look like at the end of the investigation. Finding the answer to these questions, it was hoped, would lead to a description of survival in the colony test by defining the final fate of the cells which represent the stationary end state. The experiments were to clarify to what extent the dose-response curve yields only relatively general information on radiation-induced damage, or what kind of damage is mainly and best described. This supplementary information will help to improve the interpretation of many experiments having been made with this strain. (orig./MG) [de

  6. Inactivation of Bacillus cereus vegetative cells by gastric acid and bile during in vitro gastrointestinal transit

    Directory of Open Access Journals (Sweden)

    Ceuppens Siele

    2012-10-01

    Full Text Available Abstract Background The foodborne pathogen Bacillus cereus can cause diarrhoeal food poisoning by production of enterotoxins in the small intestine. The prerequisite for diarrhoeal disease is thus survival during gastrointestinal passage. Methods Vegetative cells of 3 different B. cereus strains were cultivated in a real composite food matrix, lasagne verde, and their survival during subsequent simulation of gastrointestinal passage was assessed using in vitro experiments simulating transit through the human upper gastrointestinal tract (from mouth to small intestine. Results No survival of vegetative cells was observed, despite the high inoculum levels of 7.0 to 8.0 log CFU/g and the presence of various potentially protective food components. Significant fractions (approx. 10% of the consumed inoculum of B. cereus vegetative cells survived gastric passage, but they were subsequently inactivated by bile exposure in weakly acidic intestinal medium (pH 5.0. In contrast, the low numbers of spores present (up to 4.0 log spores/g showed excellent survival and remained viable spores throughout the gastrointestinal passage simulation. Conclusion Vegetative cells are inactivated by gastric acid and bile during gastrointestinal passage, while spores are resistant and survive. Therefore, the physiological form (vegetative cells or spores of the B. cereus consumed determines the subsequent gastrointestinal survival and thus the infective dose, which is expected to be much lower for spores than vegetative cells. No significant differences in gastrointestinal survival ability was found among the different strains. However, considerable strain variability was observed in sporulation tendency during growth in laboratory medium and food, which has important implications for the gastrointestinal survival potential of the different B. cereus strains.

  7. Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity

    Science.gov (United States)

    Thathia, Shabnam H.; Ferguson, Stuart; Gautrey, Hannah E.; van Otterdijk, Sanne D.; Hili, Michela; Rand, Vikki; Moorman, Anthony V.; Meyer, Stefan; Brown, Robert; Strathdee, Gordon

    2012-01-01

    Background Altered regulation of many transcription factors has been shown to be important in the development of leukemia. TWIST2 modulates the activity of a number of important transcription factors and is known to be a regulator of hematopoietic differentiation. Here, we investigated the significance of epigenetic regulation of TWIST2 in the control of cell growth and survival and in response to cytotoxic agents in acute lymphoblastic leukemia. Design and Methods TWIST2 promoter methylation status was assessed quantitatively, by combined bisulfite and restriction analysis (COBRA) and pyrosequencing assays, in multiple types of leukemia and TWIST2 expression was determined by quantitative reverse transcriptase polymerase chain reaction analysis. The functional role of TWIST2 in cell proliferation, survival and response to chemotherapy was assessed in transient and stable expression systems. Results We found that TWIST2 was inactivated in more than 50% of cases of childhood and adult acute lymphoblastic leukemia through promoter hypermethylation and that this epigenetic regulation was especially prevalent in RUNX1-ETV6-driven cases. Re-expression of TWIST2 in cell lines resulted in a dramatic reduction in cell growth and induction of apoptosis in the Reh cell line. Furthermore, re-expression of TWIST2 resulted in increased sensitivity to the chemotherapeutic agents etoposide, daunorubicin and dexamethasone and TWIST2 hypermethylation was almost invariably found in relapsed adult acute lymphoblastic leukemia (91% of samples hypermethylated). Conclusions This study suggests a dual role for epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia, initially through altering cell growth and survival properties and subsequently by increasing resistance to chemotherapy. PMID:22058208

  8. Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Qianqian Chen

    2017-07-01

    Full Text Available Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.

  9. Effect of dose rate on inactivation of microorganisms in spices by electron-beams and gamma-rays irradiation

    International Nuclear Information System (INIS)

    Ito, Hitoshi; Islam, Md.S.

    1994-01-01

    Total aerobic bacteria in spices used in this study were determined to be 1 x 10 6 to 6 x 10 7 per gram. A study on the inactivation of microorganisms in spices showed that doses of 6-9 kGy of EB (electron-beams) or γ-irradiation were required to reduce the total aerobic bacteria to below 10 3 per gram. However, a little increase of resistance was observed on the inactivation of total aerobic bacteria in many spices in case of EB irradiation. These differences of radiation sensitivities between EB and γ-rays was explained by dose rate effect on oxidation damage to microorganisms from the results of radiation sensitivities of Bacillus pumilus and B. megaterium spores at dry conditions. On the other hand, these high dose rate of EB irradiation suppressed the increase of peroxide values in spices at high dose irradiation up to 80 kGy. However, components of essential oils in spices were not changed even irradiated up to 50 kGy with EB and γ-rays. (author)

  10. Mechanism of reactivation of the UV-inactivated cells of Escherichia coli by cell extracts of propionic acid bacteria

    International Nuclear Information System (INIS)

    Vorob'eva, L.I.; Khodzhaev, E.Y.; Ponomareva, G.M.

    1995-01-01

    Two mechanisms of reactivation of UV-inactivated Escherichia coli cells - photoreactivation (PhR) and reactivation by the dialyzate of cell extract of propionic acid bacteria - are shown to be different but not completely additive. PhR displays an insignificant negative effect on the reactivaton by active substances (peptides) of the dialyzate, whereas reactivation by dialyzate inhibits PhR. The maximal reactivation can be attained under complete PhR followed by the protective action of dialyzate. The dialyzate protects UV-irradiated E. coli cells with PolA, UvrA, and RecA mutations and Salmonella typhimurium TA 100 (UvrB) cells, and also exerts an antimutagenic effect on S. typhimurium TA 100. Protection by dialyzate is suggested to be due to restoration of the cell division mechanism damaged by UV irradiation. 14 refs., 3 figs., 5 tabs

  11. Inactivation of Caliciviruses

    Directory of Open Access Journals (Sweden)

    Raymond Nims

    2013-03-01

    Full Text Available The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine norovirus. The caliciviruses are susceptible to wet heat inactivation at temperatures in excess of 60 °C with contact times of 30 min or greater, to UV-C inactivation at fluence ≥30 mJ/cm2, to high pressure processing >200 MPa for >5 min at 4 °C, and to certain photodynamic inactivation approaches. The enteric caliciviruses (e.g.; noroviruses display resistance to inactivation by low pH, while the non-enteric species (e.g.; feline calicivirus are much more susceptible. The caliciviruses are inactivated by a variety of chemicals, including alcohols, oxidizing agents, aldehydes, and β-propiolactone. As with inactivation of viruses in general, inactivation of caliciviruses by the various approaches may be matrix-, temperature-, and/or contact time-dependent. The susceptibilities of the caliciviruses to the various physical and chemical inactivation approaches are generally similar to those displayed by other small, non-enveloped viruses, with the exception that the parvoviruses and circoviruses may require higher temperatures for inactivation, while these families appear to be more susceptible to UV-C inactivation than are the caliciviruses.

  12. Relationship between sublethal injury and inactivation of yeast cells by the combination of sorbic acid and pulsed electric fields.

    Science.gov (United States)

    Somolinos, M; García, D; Condón, S; Mañas, P; Pagán, R

    2007-06-01

    The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log10 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.

  13. Heat inactivation of a norovirus surrogate in cell culture lysate, abalone meat, and abalone viscera.

    Science.gov (United States)

    Park, Shin Young; Bae, San-Cheong; Ha, Sang-Do

    2015-03-01

    The current study examined the effects of temperature and heat treatment duration on murine norovirus-1 (MNV-1) from both viral cell culture lysate (7-8 log10 PFU) and experimentally contaminated abalone meat and viscera (5-6 log10 PFU) as a model of human norovirus (NoV). MNV-1 titers in cell culture lysate, abalone meat, and abalone viscera were gradually reduced to 1.93-4.55, 1.79-3.00, and 2.26-3.26 log10 PFU/ml, respectively, after treatment at 70 °C for 1-10 min. Treatment at 85 °C for 1-5 min gradually reduced MNV-1 titers in abalone meat to 2.71-4.15 log10 PFU/ml. MNV-1 titers in abalone viscera were gradually reduced to 2.91-3.46 log10 PFU/ml after treatment at 85 °C for 1-3 min. No significant difference (P > 0.05) was found in MNV-1 titers in the abalone meat and viscera among treatment groups (70 °C for 5 min, 70 °C for 3 min, and 85 °C for 1 min). Complete inactivation of MNV-1 in cell culture lysate was determined at 85 °C for ≥1 min and 100 °C for ≥0.5 min. Complete inactivation of MNV-1 in abalone was determined at 100 °C for ≥0.5 min for meat, and 85 °C for 5 min and 100 °C for ≥0.5 min for viscera. At treatments at 70 °C, the Td-values (3 log reduction time) were significantly lower (P abalone meat (6.07) and viscera (10.73). Td = 3 values were not significantly different (P > 0.05) between abalone meat (1.78) and abalone viscera (1.33) at treatments at 85 °C. This study suggests that 100 °C for ≥0.5 min could potentially be used to inactivate NoV in molluscan shellfishes, including viscera.

  14. Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

    Science.gov (United States)

    Moorman, J P; Bobak, D A; Hahn, C S

    1996-06-01

    The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

  15. DNA double strand breaks as the critical type of damage with regard to inactivation of cells through ionizing radiation

    International Nuclear Information System (INIS)

    Frankenberg, D.

    1985-01-01

    This report presents the results of an investigation into the effects of ionizing radiation on eukaryotic cells, aimed at revealing the molecular mechanisms leading to cell inactivation as a result of ionizing radiation. The quantitative determination of radiation-induced double strand breaks (DSB) is done via sedimentation of the DNA released from the cells in a neutral saccharose gradient in a preparative ultracentrifuge. The 'experimental mass spectrum' of DNA molecules thus obtained, the mean number of DSB per cell is calculated using a special computer program which simulates the stochastic induction of DSB in the DNA of non-irradiated cells and links the 'simulated' mass spectrum with the 'experimental' one on the basis of the least square fit. The experimental and theoretical studies with the eukaryote yeast on the whole allow insight into the relation between energy absorption and the inactivation of irradiated cells. (orig./MG) [de

  16. Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor

    Science.gov (United States)

    Esakky, P; Hansen, D A; Drury, A M; Cusumano, A; Moley, K H

    2015-01-01

    Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line. PMID:27551479

  17. Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays

    International Nuclear Information System (INIS)

    Goodhead, D.T.

    1977-01-01

    Microdosimetric distributions for aluminium K characteristic ultrasoft X-rays and 4 He ion track intersections have been calculated and used to analyse recent biological results obtained with these radiations. Results on inactivation and mutation-induction to thioguanine resistance of both V79 Chinese hamster cells and HF19 human diploid fibroblasts in vitro were analysed in terms of the Kellerer-Rossi 'theory of dual radiation action'. The small quantum energy of the aluminium X-ray photons and the very short length of the secondary electrons which they produce highlight the inadequacy of the model. It has been shown that the model predicted r.b.e. values in conflict with those observed unless an additional variable was introduced, but that the introduction of such a variable created mathematical inconsistencies. The experimental evidence is contrary to the conventional usage and basis of the model. (author)

  18. Immunotherapy with internally inactivated virus loaded dendritic cells boosts cellular immunity but does not affect feline immunodeficiency virus infection course

    Directory of Open Access Journals (Sweden)

    Pistello Mauro

    2008-04-01

    Full Text Available Abstract Immunotherapy of feline immunodeficiency virus (FIV-infected cats with monocyte-derived dendritic cells (MDCs loaded with aldrithiol-2 (AT2-inactivated homologous FIV was performed. Although FIV-specific lymphoproliferative responses were markedly increased, viral loads and CD4+ T cell depletion were unaffected, thus indicating that boosting antiviral cell-mediated immunity may not suffice to modify infection course appreciably.

  19. Safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children.

    Science.gov (United States)

    Halperin, Scott A; Smith, Bruce; Mabrouk, Taoufik; Germain, Marc; Trépanier, Pierre; Hassell, Thomas; Treanor, John; Gauthier, Richard; Mills, Elaine L

    2002-01-15

    We performed randomized, double-blind, controlled trials to assess the safety and immunogenicity of an inactivated, Madin Darby Canine Kidney (MDCK)-derived cell line produced influenza vaccine in healthy adults (19-50 years), children (3-12 years) and the elderly (> or =65 years). We studied three lots of cell culture-derived vaccine and one lot of licensed egg-derived vaccine in healthy adults (n=462), two lots of cell culture-derived vaccine and one lot of egg-derived vaccine in seniors (n=269), and one lot of each vaccine in children (n=209). Adverse events were collected during the first 3 days post-immunization; serum was collected before and 1 month after immunization. Rates of local and systemic adverse reactions were similar with both vaccines. An injection site adverse event rated at least moderate severity was reported by 21.9% of children who received the egg-derived vaccine and 25.0% of those who received the cell culture-derived vaccine. In healthy adults the proportions were 12.1 and 15.3%, respectively and 6.7 and 6.3%, respectively in seniors. Systemic events of at least moderate severity were 12.4 and 12.5% in children, 19.8 and 13.6% in healthy adults, and 14.1 and 9.7% in seniors; none of these differences were statistically significant. The antibody response against all three viruses was similar between the two vaccines. From 83 to 100% of children, healthy adults and seniors achieved hemagglutination inhibition titers in excess of 40 post-immunization. We conclude that the cell culture-derived vaccine was safe and immunogenic in children, healthy adults and seniors.

  20. Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

    Science.gov (United States)

    Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

    2016-01-01

    Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

  1. Influence of Kupffer cell inactivation on cycloheximide-induced hepatic injury

    International Nuclear Information System (INIS)

    Kumagai, Kazuyoshi; Kiyosawa, Naoki; Ito, Kazumi; Yamoto, Takashi; Teranishi, Munehiro; Nakayama, Hiroyuki; Manabe, Sunao

    2007-01-01

    In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl 3 ) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl 3 before being treated with the same dose of CHX (GdCl 3 /CHX group). The necrotic change in the GdCl 3 /CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl 3 /CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl 3 /CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl 3 /CHX group. Thus, Kupffer cell inactivation by the GdCl 3 treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl 3 /CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment

  2. Inactivation kinetics of various chemical disinfectants on Aeromonas hydrophila planktonic cells and biofilms.

    Science.gov (United States)

    Jahid, Iqbal Kabir; Ha, Sang-Do

    2014-05-01

    The present article focuses on the inactivation kinetics of various disinfectants including ethanol, sodium hypochlorite, hydrogen peroxide, peracetic acid, and benzalkonium chloride against Aeromonas hydrophila biofilms and planktonic cells. Efficacy was determined by viable plate count and compared using a modified Weibull model. The removal of the biofilms matrix was determined by the crystal violet assay and was confirmed by field-emission scanning electron microscope. The results revealed that all the experimental data and calculated Weibull α (scale) and β (shape) parameters had a good fit, as the R(2) values were between 0.88 and 0.99. Biofilms are more resistant to disinfectants than planktonic cells. Ethanol (70%) was the most effective in killing cells in the biofilms and significantly reduced (preduction as well as the effectiveness of chemical disinfectants on biofilms. The study showed that the Weibull model could successfully be used on food and food contact surfaces to determine the exact contact time for killing biofilms-forming foodborne pathogens.

  3. Noggin inactivation affects the number and differentiation potential of muscle progenitor cells in vivo

    Science.gov (United States)

    Costamagna, Domiziana; Mommaerts, Hendrik; Sampaolesi, Maurilio; Tylzanowski, Przemko

    2016-01-01

    Inactivation of Noggin, a secreted antagonist of Bone Morphogenetic Proteins (BMPs), in mice leads, among others, to severe malformations of the appendicular skeleton and defective skeletal muscle fibers. To determine the molecular basis of the phenotype, we carried out a histomorphological and molecular analysis of developing muscles Noggin−/− mice. We show that in 18.5 dpc embryos there is a marked reduction in muscle fiber size and a failure of nuclei migration towards the cell membrane. Molecularly, the absence of Noggin results in an increased BMP signaling in muscle tissue as shown by the increase in SMAD1/5/8 phosphorylation, concomitant with the induction of BMP target genes such as Id1, 2, 3 as well as Msx1. Finally, upon removal of Noggin, the number of mesenchymal Pax7+ muscle precursor cells is reduced and they are more prone to differentiate into adipocytes in vitro. Thus, our results highlight the importance of Noggin/BMP balance for myogenic commitment of early fetal progenitor cells. PMID:27573479

  4. Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

    Science.gov (United States)

    Pohler, Petra; Müller, Meike; Winkler, Carla; Schaudien, Dirk; Sewald, Katherina; Müller, Thomas H; Seltsam, Axel

    2015-02-01

    Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients. © 2014 AABB.

  5. A novel photosensitization treatment for the inactivation of fungal spores and cells mediated by curcumin.

    Science.gov (United States)

    Al-Asmari, Fahad; Mereddy, Ram; Sultanbawa, Yasmina

    2017-08-01

    The global concerns regarding the emergence of fungicide-resistant strains and the impact of the excessive use of fungicidal practises on our health, food, and environment have increased, leading to a demand for alternative clean green technologies as treatments. Photosensitization is a treatment that utilises a photosensitiser, light and oxygen to cause cell damage to microorganisms. The effect of photosensitization mediated by curcumin on Aspergillus niger, Aspergillus flavus, Penicillium griseofulvum, Penicillium chrysogenum, Fusarium oxysporum, Candida albicans and Zygosaccharomyces bailii was investigated using three methods. The viability of spores/cells suspended in aqueous buffer using different concentrations of curcumin solution (100-1000μM) and light dose (0, 24, 48, 72 and 96J/cm 2 ) were determined. Spraying curcumin solution on inoculated surfaces of agar plates followed by irradiation and soaking spores/cells in curcumin solution prior to irradiation was also investigated. In aqueous mixtures, photosensitised spores/cells of F. oxysporum and C. albicans were inhibited at all light doses and curcumin concentrations, while inactivation of A. niger, A. flavus P. griseofulvum, P. chrysogenum and Z. bailii were highly significant (Pcurcumin at 800μM showed complete inhibition for A. niger, F. oxysporum, C. albicans and Z. bailii, while A. flavus P. griseofulvum, and P. chrysogenum reduced by 75%, 80.4% and 88.5% respectively. Soaking spores/cells with curcumin solution prior to irradiation did not have a significant effect on the percentage reduction. These observations suggest that a novel photosensitization mediated curcumin treatment is effective against fungal spores/cells and the variation of percentage reduction was dependent on curcumin concentration, light dosage and fungal species. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Relationship between Sublethal Injury and Inactivation of Yeast Cells by the Combination of Sorbic Acid and Pulsed Electric Fields▿

    OpenAIRE

    Somolinos, M.; García, D.; Condón, S.; Mañas, P.; Pagán, R.

    2007-01-01

    The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm ...

  7. Variation in RNA virus mutation rates across host cells.

    Directory of Open Access Journals (Sweden)

    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  8. Pulsed Electric Field inactivation of microbial cells: the use of ceramic layers to increase the efficiency of treatment

    International Nuclear Information System (INIS)

    Pizzichemi, M.

    2009-01-01

    The impact of Pulsed Electric Fields (PEF) on bacteria and plant or animal cells has been investigated since the early 1960s. High electric fields pulses (20-70 kV/cm, 1-10 μs) are reported to cause rupture of the cellular lipid membrane, through the mechanism of irreversible electroporation. Quantitative description of cell inactivation kinetics is based on the analysis of stability of lipid bilayers under electric fields and the thermal fluctuations associated with the production of pores. PEF has been successfully applied to inactivation of both Gram-positive and Gram-negative bacteria in many sorts of liquids, such as milk, fruit juices and liquid eggs. In all these media, the level of inactivation could reach the 5 Logs for an approximate range of pulses of 100-200, and an energy consumption of ∼ 10-100 kJ/kg. The advantages of PEF are the superior maintenance of functional and nutritional levels (if compared to traditional thermal treatment), continuous treatment and short processing times, while the current high costs of this technique make it more suitable for treatment of expensive media. We present a solution to the problem of volumes in PEF treatment through the use of high permittivity ceramics, while retaining the same inactivation efficiency and improving the duration of the electrodes.

  9. Pulsed Electric Field inactivation of microbial cells: the use of ceramic layers to increase the efficiency of treatment

    Science.gov (United States)

    Pizzichemi, M.

    2009-12-01

    The impact of Pulsed Electric Fields (PEF) on bacteria and plant or animal cells has been investigated since the early 1960s. High electric fields pulses (20-70 kV/cm, 1-10 μs) are reported to cause rupture of the cellular lipid membrane, through the mechanism of irreversible electroporation. Quantitative description of cell inactivation kinetics is based on the analysis of stability of lipid bilayers under electric fields and the thermal fluctuations associated with the production of pores. PEF has been successfully applied to inactivation of both Gram-positive and Gram-negative bacteria in many sorts of liquids, such as milk, fruit juices and liquid eggs. In all these media, the level of inactivation could reach the 5 Logs for an approximate range of pulses of 100-200, and an energy consumption of ˜ 10-100 kJ/kg. The advantages of PEF are the superior maintenance of functional and nutritional levels (if compared to traditional thermal treatment), continuous treatment and short processing times, while the current high costs of this technique make it more suitable for treatment of expensive media. We present a solution to the problem of volumes in PEF treatment through the use of high permittivity ceramics, while retaining the same inactivation efficiency and improving the duration of the electrodes.

  10. Pulsed Electric Field inactivation of microbial cells: the use of ceramic layers to increase the efficiency of treatment

    Energy Technology Data Exchange (ETDEWEB)

    Pizzichemi, M. [Physics Department, University of Milano - Bicocca (Italy)

    2009-12-15

    The impact of Pulsed Electric Fields (PEF) on bacteria and plant or animal cells has been investigated since the early 1960s. High electric fields pulses (20-70 kV/cm, 1-10 mus) are reported to cause rupture of the cellular lipid membrane, through the mechanism of irreversible electroporation. Quantitative description of cell inactivation kinetics is based on the analysis of stability of lipid bilayers under electric fields and the thermal fluctuations associated with the production of pores. PEF has been successfully applied to inactivation of both Gram-positive and Gram-negative bacteria in many sorts of liquids, such as milk, fruit juices and liquid eggs. In all these media, the level of inactivation could reach the 5 Logs for an approximate range of pulses of 100-200, and an energy consumption of approx 10-100 kJ/kg. The advantages of PEF are the superior maintenance of functional and nutritional levels (if compared to traditional thermal treatment), continuous treatment and short processing times, while the current high costs of this technique make it more suitable for treatment of expensive media. We present a solution to the problem of volumes in PEF treatment through the use of high permittivity ceramics, while retaining the same inactivation efficiency and improving the duration of the electrodes.

  11. Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation.

    LENUS (Irish Health Repository)

    Whelan, M C

    2012-01-31

    Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm(3) tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

  12. High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

    Science.gov (United States)

    Kafka, T. A.; Reitermayer, D.; Lenz, C. A.; Vogel, R. F.

    2017-07-01

    Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.

  13. Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold

    Science.gov (United States)

    Mazzer, Alice R.; Perraud, Xavier; Halley, Jennifer; O’Hara, John; Bracewell, Daniel G.

    2015-01-01

    Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested. PMID:26346187

  14. A model for the induction of DNA damages and their evolution into cell clonogenic inactivation

    International Nuclear Information System (INIS)

    Yamaguchi, Hiroshi; Ohara, Hiroshi; Waker, A.J.

    2006-01-01

    The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 base-pairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for AT-cell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7-4.3 nm and the other filling all space with a depth 4.6-4.9 nm. (author)

  15. Mechanism of photodynamic inactivation of hepatocarcinoma cells with sulfonated aluminum phthalocyanine

    Science.gov (United States)

    Yu, Hong-Yu; Dong, Rong-Chun; Chen, Ji-Yao; Cai, Huai-Xin

    1993-03-01

    The mechanism of photodynamic therapy (PDT) with sulfonated aluminum phthalocyanine (AlSPC) studied with the human hepatocellular carcinoma cell line in culture is reported herein. Photofrin II (PII) was chosen as the control photosensitizer of AlSPC. Deuterium oxide (D2O), an enhancer of singlet oxygen (1O2); 1,3-diphenylisobenzofuran (DPBF), a quencher of 1O2: glycerol, a quencher of OH radical (OH(DOT)); superoxide dismutase (SOD), a quencher of O2- radical (O2-(DOT)); diethyldithiocarbamate (DDC), an inhibitor of SOD and glutathione peroxidase; were introduced into both the processes of photodynamic inactivation of human liver cancer cells in culture with AlSPC (AlSPC-PDT) and with PII (PII-PDT). The results suggest that: 1O2 is dominantly involved in both PII-PDT and AlSPC-PDT; O2-(DOT) is involved in AlSPC-PDT in a lower degree than 1O2, while almost not involved in PII-PDT; OH(DOT) is involved in PII-PDT in a lower degree than 1O2, while almost not involved in AlSPC-PDT.

  16. Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

    Science.gov (United States)

    Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

    2013-12-01

    Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

  17. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

    Science.gov (United States)

    Sun, Xiaofei; Park, Craig B; Deng, Wenbo; Potter, S Steven; Dey, Sudhansu K

    2016-04-01

    Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. © FASEB.

  18. Rate-limiting events in hyperthermic cell killing

    International Nuclear Information System (INIS)

    Landry, J.; Marceau, N.

    1978-01-01

    The inactivation rate of HeLa cells for temperatures ranging from 41 to 55 0 C and treatment durations varying from 2 to 300 min was analyzed in thermodynamic terms by considering the dependence of cell free energy (ΔG + ) on temperature. Within this temperature range the loss of proliferative capacity exhibits a complex temperature dependence which is characterized by entropy and enthalpy values that gradually decrease as temperature increases. This complex process of heat-induced cell killing was postulated to be the result of a series of reactions, each of them being alternatively rate limiting within a certain temperature range. From this kinetic scheme a mathematical model was derived and, in the case of HeLa cells, the use of a least-squares search parameter procedure (as applied to the derived survival regression function) demonstrated that three such sequential reactions were sufficient to explain all experimental data points obtained within the 41 to 55 0 C range. The proposed model was also shown to be adequate for explaining survival data of HeLa cells exposed to nanosecond heat pulses of infrared laser energy. Considerations of thermodynamic properties of known biochemical reactions suggest plausible rate-limiting events in hyperthermic cell killing

  19. Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea.

    Science.gov (United States)

    Chen, Qianqian; Quan, Yizhou; Wang, Naitao; Xie, Chengying; Ji, Zhongzhong; He, Hao; Chai, Renjie; Li, Huawei; Yin, Shankai; Chin, Y Eugene; Wei, Xunbin; Gao, Wei-Qiang

    2017-07-11

    Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment. Copyright © 2017 International Society for Stem Cell Research. Published by Elsevier Inc. All rights reserved.

  20. Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells

    Directory of Open Access Journals (Sweden)

    Verena Staudacher

    2018-04-01

    Full Text Available Redox-sensitive green fluorescent protein 2 (roGFP2 is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis. Keywords: Peroxiredoxin, Redox sensor, roGFP2, H2O2, Plasmodium falciparum

  1. Inactivation of Ricin Toxin by Nanosecond Pulsed Electric Fields Including Evidences from Cell and Animal Toxicity

    Science.gov (United States)

    Wei, Kai; Li, Wei; Gao, Shan; Ji, Bin; Zang, Yating; Su, Bo; Wang, Kaile; Yao, Maosheng; Zhang, Jue; Wang, Jinglin

    2016-01-01

    Ricin is one of the most toxic and easily produced plant protein toxin extracted from the castor oil plant, and it has been classified as a chemical warfare agent. Here, nanosecond pulsed electric fields (nsPEFs) at 30 kV/cm (pulse durations: 10 ns, 100 ns, and 300 ns) were applied to inactivating ricin up to 4.2 μg/mL. To investigate the efficacy, cells and mice were tested against the ricin treated by the nsPEFs via direct intraperitoneal injection and inhalation exposure. Results showed that nsPEFs treatments can effectively reduce the toxicity of the ricin. Without the nsPEFs treatment, 100% of mice were killed upon the 4 μg ricin injection on the first day, however 40% of the mice survived the ricin treated by the nsPEFs. Compared to injection, inhalation exposure even with higher ricin dose required longer time to observe mice fatality. Pathological observations revealed damages to heart, lung, kidney, and stomach after the ricin exposure, more pronounced for lung and kidney including severe bleeding. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and circular dichroism (CD) analyses revealed that although the primary structure of ricin was not altered, its secondary structures (beta-sheet and beta-turn) underwent transition upon the nsPEFs treatment. PMID:26728251

  2. SETD2 and PBRM1 inactivation in the development of clear cell renal cell carcinoma

    NARCIS (Netherlands)

    Li, Jun

    2016-01-01

    Kidney cancer of the clear cell type is often lethal and causes more than 100,000 deaths worldwide every year. Understanding the biology of this cancer type may help to develop better ways to diagnose and treat it. Damage in DNA (genes) is present in all cancer cells and clear cell kidney cancer is

  3. Can microbial cells develop resistance to oxidative stress in antimicrobial photodynamic inactivation?

    Science.gov (United States)

    Kashef, Nasim; Hamblin, Michael R

    2017-03-01

    Infections have been a major cause of disease throughout the history of humans on earth. With the introduction of antibiotics, it was thought that infections had been conquered. However, bacteria have been able to develop resistance to antibiotics at an exponentially increasing rate. The growing threat from multi-drug resistant organisms calls for intensive action to prevent the emergence of totally resistant and untreatable infections. Novel, non-invasive, non-antibiotic strategies are needed that act more efficiently and faster than current antibiotics. One promising alternative is antimicrobial photodynamic inactivation (APDI), an approach that produces reactive oxygen species when dyes and light are combined. So far, it has been questionable if bacteria can develop resistance against APDI. This review paper gives an overview of recent studies concerning the susceptibility of bacteria towards oxidative stress, and suggests possible mechanisms of the development of APDI-resistance that should at least be addressed. Some ways to potentiate APDI and also to overcome future resistance are suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Microbial Inactivation by Ultrasound Assisted Supercritical Fluids

    Science.gov (United States)

    Benedito, Jose; Ortuño, Carmen; Castillo-Zamudio, Rosa Isela; Mulet, Antonio

    A method combining supercritical carbon dioxide (SC-CO2) and high power ultrasound (HPU) has been developed and tested for microbial/enzyme inactivation purposes, at different process conditions for both liquid and solid matrices. In culture media, using only SC-CO2, the inactivation rate of E. coli and S. cerevisiae increased with pressure and temperature; and the total inactivation (7-8 log-cycles) was attained after 25 and 140 min of SC-CO2 (350 bar, 36 °C) treatment, respectively. Using SC-CO2+HPU, the time for the total inactivation of both microorganisms was reduced to only 1-2 min, at any condition selected. The SC-CO2+HPU inactivation of both microorganisms was slower in juices (avg. 4.9 min) than in culture media (avg. 1.5 min). In solid samples (chicken, turkey ham and dry-cured pork cured ham) treated with SC-CO2 and SC-CO2+HPU, the inactivation rate of E. coli increased with temperature. The application of HPU to the SC-CO2 treatments accelerated the inactivation rate of E. coli and that effect was more pronounced in treatments with isotonic solution surrounding the solid food samples. The application of HPU enhanced the SC-CO2 inactivation mechanisms of microorganisms, generating a vigorous agitation that facilitated the CO2 solubilization and the mass transfer process. The cavitation generated by HPU could damage the cell walls accelerating the extraction of vital constituents and the microbial death. Thus, using the combined technique, reasonable industrial processing times and mild process conditions could be used which could result into a cost reduction and lead to the minimization in the food nutritional and organoleptic changes.

  5. Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin.

    Science.gov (United States)

    Poma, A; Miranda, M; Spanò, L

    1998-10-01

    The cytotoxicity and inhibitory effect on proliferation of the type 1 ribosome-inactivating protein luffin purified from the seeds of Luffa aegyptiaca were investigated both in human metastatic melanoma cells and in murine Ehrlich ascites tumour cells. Results indicate that luffin from the seeds of Luffa aegyptiaca is cytotoxic to the cell lines tested, with approximately 10 times greater potency in Ehrlich cells. Luffin was found to induce an increase in cytosolic oligonucleosome-bound DNA in both melanoma and Ehrlich ascites tumour cells, the level of DNA fragmentation in the former cell line being higher than in the latter. Experiments with melanoma cells indicate that an increase in cytosolic nucleosomes could be supportive of apoptosis as the type of cell death induced by luffin.

  6. Involvement of Atm and Trp53 in neural cell loss due to Terf2 inactivation during mouse brain development.

    Science.gov (United States)

    Kim, Jusik; Choi, Inseo; Lee, Youngsoo

    2017-11-01

    Maintenance of genomic integrity is one of the critical features for proper neurodevelopment and inhibition of neurological diseases. The signals from both ATM and ATR to TP53 are well-known mechanisms to remove neural cells with DNA damage during neurogenesis. Here we examined the involvement of Atm and Atr in genomic instability due to Terf2 inactivation during mouse brain development. Selective inactivation of Terf2 in neural progenitors induced apoptosis, resulting in a complete loss of the brain structure. This neural loss was rescued partially in both Atm and Trp53 deficiency, but not in an Atr-deficient background in the mouse. Atm inactivation resulted in incomplete brain structures, whereas p53 deficiency led to the formation of multinucleated giant neural cells and the disruption of the brain structure. These giant neural cells disappeared in Lig4 deficiency. These data demonstrate ATM and TP53 are important for the maintenance of telomere homeostasis and the surveillance of telomere dysfunction during neurogenesis.

  7. High-rate lithium thionyl chloride cells

    Science.gov (United States)

    Goebel, F.

    1982-03-01

    A high-rate C cell with disc electrodes was developed to demonstrate current rates which are comparable to other primary systems. The tests performed established the limits of abuse beyond which the cell becomes hazardous. Tests include: impact, shock, and vibration tests; temperature cycling; and salt water immersion of fresh cells.

  8. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  9. Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

    Science.gov (United States)

    Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

    2011-07-15

    Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

  10. New Treatment Options for Osteosarcoma - Inactivation of Osteosarcoma Cells by Cold Atmospheric Plasma.

    Science.gov (United States)

    Gümbel, Denis; Gelbrich, Nadine; Weiss, Martin; Napp, Matthias; Daeschlein, Georg; Sckell, Axel; Ender, Stephan A; Kramer, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

    2016-11-01

    Cold atmospheric plasma has been shown to inhibit tumor cell growth and induce tumor cell death. The aim of the study was to investigate the effects of cold atmospheric plasma treatment on proliferation of human osteosarcoma cells and to characterize the underlying cellular mechanisms. Human osteosarcoma cells (U2-OS and MNNG/HOS) were treated with cold atmospheric plasma and seeded in culture plates. Cell proliferation, p53 and phospho-p53 protein expression and nuclear morphology were assessed. The treated human osteosarcoma cell lines exhibited attenuated proliferation rates by up to 66%. The cells revealed an induction of p53, as well as phospho-p53 expression, by 2.3-fold and 4.5-fold, respectively, compared to controls. 4',6-diamidino-2-phenylindole staining demonstrated apoptotic nuclear condensation following cold atmospheric plasma treatment. Cold atmospheric plasma treatment significantly attenuated cell proliferation in a preclinical in vitro osteosarcoma model. The resulting increase in p53 expression and phospho-activation in combination with characteristic nuclear changes indicate this was through induction of apoptosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Wen-Hsiung Chan

    2008-05-01

    Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

  12. Production of DNA strand breaks by ionizing radiation of different quality and their consequences for cell inactivation

    International Nuclear Information System (INIS)

    Kampf, G.

    1983-07-01

    The production of single- and double-strand breaks (DSB) in the DNA of Chinese hamster cells (V 79) was studied by use of 11 radiation qualities, with some also under hypoxic conditions. The aim was to find relations between the induction of lesions on the molecular level and the expression of this damage on the cellular level. The results suggest that release of DNA from the nuclear-membrane complex, induction of chromosome breaks, and cell inactivation are triggered by DSB. However, not simply a certain number of DSB in the DNA of the nucleus, but their cooperation within a small structural section of DNA is required for cell inactivation. Such sections may be the membrane-associated superstructure units. DSB produced under hypoxic conditions show a greater effectiveness than those produced under oxic conditions. The investigations with eukaryotic cells and bacteria suggest that not the entire DNA of all organisms but a structural unit common to them represents the critical target for radiation action. (author)

  13. Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling.

    Directory of Open Access Journals (Sweden)

    Sang-Hun Kang

    Full Text Available BACKGROUND: The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML cell line U937. METHODOLOGY/PRINCIPAL FINDINGS: Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose polymerase (PARP in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x(L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2, the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP SH2 domain-containing phosphatase (SHP-1, and the addition of sodium pervanadate (a PTP inhibitor prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.

  14. Apc inactivation, but not obesity, synergizes with Pten deficiency to drive intestinal stem cell-derived tumorigenesis.

    Science.gov (United States)

    Tabrizian, Tahmineh; Wang, Donghai; Guan, Fangxia; Hu, Zunju; Beck, Amanda P; Delahaye, Fabien; Huffman, Derek M

    2017-06-01

    Obesity is a major risk factor for colorectal cancer and can accelerate Lgr5+ intestinal stem cell (ISC)-derived tumorigenesis after the inactivation of Apc However, whether non-canonical pathways involving PI3K-Akt signaling in ISCs can lead to tumor formation, and if this can be further exacerbated by obesity is unknown. Despite the synergy between Pten and Apc inactivation in epithelial cells on intestinal tumor formation, their combined role in Lgr5+-ISCs, which are the most rapidly dividing ISC population in the intestine, is unknown. Lgr5+-GFP mice were provided low-fat diet (LFD) or high-fat diet (HFD) for 8 months, and the transcriptome was evaluated in Lgr5+-ISCs. For tumor studies, Lgr5+-GFP and Lgr5+-GFP- Pten flox/flox mice were tamoxifen treated to inactivate Pten in ISCs and provided LFD or HFD until 14-15 months of age. Finally, various combinations of Lgr5+-ISC-specific, Apc- and Pten -deleted mice were generated and evaluated for histopathology and survival. HFD did not overtly alter Akt signaling in ISCs, but did increase other metabolic pathways. Pten deficiency, but not HFD, increased BrdU-positive cells in the small intestine ( P  Apc deficiency synergistically increased proliferative markers, tumor pathology and mortality, in a dose-dependent fashion ( P  Apc deficiency in ISCs synergistically increases proliferation, tumor formation and mortality. Thus, aberrant Wnt/β-catenin, rather than PI3K-Akt signaling, is requisite for obesity to drive Lgr5+ ISC-derived tumorigenesis. © 2017 Society for Endocrinology.

  15. Bortezomib induces apoptosis and suppresses cell growth and metastasis by inactivation of Stat3 signaling in chondrosarcoma.

    Science.gov (United States)

    Bao, Xing; Ren, Tingting; Huang, Yi; Ren, Chongmin; Yang, Kang; Zhang, Hongliang; Guo, Wei

    2017-02-01

    Bortezomib, formerly known as PS341, is a novel proteasome inhibitor with in vitro and in vivo antineoplastic effects in many malignancies. However, diverse antitumor mechanisms of bortezomib have been identified in many investigations and preclinical studies. Understanding the molecular and cellular mechanisms through which bortezomib acts will improve the therapeutic utility of this drug in different cancer types. In the present study, we investigated the in vitro and in vivo effects of bortezomib on chondrosarcoma. Bortezomib selectively inhibited cell growth in chondrosarcoma cells but not in normal articular cartilage cells. In addition to growth inhibition, apoptosis and cell cycle arrest, bortezomib triggered alleviation of migratory and invasive properties of chondrosarcoma cells. Mechanistically, signal transducer and activator of transcription 3 (Stat3) and its downstream targets Bcl-2, cyclin D1 and c-Myc was inactivated by bortezomib treatment. Accordingly, small interfering RNA (siRNA)-mediated Stat3 knockdown enhanced bortezomib-induced apoptosis, and concomitantly enhanced the inhibitory effect of bortezomib on cell viability, migration and invasion. Moreover, while Slug, MMP9, MMP2, CD44, N-cadherin and vimentin, the mesenchymal cell markers, were repressed by bortezomib concomitant increased expression of E-cadherin was observed. In vivo, bortezomib downregulated Stat3 activity and mesenchymal cell marker expression, induced apoptosis and inhibition of metastasis and tumor growth. Together, inactivation of Stat3 signaling contributes to bortezomib-induced inhibition of tumor growth, migration and invation on chondrosarcoma. Bortezomib demonstrates an antineoplastic role on chondrosarcoma both in vitro and in vivo. These beneficial effects can be explained by bortezomib-mediated Stat3 supression. The present study suggests a promising therapeutics target in chondrosarcoma and probably in other kinds of metastatic malignant tumors.

  16. Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule-1 and enhances natural killer cell sensitivity on cancer cells.

    Science.gov (United States)

    Li, Simin; Nishikawa, Tomoyuki; Kaneda, Yasufumi

    2017-12-01

    We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) has multiple anticancer effects, including induction of cancer-selective cell death and activation of anticancer immunity. The HVJ-E stimulates dendritic cells to produce cytokines and chemokines such as β-interferon, interleukin-6, chemokine (C-C motif) ligand 5, and chemokine (C-X-C motif) ligand 10, which activate both CD8 + T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ-E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ-E induced the production of intercellular adhesion molecule-1 (ICAM-1, CD54), a ligand of lymphocyte function-associated antigen 1, in several cancer cell lines through the activation of nuclear factor-κB downstream of retinoic acid-inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM-1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM-1 in MDA-MB-231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM-1-depleted MDA-MB-231 cells. In addition, HVJ-E suppressed tumor growth in MDA-MB-231 tumor-bearing SCID mice, and the HVJ-E antitumor effect was impaired when NK cells were depleted by treatment with the anti-asialo GM1 antibody. Our findings suggest that HVJ-E enhances NK cell sensitivity against cancer cells by increasing ICAM-1 expression on the cancer cell surface. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  17. Photodynamic inactivation of rubella virus enhances recombination with a latent virus of a baby hamster kidney cell line BHK21

    International Nuclear Information System (INIS)

    Yamamoto, Nobuto; Urade, Masahiro

    1989-01-01

    Rubella virus is very sensitive to photodynamic action. When tested with 1.2 x 10 -5 M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m 2 , inactivation kinetics showed a linear single hit curve with a k value of 1.48 min -1 . Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R-virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV-treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV-RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti-rubella serum nor by anti-latent virus serum while type 2 hybrid is neutralized by anti-latent virus serum. (author)

  18. Modification of cell growth rate by irradiation

    International Nuclear Information System (INIS)

    Itoh, Hisao; Takemasa, Kazuhiko; Nishiguchi, Iku; Ka, Wei-Jei; Kutsuki, Shoji; Hashimoto, Shozo

    1993-01-01

    The effect of irradiation on the proliferation kinetics of the monolayer cells has been studied. Two human cell lines with different doubling times (HeLa-P and RMUG) and two clones that have the same radiosensitivity but different doubling times (HeLa-R and HeLa-S) were irradiated with a daily dose of 2 Gy for 6 days. The number of the clonogenic cells/dish was calculated by multiplying the number of total cell/dish by the survival fraction. In the rapidly growing cells (HeLa-P, HeLa-R), the number of the clonogenic cells was not decreased by the first two fractionated irradiations, but decreased thereafter at a similar rate as by single-dose fractionation, whereas the clonogenic cell number decreased from the first fractionated irradiation in the slowly growing cells (RMUG, HeLa-S). When the proliferation of clonogenic cell number increased along with a similar growth rates that was seen in all other types of cells. Further, no correlation was seen between the growth rates of cells without irradiation and cells that received irradiation. This latter result suggests that the slow growth rate of non-irradiated cells may not be the predictive factor of the tumor cure and the interruption of radiotherapy may reduce the beneficial effect of this treatment even in slow growing tumors. (author)

  19. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1981-01-01

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

  20. DNA fork displacement rates in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kapp, L.N.; Painter, R.B. (California Univ., San Francisco (USA). Lab. of Radiobiology)

    1981-11-27

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 ..mu..m/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

  1. d,l-Sulforaphane Induces ROS-Dependent Apoptosis in Human Gliomablastoma Cells by Inactivating STAT3 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ziwei Miao

    2017-01-01

    Full Text Available d,l-Sulforaphane (SFN, a synthetic analogue of broccoli-derived isomer l-SFN, exerts cytotoxic effects on multiple tumor cell types through different mechanisms and is more potent than the l-isomer at inhibiting cancer growth. However, the means by which SFN impairs glioblastoma (GBM cells remains poorly understood. In this study, we investigated the anti-cancer effect of SFN in GBM cells and determined the underlying molecular mechanisms. Cell viability assays, flow cytometry, immunofluorescence, and Western blot results revealed that SFN could induced apoptosis of GBM cells in a dose- and time-dependent manner, via up-regulation of caspase-3 and Bax, and down-regulation of Bcl-2. Mechanistically, SFN treatment led to increase the intracellular reactive oxygen species (ROS level in GBM cells. Meanwhile, SFN also suppressed both constitutive and IL-6-induced phosphorylation of STAT3, and the activation of upstream JAK2 and Src tyrosine kinases, dose- and time-dependently. Moreover, blockage of ROS production by using the ROS inhibitor N-acetyl-l-cysteine totally reversed SFN-mediated down-regulation of JAK2/Src-STAT3 signaling activation and the subsequent effects on apoptosis by blocking the induction of apoptosis-related genes in GBM cells. Taken together, our data suggests that SFN induces apoptosis in GBM cells via ROS-dependent inactivation of STAT3 phosphorylation. These findings motivate further evaluation of SFN as a cancer chemopreventive agent in GBM treatment.

  2. Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells

    Science.gov (United States)

    Bauer, Georg

    2015-01-01

    Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of

  3. Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells.

    Science.gov (United States)

    Bauer, Georg

    2015-12-01

    Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of

  4. Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

    Science.gov (United States)

    Zhang, Yali; Guo, Zonglou; Xu, Lihong

    2014-03-01

    The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Cell inactivation studies on yeast cells under euoxic and hypoxic condition using electron beam from microtron accelerator

    International Nuclear Information System (INIS)

    Praveen Joseph; Santhosh Acharya; Ganesh Sanjeev; Narayana, Y.; Bhat, N.N.

    2011-01-01

    In the case of sparsely ionizing radiation such as electron, the dose rate and the pattern of energy deposition of the radiation are the important physical factors which can affect the amount of damage in living cells. In the present study, the differences in the cell survival efficiency and dose rate effect in diploid yeast strains Saccharomyces cerevisiae X2180 and Saccharomyces cerevisiae D7 under euoxic and hypoxic condition have been quantified. Irradiation was carried out using 8 MeV pulsed electron beam from Microtron accelerator. The dose per pulse and pulse width of the beam used was 0.6 Gy and 2.3 μs, respectively, which correspond to an instantaneous dose rate of 2.6 x 10 5 Gy s -1 . For survival studies doses were delivered at a rate of 50 pulses per second (an average dose rate of 1,800 Gy s -1 ). Fricke and alanine dosimeters were used to measure the dose delivered to the sample. A significant difference in the dose response has been observed under euoxic and hypoxic condition. Dose rate effect has been studied by changing the pulse repetition rate of the Microtron and the dose rate used was from 180 to 1800 Gy min -1 . A significant dose rate effect was observed under euoxic condition for Saccharomyces cerevisiae X2180 but the same was absent under hypoxic condition. The dose rate effect was absent for Saccharomyces cerevisiae D7 under both irradiation condition. The survival curves are found to be sigmoidal in shape under both condition but with a wider shoulder under hypoxic condition. The D 0 value and the Oxygen Enhancement Ratio (OER) at that point have been derived. (author)

  6. Bio-inactivation of human malignant cells through highly responsive diluted colloidal suspension of functionalized magnetic iron oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Roberta V. [Federal Center of Technological Education of Minas Gerais, Department of Materials (Brazil); Silva-Caldeira, Priscila P. [Federal Center of Technological Education of Minas Gerais, Department of Chemistry (Brazil); Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D. [Federal University of Minas Gerais (UFMG), Department of Chemistry – ICEx (Brazil); Ardisson, José D. [Nuclear Technology Development Center (CDTN) (Brazil); Domingues, Rosana Z., E-mail: rosanazd@yahoo.com.br, E-mail: rosanazd@ufmg.br [Federal University of Minas Gerais (UFMG), Department of Chemistry – ICEx (Brazil)

    2016-04-15

    Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 10{sup 3} mg L{sup −1}, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K

  7. Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species

    Science.gov (United States)

    Quan, Juan-Hua; Kang, Byung-Hun; Yang, Jung-Bo; Rhee, Yun-Ee; Noh, Heung-Tae; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min

    2017-01-01

    Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS), and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5) for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC), reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway. PMID:29410962

  8. Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species

    Directory of Open Access Journals (Sweden)

    Juan-Hua Quan

    2017-01-01

    Full Text Available Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS, and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5 for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC, reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway.

  9. Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

    Directory of Open Access Journals (Sweden)

    Alan Yung-Chih Hu

    Full Text Available BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK cells grown in a serum-free (SF medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

  10. The SKINT1-like gene is inactivated in hominoids but not in all primate species: implications for the origin of dendritic epidermal T cells.

    Directory of Open Access Journals (Sweden)

    Rania Hassan Mohamed

    Full Text Available Dendritic epidermal T cells, which express an invariant Vγ5Vδ1 T-cell receptor and account for 95% of all resident T cells in the mouse epidermis, play a critical role in skin immune surveillance. These γδ T cells are generated by positive selection in the fetal thymus, after which they migrate to the skin. The development of dendritic epidermal T cells is critically dependent on the Skint1 gene expressed specifically in keratinocytes and thymic epithelial cells, suggesting an indispensable role for Skint1 in the selection machinery for specific intraepithelial lymphocytes. Phylogenetically, rodents have functional SKINT1 molecules, but humans and chimpanzees have a SKINT1-like (SKINT1L gene with multiple inactivating mutations. In the present study, we analyzed SKINT1L sequences in representative primate species and found that all hominoid species have a common inactivating mutation, but that Old World monkeys such as olive baboons, green monkeys, cynomolgus macaques and rhesus macaques have apparently functional SKINT1L sequences, indicating that SKINT1L was inactivated in a common ancestor of hominoids. Interestingly, the epidermis of cynomolgus macaques contained a population of dendritic-shaped γδ T cells expressing a semi-invariant Vγ10/Vδ1 T-cell receptor. However, this population of macaque T cells differed from rodent dendritic epidermal T cells in that their Vγ10/Vδ1 T-cell receptors displayed junctional diversity and expression of Vγ10 was not epidermis-specific. Therefore, macaques do not appear to have rodent-type dendritic epidermal T cells despite having apparently functional SKINT1L. Comprehensive bioinformatics analysis indicates that SKINT1L emerged in an ancestor of placental mammals but was inactivated or lost multiple times in mammalian evolution and that Skint1 arose by gene duplication in a rodent lineage, suggesting that authentic dendritic epidermal T cells are presumably unique to rodents.

  11. Inactivation of stable viruses in cell culture facilities by peracetic acid fogging.

    Science.gov (United States)

    Gregersen, Jens-Peter; Roth, Bernhard

    2012-07-01

    Looking for a robust and simple method to replace formaldehyde fumigation for the disinfection of virus-handling laboratories and facilities, we tested peracetic acid fogging as a method to inactivate stable viruses under practical conditions. Peracetic acid/hydrogen peroxide (5.8%/27.5%, 2.0 mL/m³) was diluted in sufficient water to achieve ≥ 70% relative humidity and was vaporized as peracetic acid fog in various positions in the laboratory. After vaporization, a 60 min exposure time, and venting of the laboratory, no residual virus was detected on any of the carriers (detection limit disinfection runs within 12 months, no damage or functional impairment of electrical and electronic equipment was noted. Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  12. The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

    Science.gov (United States)

    Papanikolaou, Eleni; Georgomanoli, Maria; Stamateris, Evangelos; Panetsos, Fottes; Karagiorga, Markisia; Tsaftaridis, Panagiotis; Graphakos, Stelios

    2012-01-01

    Abstract To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro. PMID:21875313

  13. Von Hippel-Lindau (VHL inactivation in sporadic clear cell renal cancer: associations with germline VHL polymorphisms and etiologic risk factors.

    Directory of Open Access Journals (Sweden)

    Lee E Moore

    2011-10-01

    Full Text Available Renal tumor heterogeneity studies have utilized the von Hippel-Lindau VHL gene to classify disease into molecularly defined subtypes to examine associations with etiologic risk factors and prognosis. The aim of this study was to provide a comprehensive analysis of VHL inactivation in clear cell renal tumors (ccRCC and to evaluate relationships between VHL inactivation subgroups with renal cancer risk factors and VHL germline single nucleotide polymorphisms (SNPs. VHL genetic and epigenetic inactivation was examined among 507 sporadic RCC/470 ccRCC cases using endonuclease scanning and using bisulfite treatment and Sanger sequencing across 11 CpG sites within the VHL promoter. Case-only multivariate analyses were conducted to identify associations between alteration subtypes and risk factors. VHL inactivation, either through sequence alterations or promoter methylation in tumor DNA, was observed among 86.6% of ccRCC cases. Germline VHL SNPs and a haplotype were associated with promoter hypermethylation in tumor tissue (OR = 6.10; 95% CI: 2.28-16.35, p = 3.76E-4, p-global = 8E-5. Risk of having genetic VHL inactivation was inversely associated with smoking due to a higher proportion of wild-type ccRCC tumors [former: OR = 0.70 (0.20-1.31 and current: OR = 0.56 (0.32-0.99; P-trend = 0.04]. Alteration prevalence did not differ by histopathologic characteristics or occupational exposure to trichloroethylene. ccRCC cases with particular VHL germline polymorphisms were more likely to have VHL inactivation through promoter hypermethylation than through sequence alterations in tumor DNA, suggesting that the presence of these SNPs may represent an example of facilitated epigenetic variation (an inherited propensity towards epigenetic variation in renal tissue. A proportion of tumors from current smokers lacked VHL alterations and may represent a biologically distinct clinical entity from inactivated cases.

  14. The effect of deep frying or conventional oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs

    Science.gov (United States)

    We investigated the effects deep frying or oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs. A finely-ground veal and/or a beef-pork-veal mixture were inoculated (ca. 7.0 log CFU/g) with an eight-strain, genetically-marked cocktail of rifampicin-res...

  15. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    Science.gov (United States)

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-08

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade.

  16. Aberrant patterns of X chromosome inactivation in a new line of human embryonic stem cells established in physiological oxygen concentrations.

    Science.gov (United States)

    de Oliveira Georges, Juliana Andrea; Vergani, Naja; Fonseca, Simone Aparecida Siqueira; Fraga, Ana Maria; de Mello, Joana Carvalho Moreira; Albuquerque, Maria Cecília R Maciel; Fujihara, Litsuko Shimabukuro; Pereira, Lygia Veiga

    2014-08-01

    One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.

  17. Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling☆

    Science.gov (United States)

    Riethmüller, Michaela; Burger, Nils; Bauer, Georg

    2015-01-01

    Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2.) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling. PMID:26225731

  18. Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling.

    Science.gov (United States)

    Riethmüller, Michaela; Burger, Nils; Bauer, Georg

    2015-12-01

    Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

    Science.gov (United States)

    Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

    2014-01-01

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

  20. Variation in cisplatinum sensitivity is not associated with Fanconi Anemia/BRCA pathway inactivation in head and neck squamous cell carcinoma cell lines.

    Science.gov (United States)

    Snyder, Eric R; Ricker, Justin L; Chen, Zhong; Waes, Carter Van

    2007-01-08

    Fanconi Anemia has recently been associated with a high risk of head and neck squamous cell carcinoma (HNSCC). Inactivation of the Fanconi Anemia (FANC-BRCA) pathway via promoter methylation of the FANCF gene has been proposed to be responsible for variation in cisplatinum (CDDP) sensitivity seen in ovarian and HNSCCs. Promoter methylation of the FANCF gene has been observed in 15% of HNSCC specimens, but the relationship to FANC pathway activation and CDDP sensitivity has not been reported. In the present study, 10 HNSCC cell lines were examined for expression of nine genes involved in the FANC-BRCA pathway by RT-PCR: FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, BRCA1 and BRCA2. FANC pathway function was evaluated by western blotting for FANCD2 mono-ubiquitination. All of the cell lines were also analyzed for variation in CDDP cytotoxicity. While significant differences were found in CDDP cytotoxicity, Fanconi pathway defects are an infrequent cause, as no evidence of transcriptional down-regulation of FANCF or other FANC mRNAs, or functional FANC-BRCA pathway defects were observed. These findings suggest that the variation in CDDP sensitivity of many HNSCCs is most frequently due to factors other than FANC-BRCA pathway inactivation.

  1. Identification of Key Factors Involved in the Biosorption of Patulin by Inactivated Lactic Acid Bacteria (LAB Cells.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available The purpose of this study was to identify the key factors involved in patulin adsorption by heat-inactivated lactic acid bacteria (LAB cells. For preventing bacterial contamination, a sterilization process was involved in the adsorption process. The effects of various physical, chemical, and enzymatic pre-treatments, simultaneous treatments, and post-treatments on the patulin adsorption performances of six LAB strains were evaluated. The pre-treated cells were characterized by scanning electron microscopy (SEM. Results showed that the removal of patulin by viable cells was mainly based on adsorption or degradation, depending on the specific strain. The adsorption abilities were widely increased by NaOH and esterification pre-treatments, and reduced by trypsin, lipase, iodate, and periodate pre-treatments. Additionally, the adsorption abilities were almost maintained at pH 2.2-4.0, and enhanced significantly at pH 4.0-6.0. The effects of sodium and magnesium ions on the adsorption abilities at pH 4 were slight and strain-specific. A lower proportion of patulin was released from the strain with higher adsorption ability. Analyses revealed that the physical structure of peptidoglycan was not a principal factor. Vicinal OH and carboxyl groups were not involved in patulin adsorption, while alkaline amino acids, thiol and ester compounds were important for patulin adsorption. Additionally, besides hydrophobic interaction, electrostatic interaction also participated in patulin adsorption, which was enhanced with the increase in pH (4.0-6.0.

  2. Identification of Key Factors Involved in the Biosorption of Patulin by Inactivated Lactic Acid Bacteria (LAB) Cells.

    Science.gov (United States)

    Wang, Ling; Wang, Zhouli; Yuan, Yahong; Cai, Rui; Niu, Chen; Yue, Tianli

    2015-01-01

    The purpose of this study was to identify the key factors involved in patulin adsorption by heat-inactivated lactic acid bacteria (LAB) cells. For preventing bacterial contamination, a sterilization process was involved in the adsorption process. The effects of various physical, chemical, and enzymatic pre-treatments, simultaneous treatments, and post-treatments on the patulin adsorption performances of six LAB strains were evaluated. The pre-treated cells were characterized by scanning electron microscopy (SEM). Results showed that the removal of patulin by viable cells was mainly based on adsorption or degradation, depending on the specific strain. The adsorption abilities were widely increased by NaOH and esterification pre-treatments, and reduced by trypsin, lipase, iodate, and periodate pre-treatments. Additionally, the adsorption abilities were almost maintained at pH 2.2-4.0, and enhanced significantly at pH 4.0-6.0. The effects of sodium and magnesium ions on the adsorption abilities at pH 4 were slight and strain-specific. A lower proportion of patulin was released from the strain with higher adsorption ability. Analyses revealed that the physical structure of peptidoglycan was not a principal factor. Vicinal OH and carboxyl groups were not involved in patulin adsorption, while alkaline amino acids, thiol and ester compounds were important for patulin adsorption. Additionally, besides hydrophobic interaction, electrostatic interaction also participated in patulin adsorption, which was enhanced with the increase in pH (4.0-6.0).

  3. Free radical inactivation of trypsin

    International Nuclear Information System (INIS)

    Cudina, Ivana; Jovanovic, S.V.

    1988-01-01

    Reactivities of free radical oxidants, radical OH, Br2-anion radical and Cl 3 COO radical and a reductant, CO2-anion radical, with trypsin and reactive protein components were determined by pulse radiolysis of aqueous solutions at pH 7, 20 0 C. Highly reactive free radicals, radical OH, Br2-anion radical and CO2-anion radical, react with trypsin at diffusion controlled rates. Moderately reactive trichloroperoxy radical, k(Cl 3 COO radical + trypsin) preferentially oxidizes histidine residues. The efficiency of inactivation of trypsin by free radicals is inversely proportional to their reactivity. The yields of inactivation of trypsin by radical OH, Br2-anion radical and CO2-anion radical are low, G(inactivation) = 0.6-0.8, which corresponds to ∼ 10% of the initially produced radicals. In contrast, Cl 3 COO radical inactivates trypsin with ∼ 50% efficiency, i.e. G(inactivation) = 3.2. (author)

  4. Responses of rat R-1 cells to low dose rate gamma radiation and multiple daily dose fractions

    International Nuclear Information System (INIS)

    Kal, H.B.; Bijman, J.Th.

    1981-01-01

    Multifraction irradiation may offer the same therapeutic gain as continuous irradiation. Therefore, a comparison of the efficacy of low dose rate irradiation and multifraction irradiation was the main objective of the experiments to be described. Both regimens were tested on rat rhabdomyosarcoma (R-1) cells in vitro and in vivo. Exponentially growing R-1 cells were treated in vitro by a multifraction irradiation procedure with dose fractions of 2 Gy gamma radiation and time intervals of 1 to 3 h. The dose rate was 1.3 Gy.min -1 . The results indicate that multifractionation of the total dose is more effective with respect to cell inactivation than continuous irradiation. (Auth.)

  5. Comparison of glycerolisation with cryopreservation methods on HIV-1 inactivation

    International Nuclear Information System (INIS)

    Van Baare, J.; Pagnon, J.; Cameron, P.; Vardaxis, N.; Middlekoop, E.; Crowe, S.

    1999-01-01

    Cryopreservation and glycerolisation are two successful long-term preservation methods for human cadaveric donor skin, which is used in the treatment of bum patients. High concentrations of glycerol has been shown to be antibacterial and virucidal. Because fear of possible transmission of HIV-1 following allograft transplantation, this study was undertaken to investigate whether HIV can be effectively eliminated from skin explants. HIV-1 Ba-L, which has been shown to infect monocytes in skin explants and also dendritic cells, was. For the experiments we used cell-free virus, exogenously HIV infected peripheral blood mononuclear cells (PBMCs) and exogenously HIV infected cadaver split skin. Different concentrations of glycerol at various temperatures and the glycerolisation procedure as used by the Euro Skin Bank were used to determine the effects on HIV-1 Ba-L infectivity. For the cryopreservation technique we used 10% DMSO and a controlled rate freezer. HIV-1 Ba-L transfer was determined by adding uninfected PBMCs to the infected material and reverse transcriptase was measured. Cell-free HIV-1 Ba-L was not inactivated by 50% glycerol but was effectively inactivated within 30 minutes by 70% and 85% glycerol at 4 degree C, room temperature and 37 degree C. In contrast, cell-free HIV-1 Ba-L was not inactivated by cryopreservation. Most importantly, we have shown that HIV-1 Ba-L present in split skin is inactivated by incubating skin in 70% glycerol for three hours at 37-C. HIV in exogenously infected skin was not inactivated by cryopreservation. High concentrations of glycerol effectively inactivates free HIV-1 Ba-L and intracellular HIV-1 Ba-L. Also the current glycerolisation procedure carried out by the Euro Skin Bank effectively inactivates infectious virus. However, the cryopreservation technique did not show any reduction in HIV-1 Ba-L infectivity

  6. Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

    Energy Technology Data Exchange (ETDEWEB)

    Takagi, N. (Hokkaido Univ., Sapporo (Japan))

    1988-04-01

    By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method the authors studied reactivation of the inactivated X chromosome (X{sub i}) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. Synchronization of the late replicating S chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the X{sub i}, which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using ({sup 3}H)thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the X{sub i}. Most probably reactivation of the X{sub i} is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of X{sub i}-specific factors by mitoses may be involved in this process.

  7. Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

    International Nuclear Information System (INIS)

    Takagi, N.

    1988-01-01

    By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method the authors studied reactivation of the inactivated X chromosome (X i ) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. Synchronization of the late replicating S chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the X i , which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using [ 3 H]thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the X i . Most probably reactivation of the X i is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of X i -specific factors by mitoses may be involved in this process

  8. THE CYTOKINES SYNTHESIS IN VITRO IN THE TICK-BORNE ENCEPHALITIS VIRUS INFECTED CELLS AND IN THE PRESENCE OF INACTIVATED VACCINE

    Directory of Open Access Journals (Sweden)

    M. V. Mesentseva

    2014-01-01

    Full Text Available Abstract. Tick-borne encephalitis (TBE is severe neuroinfectious disease with involvement of immune mechanisms in pathogenesis. Comparative analysis of synthesis of key cytokines had been performed for the TBE virus (TBEV infected cells and in the presence of inactivated vaccine against TBE in vitro. Persistent TBEV infection of immortal tissue culture of human larynx cancer cells caused transcription activation of interferons IFNα, IFNγ, IFNλ1, interleukins IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12, tumour necrosis factor TNFα as well as one of apoptosis factors Fas. Comparison of transcription and production of cytokines revealed that the TBEV infection resulted in posttranscription Th1 shift of cytokine response. In the presence of inactivated vaccine against TBE based on the same strain Sofjin of the TBEV activation of transcription of cytokines IFNα, IFNλ1, IL-4, IL-10 was also observed as after the TBEV infection that together with an additional stimulation of GM-CSF production might serve as an evidence of Th2 response. Involvement of IFNIII type (IFNλ1 both during persistent infection and after addition of inactivated vaccines was found in the first time. Differences in dynamics of cytokines IL-2, IL-8, IL-10, IL-12, TNFα response during the TBEV infection and in the presence of inactivated vaccine are described.

  9. Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

    Science.gov (United States)

    Bauer, Georg; Zarkovic, Neven

    2015-04-01

    Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Tracing the spatiotemporally resolved inactivation of optically arranged bacteria by photofunctional microparticles at the single-cell level (Conference Presentation)

    Science.gov (United States)

    Barroso Peña, Alvaro; Grüner, Malte; Forbes, Taylor; Denz, Cornelia; Strassert, Cristian A.

    2016-09-01

    Antimicrobial Photodynamic Inactivation (PDI) represents an attractive alternative in the treatment of infections by antibiotic-resistant pathogenic bacteria. In PDI a photosensitizer (PS) is administered to the site of the biological target in order to generate cytotoxic singlet oxygen which reacts with the biological membrane upon application of harmless visible light. Established methods for testing the photoinduced cytotoxicity of PSs rely on the observation of the whole bacterial ensemble providing only a population-averaged information about the overall produced toxicity. However, for a deeper understanding of the processes that take place in PDI, new methods are required that provide simultaneous regulation of the ROS production, monitoring the subsequent damage induced in the bacteria cells, and full control of the distance between the bacteria and the center of the singlet oxygen production. Herein we present a novel method that enables the quantitative spatio-time-resolved analysis at the single cell level of the photoinduced damage produced by transparent microspheres functionalized with PSs. For this purpose, a methodology was introduced to monitor phototriggered changes with spatiotemporal resolution employing holographic optical tweezers and functional fluorescence microscopy. The defined distance between the photoactive particles and individual bacteria can be fixed under the microscope before the photosensitization process, and the photoinduced damage is monitored by tracing the fluorescence turn-on of a suitable marker. Our methodology constitutes a new tool for the in vitro design and analysis of photosensitizers, as it enables a quantitative response evaluation of living systems towards oxidative stress.

  11. Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

    Science.gov (United States)

    Molenaar, Jan J.; Ebus, Marli E.; Geerts, Dirk; Koster, Jan; Lamers, Fieke; Valentijn, Linda J.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.

    2009-01-01

    Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics. PMID:19525400

  12. Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Shao-Qing Kuang

    Full Text Available The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL. We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2'-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.

  13. Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Kuang, Shao-Qing; Fang, Zhihong; Zweidler-McKay, Patrick A; Yang, Hui; Wei, Yue; Gonzalez-Cervantes, Emilio A; Boumber, Yanis; Garcia-Manero, Guillermo

    2013-01-01

    The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA)/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL). We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2'-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.

  14. Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

    Science.gov (United States)

    Shao, Genbao; Lai, Wensheng; Wan, Xiaolei; Xue, Jing; Wei, Ye; Jin, Jie; Zhang, Liuping; Lin, Qiong; Shao, Qixiang; Zou, Shengqiang

    2017-05-01

    Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclin D1. Significantly, blockage of epidermal growth factor receptor (EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

  15. Pathogen inactivation by riboflavin and ultraviolet light illumination accelerates the red blood cell storage lesion and promotes eryptosis.

    Science.gov (United States)

    Qadri, Syed M; Chen, Deborah; Schubert, Peter; Perruzza, Darian L; Bhakta, Varsha; Devine, Dana V; Sheffield, William P

    2017-03-01

    Pathogen reduction treatment using riboflavin and ultraviolet light illumination (Mirasol) effectively reduces the risk of transfusion-transmitted infections. This treatment is currently licensed for only platelets and plasma products, while its application to whole blood (WB) to generate pathogen-inactivated red blood cells (RBCs) is under development. RBC storage lesion, constituting numerous morphologic and biochemical changes, influences RBC quality and limits shelf life. Stored RBCs further show enhanced susceptibility to RBC programmed cell death (eryptosis) characterized by increased cytosolic Ca 2+ -provoked membrane phosphatidylserine (PS) externalization. Using a "pool-and-split" approach, we examined multiple variables of RBC storage lesion and eryptosis in RBC units, derived from Mirasol-treated or untreated WB, after 4 to 42 days of storage, under blood bank conditions. In comparison to untreated RBC units, Mirasol treatment significantly altered membrane microvesiculation, supernatant hemoglobin, osmotic fragility, and intracellular adenosine triphosphate levels but did not influence membrane CD47 expression and 2,3-diphosphoglycerate levels. Mirasol-treated RBCs showed significantly higher PS exposure after 42, but not after not more than 21, days of storage, which was accompanied by enhanced cytosolic Ca 2+ activity, ceramide abundance, and oxidative stress, but not p38 kinase activation. Mirasol treatment significantly augmented PS exposure, Ca 2+ entry, and protein kinase C activation after energy depletion, a pathophysiologic cell stressor. Mirasol-treated RBCs were, however, more resistant to cell shrinkage. Prolonged storage of Mirasol-treated RBCs significantly increases the proportion of eryptotic RBCs, while even short-term storage enhances the susceptibility of RBCs to stress-induced eryptosis, which could reduce posttransfusion RBC recovery in patients. © 2016 AABB.

  16. Inactivation of HTB63 human melanoma cells by irradiation with protons and gamma rays.

    Science.gov (United States)

    Ristic-Fira, Aleksandra; Petrovic, Ivan; Todorovic, Danijela; Koricanac, Lela; Vujèic, Miroslava; Demajo, Miroslav; Sabini, Gabriella; Cirrone, Pablo; Cuttone, Giacomo

    2004-12-01

    The effects of single irradiation with gamma rays and protons on HTB63 human melanoma cell growth were compared. The exponentially growing cells were irradiated with gamma rays or protons using doses ranging from 2-20 Gy. At 48 h of post-irradiation incubation under standard conditions, cell survival and induction of apoptotic cell death were examined. The best effect of the single irradiation with gamma rays was the reduction of cell growth by up to 26% (p=0.048, irradiation vs. control), obtained using the dose of 16 Gy. The same doses of proton irradiation, having energy at the target of 22.6 MeV, significantly inhibited melanoma cell growth. Doses of 12 and 16 Gy of protons provoked growth inhibition of 48.9% (p=0.003, irradiation vs. control) and 51.2% (p=0.012, irradiation vs. control) respectively. Irradiation with 12 and 16 Gy protons, compared to the effects of the same doses of gamma rays, significantly reduced melanoma cell growth (p=0.015 and p=0.028, protons vs. gamma rays, respectively). Estimated RBEs for growth inhibition of HTB63 cells ranged from 1.02 to 1.45. The electrophoretical analyses of DNA samples and flow cytometric evaluation have shown a low percentage of apoptotic cells after both types of irradiation. The better inhibitory effect achieved by protons in contrast to gamma rays, can be explained considering specific physical properties of protons, especially taking into account the highly localized energy deposition (high LET).

  17. Epigenetic inactivation of SPINT2 is associated with tumor suppressive function in esophageal squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Dongli [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); The Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Fan, Qingxia [The Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Chen, Xinfeng; Li, Feng [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Wang, Liping [The Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Huang, Lan [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Dong, Wenjie; Chen, Xiaoqi [The Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Zhang, Zhen [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Liu, Jinyan; Wang, Fei [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); The School of Life Sciences, Zhengzhou University, Zhengzhou 450052, Henan (China); Wang, Meng [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); The Department of Gastroenterology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); Zhang, Bin [The Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan (China); The Department of Hematology/Oncology, School of Medicine, Northwestern University, Chicago 60611 (United States); and others

    2014-03-10

    Hepatocyte growth factor activator inhibitor type 2 (SPINT2), a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor suppressor gene silenced by promoter methylation. We aimed to investigate whether SPINT2 might act as an esophageal squamous cell carcinoma (ESCC) tumor suppressor gene. Four ESCC cell lines, Fifty-two ESCC tissues and twenty-nine neighboring non-cancerous tissues were included in this study. The expression of SPINT2 was monitored by real time PCR. Bisulfite genomic sequencing and methylation-specific PCR were used to analyze methylation status. The effect of SPINT2 on cell proliferation and apoptosis in EC109 and EC9706 cells was observed by CCK-8 assay and flow cytometric analysis. We found that silencing of SPINT2 was associated with promoter methylation in ESCC cell lines. The densely methylated SPINT2 promoter region was confirmed by bisulfite genomic sequencing. Ectopic expression of SPINT2 inhibited cell proliferation through inducing cell apoptosis in vitro. Furthermore, methylation-specific PCR analysis revealed that SPINT2 promoter methylation was prominent in carcinoma tissues (52.08%) compared with neighboring non-cancerous tissues (22.58%). Kaplan–Meier analysis showed that patients with SPINT2 hypermethylation had shorter survival time. The tumor suppressor gene of SPINT2 is commonly silenced by promoter hypermethylation in human ESCC and SPINT2 hypermethylation is correlated with poor overall survival, implicating SPINT2 is an underlying prognostic marker for human ESCC. - Highlights: • We firstly found SPINT2 gene may be transcriptionally repressed by promoter hypermethylation in ESCC cells. • SPINT2 overexpressing cells induced proliferation inhibition through promoting apoptosis. • mRNA expression of SPINT2 was significantly higher in ESCC tissues than in neighboring non-cancerous tissues. • Promoter hypermethylation of SPINT2 is significantly linked to TNM stage and poor overall survival.

  18. Chromosomal Aberrations in Normal and AT Cells Exposed to High Dose of Low Dose Rate Irradiation

    Science.gov (United States)

    Kawata, T.; Shigematsu, N.; Kawaguchi, O.; Liu, C.; Furusawa, Y.; Hirayama, R.; George, K.; Cucinotta, F.

    2011-01-01

    Ataxia telangiectasia (A-T) is a human autosomally recessive syndrome characterized by cerebellar ataxia, telangiectases, immune dysfunction, and genomic instability, and high rate of cancer incidence. A-T cell lines are abnormally sensitive to agents that induce DNA double strand breaks, including ionizing radiation. The diverse clinical features in individuals affected by A-T and the complex cellular phenotypes are all linked to the functional inactivation of a single gene (AT mutated). It is well known that cells deficient in ATM show increased yields of both simple and complex chromosomal aberrations after high-dose-rate irradiation, but, less is known on how cells respond to low-dose-rate irradiation. It has been shown that AT cells contain a large number of unrejoined breaks after both low-dose-rate irradiation and high-dose-rate irradiation, however sensitivity for chromosomal aberrations at low-dose-rate are less often studied. To study how AT cells respond to low-dose-rate irradiation, we exposed confluent normal and AT fibroblast cells to up to 3 Gy of gamma-irradiation at a dose rate of 0.5 Gy/day and analyzed chromosomal aberrations in G0 using fusion PCC (Premature Chromosomal Condensation) technique. Giemsa staining showed that 1 Gy induces around 0.36 unrejoined fragments per cell in normal cells and around 1.35 fragments in AT cells, whereas 3Gy induces around 0.65 fragments in normal cells and around 3.3 fragments in AT cells. This result indicates that AT cells can rejoin breaks less effectively in G0 phase of the cell cycle? compared to normal cells. We also analyzed chromosomal exchanges in normal and AT cells after exposure to 3 Gy of low-dose-rate rays using a combination of G0 PCC and FISH techniques. Misrejoining was detected in the AT cells only? When cells irradiated with 3 Gy were subcultured and G2 chromosomal aberrations were analyzed using calyculin-A induced PCC technique, the yield of unrejoined breaks decreased in both normal and AT

  19. Cell inactivation by ultrasoft X-rays: Role of inner shell ionization

    International Nuclear Information System (INIS)

    Abel, F.; du Penhoat, H.; Despiney-Bailly, I.

    1997-01-01

    The biological effect of K-electron removal in C, N, O atoms of the DNA needs to be systematically studied. A calculation of the RBE of X-rays around K-edges was performed to test experimental feasibility. Results obtained motivated an experimental study of the lethal effectiveness of C-K ionizations and experiments were performed with monochronized synchrotron radiation. Cell survival was studied with a V79 chinese hamster cell line, using cells grown as monolayers on mylar foil. The linear quadratic shape survival curves obtained for 250 eV and 340 eV radiation are discussed. (author)

  20. Dose rate effect on low-dose hyper-radiosensitivity with cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Geon-Min; Kim, Eun-Hee [Seoul National University, Seoul (Korea, Republic of)

    2016-10-15

    Low-dose hyper-radiosensitivity (HRS) is the phenomenon that mammalian cells exhibit higher sensitivity to radiation at low doses (< 0.5 Gy) than expected by the linear-quadratic model. At doses above 0.5Gy, the cellular response is recovered to the level expected by the linear-quadratic model. This transition is called the increased radio-resistance (IRR). HRS was first verified using Chinese hamster V79 cells in vitro by Marples and has been confirmed in studies with other cell lines including human normal and tumor cells. HRS is known to be induced by inactivation of ataxia telangiectasia-mutated (ATM), which plays a key role in repairing DNA damages. Considering the connection between ATM and HRS, one can infer that dose rate may affect cellular response regarding HRS at low doses. In this study, we quantitated the effect of dose rate on HRS by clonogenic assay with normal and tumor cells. The HRS of cells at low dose exposures is a phenomenon already known. In this study, we observed HRS of rat normal diencephalon cells and rat gliosarcoma cells at doses below 1 Gy. In addition, we found that dose rate mattered. HRS occurred at low doses, but only when total dose was delivered at a rate below certain level.

  1. Biphasic voltage-dependent inactivation of human NaV 1.3, 1.6 and 1.7 Na+ channels expressed in rodent insulin-secreting cells.

    Science.gov (United States)

    Godazgar, Mahdieh; Zhang, Quan; Chibalina, Margarita V; Rorsman, Patrik

    2018-05-01

    Na + current inactivation is biphasic in insulin-secreting cells, proceeding with two voltage dependences that are half-maximal at ∼-100 mV and -60 mV. Inactivation of voltage-gated Na + (Na V ) channels occurs at ∼30 mV more negative voltages in insulin-secreting Ins1 and primary β-cells than in HEK, CHO or glucagon-secreting αTC1-6 cells. The difference in inactivation between Ins1 and non-β-cells persists in the inside-out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary β-cells, but not in HEK cells, inactivation of a single Na V subtype is biphasic and follows two voltage dependences separated by 30-40 mV. We propose that Na V channels adopt different inactivation behaviours depending on the local membrane environment. Pancreatic β-cells are equipped with voltage-gated Na + channels that undergo biphasic voltage-dependent steady-state inactivation. A small Na + current component (10-15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na + channel component is completely inactivated at -90 to -80 mV and is therefore inactive in the β-cell. It has been proposed that the biphasic inactivation reflects the contribution of different Na V α-subunits. We tested this possibility by expression of TTX-resistant variants of the Na V subunits found in β-cells (Na V 1.3, Na V 1.6 and Na V 1.7) in insulin-secreting Ins1 cells and in non-β-cells (including HEK and CHO cells). We found that all Na V subunits inactivated at 20-30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. Na V 1.7 inactivated at 15--20 mV more negative membrane potentials than Na V 1.3 and Na V 1.6 in Ins1 cells but this small difference is insufficient to solely

  2. Chronic action of gamma-radiation on growing cell population of the yeast Saccharomyces cerevisiae at various dose rates

    International Nuclear Information System (INIS)

    Zyuzikov, N.A.; Petin, V.G.

    1996-01-01

    Experimental data on the processes of division and death of haploid and diploid yeast Saccharomyces cerevisiae of wild type and of their radiosensitive mutants exposed under optimal for reproduction conditions to chronic gamma-radiation at various dose rates are presented. It is shown that the dependence of the integral division/death process in time was exponential for all the studied strains. With dose rate increasing, the duration of the lag period and the probability of cell inactivation increased, while the multiplication rate decreased. These processes, for equal dose rates, were more expressed for the radiosensitive mutants

  3. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Celine J. Granier

    2014-08-01

    Full Text Available PDCD2 (programmed cell death domain 2 is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs and embryonic fibroblasts (MEFs. We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse.

  4. Photoreactivation of cells and phages inactivated by UV of ecological wave-lengths

    International Nuclear Information System (INIS)

    Samojlova, K.A.; Yanovska, Eh.; Vizdalova, M.; Ceskoslovenska Akademie Ved, Brno. Biofysikalni Ustav)

    1979-01-01

    It has been found that the photoreactivity of infusoria Paramecium caudatum and bacteria Escherichia coli is high and practically similar if they are irradiated with short-wave (254 nm) and mean-wave (300-315 nm) UV radiation. The cells damaged with long-wave (315-400 nm) UV rays are not photoactivated. The latter is caused by the appearance of nonphotoreactivated damages since the phages jrradiated with the same UV rays are reactivated extremely weakly in the intact cells of bacteria (phage T7) or are not reactivated at all (phage lambdasub(c1 857))

  5. Systems biology modeling reveals a possible mechanism of the tumor cell death upon oncogene inactivation in EGFR addicted cancers.

    Directory of Open Access Journals (Sweden)

    Jian-Ping Zhou

    Full Text Available Despite many evidences supporting the concept of "oncogene addiction" and many hypotheses rationalizing it, there is still a lack of detailed understanding to the precise molecular mechanism underlying oncogene addiction. In this account, we developed a mathematic model of epidermal growth factor receptor (EGFR associated signaling network, which involves EGFR-driving proliferation/pro-survival signaling pathways Ras/extracellular-signal-regulated kinase (ERK and phosphoinositol-3 kinase (PI3K/AKT, and pro-apoptotic signaling pathway apoptosis signal-regulating kinase 1 (ASK1/p38. In the setting of sustained EGFR activation, the simulation results show a persistent high level of proliferation/pro-survival effectors phospho-ERK and phospho-AKT, and a basal level of pro-apoptotic effector phospho-p38. The potential of p38 activation (apoptotic potential due to the elevated level of reactive oxygen species (ROS is largely suppressed by the negative crosstalk between PI3K/AKT and ASK1/p38 pathways. Upon acute EGFR inactivation, the survival signals decay rapidly, followed by a fast increase of the apoptotic signal due to the release of apoptotic potential. Overall, our systems biology modeling together with experimental validations reveals that inhibition of survival signals and concomitant release of apoptotic potential jointly contribute to the tumor cell death following the inhibition of addicted oncogene in EGFR addicted cancers.

  6. Oxygen-Glucose Deprivation Induces G2/M Cell Cycle Arrest in Brain Pericytes Associated with ERK Inactivation.

    Science.gov (United States)

    Wei, Wenjie; Yu, Zhiyuan; Xie, Minjie; Wang, Wei; Luo, Xiang

    2017-01-01

    Growing evidence has revealed that brain pericytes are multifunctional and contribute to the pathogenesis of a number of neurological disorders. However, the role of pericytes in cerebral ischemia, and especially the pathophysiological alterations in pericytes, remains unclear. In the present study, our aim was to determine whether the proliferation of pericytes is affected by cerebral ischemia and, if so, to identify the underlying mechanism(s). Cultured brain pericytes subjected to oxygen-glucose deprivation (OGD) were used as our model of cerebral ischemia; the protein expression levels of cyclin D1, cyclin E, cdk4, and cyclin B1 were determined by Western blot analysis, and cell cycle analysis was assessed by flow cytometry. The OGD treatment reduced the brain pericyte proliferation by causing G2/M phase arrest and downregulating the protein levels of cyclin D1, cyclin E, cdk4, and cyclin B1. Further studies demonstrated a simultaneous decrease in the activity of extracellular regulated protein kinases (ERK), suggesting a critical role of the ERK signaling cascade in the inhibition of OGD-induced pericyte proliferation. We suggest that OGD inhibition of the proliferation of brain pericytes is associated with the inactivation of the ERK signaling pathway, which arrests them in the G2/M phase.

  7. Inactivation of Escherichia coli in broth and sausage by combined high pressure and Lactobacillus casei cell extract.

    Science.gov (United States)

    Chung, Hyun-Jung; Yousef, Ahmed E

    2010-10-01

    The purpose of this study was to investigate the effect of combined high pressure and Lactobacillus casei cell extract (CE) on Escherichia coli O157 strains with variation in pressure resistance in broth and sausage. Pressure-resistant (O157:H7 and O157:H12) and -sensitive (O157-M1 and O157-M2) E. coli strains were used. Pressure treatment at 350 MPa for 20 min in broth caused 1.1-1.2 logs reduction in O157:H12 and O157:H7 and 4.1-5.5 logs reduction in the O157-M1 and O157-M2. When high pressure was treated in the presence of CE (32 CEAU/mL), the combination treatment caused a significant inactivation in the pressure-resistant O157:H7 strains resulting in the viability loss of 4.3-4.6 logs and the synergistic effect increased with increase in treatment time (p casei CE may cause considerable damage to cellular components of E. coli during the high pressure treatment. The synergy between high pressure processing and Lb. casei OSY-LB6A CE against pressure-resistant E. coli O157 strains suggests the feasibility of using this combination to minimize the risk of transmission of E. coli O157 by food.

  8. Jolkinolide B inhibits glycolysis by downregulating hexokinase 2 expression through inactivating the Akt/mTOR pathway in non-small cell lung cancer cells.

    Science.gov (United States)

    Gao, Xiang; Han, Han

    2018-06-01

    Jolkinolide B (JB), a bioactive compound isolated from herbal medicine, has been found to inhibit tumor growth by altering glycolysis. However, whether glycolysis is influenced by JB in non-small cell lung cancer (NSCLC) cells and the mechanism remain unknown. The aim of the present study was to evaluate the effect of JB on the glycolysis in NSCLC cells and the underlying molecular mechanism. The results showed that JB treatment inhibited cell viability of A549 and H1299 cells in a concentration-dependent manner. JB reduced the glucose consumption, lactate production, and HK2 expression. The expressions of p-Akt and p-mTOR were also decreased by JB treatment. Knockdown of HK2 reduced glucose consumption and lactate production. Inhibition of the Akt/mTOR pathway decreased HK2 expression and inhibited glycolysis. In conclusion, the results indicated that JB inhibits glycolysis by down-regulating HK2 expression through inactivating the Akt/mTOR pathway in NSCLC cells, suggesting that JB might be a potential therapeutic agent for the treatment of NSCLC. © 2018 Wiley Periodicals, Inc.

  9. A New Adjuvant Combined with Inactivated Influenza Enhances Specific CD8 T Cell Response in Mice and Decreases Symptoms in Swine Upon Challenge.

    Science.gov (United States)

    Bouguyon, Edwige; Goncalves, Elodie; Shevtsov, Alexander; Maisonnasse, Pauline; Remyga, Stepan; Goryushev, Oleg; Deville, Sebastien; Bertho, Nicolas; Ben Arous, Juliette

    2015-11-01

    Vaccination is the most effective way to control swine influenza virus (SIV) in the field. Classical vaccines are based on inactivated antigens formulated with an oil emulsion or a polymeric adjuvant. Standard adjuvants enhance the humoral response and orient the immune response toward a Th2 response. An important issue is that current vaccines do not protect against new strains. One approach to improve cross-protection is to enhance Th1 and cytotoxic responses. The development of adjuvants orienting the immune response of inactivated vaccines toward Th1/Cytotoxic responses would be highly beneficial. This study shows that the water in oil in water emulsion adjuvant Montanide™ ISA 201 VG allows the induction of anti-influenza CD8 T cell in mice and induces homologous protection against an H1N1 challenge in swine. Such adjuvants that induce both humoral and cell-mediated immunity could improve the protection conferred by SIV vaccines in the field.

  10. Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/- mice by deregulation of Gap43-dependent granule cell precursor migration.

    Directory of Open Access Journals (Sweden)

    Daniel Haag

    Full Text Available Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/- mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/- mice with mice lacking inducible nitric oxide synthase (Nos2 to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/- Nos2(-/- mice compared to Ptch1(+/- Nos2(+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+ Nos2(-/- mice but not from Ptch1(+/- Nos2(-/- mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43. Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+ Nos2(-/- mice but increased in Ptch1(+/- Nos2(-/ (- mice relative to Ptch1(+/- Nos2(+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/- mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during

  11. Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation.

    Science.gov (United States)

    Hayes, Tyler F; Benaich, Nathan; Goldie, Stephen J; Sipilä, Kalle; Ames-Draycott, Ashley; Cai, Wenjun; Yin, Guangliang; Watt, Fiona M

    2016-12-01

    Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  12. Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade.

    Science.gov (United States)

    Gu, Yi-Feng; Cohn, Shannon; Christie, Alana; McKenzie, Tiffani; Wolff, Nicholas; Do, Quyen N; Madhuranthakam, Ananth J; Pedrosa, Ivan; Wang, Tao; Dey, Anwesha; Busslinger, Meinrad; Xie, Xian-Jin; Hammer, Robert E; McKay, Renée M; Kapur, Payal; Brugarolas, James

    2017-08-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by BAP1 and PBRM1 mutations, which are associated with tumors of different grade and prognosis. However, whether BAP1 and PBRM1 loss causes ccRCC and determines tumor grade is unclear. We conditionally targeted Bap1 and Pbrm1 (with Vhl ) in the mouse using several Cre drivers. Sglt2 and Villin proximal convoluted tubule drivers failed to cause tumorigenesis, challenging the conventional notion of ccRCC origins. In contrast, targeting with PAX8, a transcription factor frequently overexpressed in ccRCC, led to ccRCC of different grades. Bap1 -deficient tumors were of high grade and showed greater mTORC1 activation than Pbrm1 -deficient tumors, which exhibited longer latency. Disrupting one allele of the mTORC1 negative regulator, Tsc1 , in Pbrm1 -deficient kidneys triggered higher grade ccRCC. This study establishes Bap1 and Pbrm1 as lineage-specific drivers of ccRCC and histologic grade, implicates mTORC1 as a tumor grade rheostat, and suggests that ccRCCs arise from Bowman capsule cells. Significance: Determinants of tumor grade and aggressiveness across cancer types are poorly understood. Using ccRCC as a model, we show that Bap1 and Pbrm1 loss drives tumor grade. Furthermore, we show that the conversion from low grade to high grade can be promoted by activation of mTORC1. Cancer Discov; 7(8); 900-17. ©2017 AACR. See related commentary by Leung and Kim, p. 802 This article is highlighted in the In This Issue feature, p. 783 . ©2017 American Association for Cancer Research.

  13. Heat Inactivation of Garlic (Allium sativum) Extract Abrogates Growth Inhibition of HeLa Cells.

    Science.gov (United States)

    Chintapalli, Renuka; Murray, Matthew J J; Murray, James T

    2016-07-01

    The potential anticancer properties of garlic (Allium sativum) may depend on the method of preparation and its storage. Storage of garlic has not been thoroughly investigated to determine whether anticancer properties are retained. Garlic was prepared and processed to mimic normal options for storage and preparation for consumption. Cytotoxicity was determined by crystal violet assay and mechanisms of cytotoxicity were established by microscopy, SDS-PAGE, and Western immunoblotting. Significant (P < 0.0001) cytotoxicity was observed in all preparations, except with boiled (cooked) garlic. Depending on the method of storage, garlic extract induced either type I or type II programmed cell death, detectable by caspase 9 cleavage, or Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage and LC3-II accumulation, respectively. The conflicting literature on the anticancer properties of garlic may be explained by differences in processing and storage. This study has highlighted that the potency of the antiproliferative properties of cooked garlic, compared to the uncooked form, is diminished in HeLa cells.

  14. TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

    International Nuclear Information System (INIS)

    Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

    2006-01-01

    Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

  15. A cell-death-defying factor, anamorsin mediates cell growth through inactivation of PKC and p38MAPK

    International Nuclear Information System (INIS)

    Saito, Yuri; Shibayama, Hirohiko; Tanaka, Hirokazu; Tanimura, Akira; Kanakura, Yuzuru

    2011-01-01

    Research highlights: → Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. → Biological mechanisms of AM functions have not been elucidated yet. → PKCθ , PKCδ and p38MAPK were more phosphorylated in AM deficient MEF cells. → AM may negatively regulates PKCs and p38MAPK in MEF cells. -- Abstract: Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.

  16. Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice

    Directory of Open Access Journals (Sweden)

    Yi Liu

    2017-04-01

    Full Text Available Summary: Natural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC function. Latexin (Lxn is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1 was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit. : In this article, Liang and colleagues show that loss of latexin in vivo expands the HSC population and increases their survival and engraftment. Latexin regulates HSC function and hematopoiesis via the Thbs1 signaling pathway. Keywords: latexin, hematopoietic stem cell, repopulating advantage, expansion, survival, thrombospondin 1

  17. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    International Nuclear Information System (INIS)

    Karentz, D.; Cleaver, J.E.

    1986-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia

  18. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...

  19. Direct and indirect inactivation of tumor cell protective catalase by salicylic acid and anthocyanidins reactivates intercellular ROS signaling and allows for synergistic effects.

    Science.gov (United States)

    Scheit, Katrin; Bauer, Georg

    2015-03-01

    Salicylic acid and anthocyanidins are known as plant-derived antioxidants, but also can provoke paradoxically seeming prooxidant effects in vitro. These prooxidant effects are connected to the potential of salicylic acid and anthocyanidins to induce apoptosis selectively in tumor cells in vitro and to inhibit tumor growth in animal models. Several epidemiological studies have shown that salicylic acid and its prodrug acetylsalicylic acid are tumor-preventive for humans. The mechanism of salicylic acid- and anthocyanidin-dependent antitumor effects has remained enigmatic so far. Extracellular apoptosis-inducing reactive oxygen species signaling through the NO/peroxynitrite and the HOCl signaling pathway specifically induces apoptosis in transformed cells. Tumor cells have acquired resistance against intercellular reactive oxygen species signaling through expression of membrane-associated catalase. Here, we show that salicylic acid and anthocyanidins inactivate tumor cell protective catalase and thus reactive apoptosis-inducing intercellular reactive oxygen species signaling of tumor cells and the mitochondrial pathway of apoptosis Salicylic acid inhibits catalase directly through its potential to transform compound I of catalase into the inactive compound II. In contrast, anthocyanidins provoke a complex mechanism for catalase inactivation that is initiated by anthocyanidin-mediated inhibition of NO dioxygenase. This allows the formation of extracellular singlet oxygen through the reaction between H(2)O(2) and peroxynitrite, amplification through a caspase8-dependent step and subsequent singlet oxygen-mediated inactivation of catalase. The combination of salicylic acid and anthocyanidins allows for a remarkable synergistic effect in apoptosis induction. This effect may be potentially useful to elaborate novel therapeutic approaches and crucial for the interpretation of epidemiological results related to the antitumor effects of secondary plant compounds. © The

  20. Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

    2017-12-01

    Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

  1. Inactivation of Staphylococcus aureus and Enterococcus faecalis by a direct-current, cold atmospheric-pressure air plasma microjet☆

    Science.gov (United States)

    Tian, Ye; Sun, Peng; Wu, Haiyan; Bai, Na; Wang, Ruixue; Zhu, Weidong; Zhang, Jue; Liu, Fuxiang

    2010-01-01

    Objective A direct-current, cold atmospheric-pressure air plasma microjet (PMJ) was performed to inactivate Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) in air. The process of sterilization and morphology of bacteria was observed. We wish to know the possible inactivation mechanisms of PMJ and explore a potential application in dental and other temperature sensitive treatment. Methods In this study, we employed a direct current, atmospheric pressure, cold air PMJ to inactivate bacterias. Scanning electron microscopy was employed to evaluate the morphology of S. aureus and showed rupture of cell walls after the plasma treatment and Optical emission spectrum (OES) were used to understand the possible inactivation mechanisms of PMJ. Results The inactivation rates could reach 100% in 5 min. When the distance between the exit nozzle of the PMJ device and Petri dish was extended from 1 cm to 3 cm, effective inactivation was also observed with a similar inactivation curve. Conclusion The inactivation of bacteria is attributed to the abundant reactive oxygen and nitrogen species, as well as ultroviolet radiation in the plasma. Different life spans and defensibilities of these killing agents may hold the key to understanding the different inactivation curves at different treatment distances. PMID:23554639

  2. Inactivation of Staphylococcus aureus and Enterococcus faecalis by a direct-current, cold atmospheric-pressure air plasma microjet.

    Science.gov (United States)

    Tian, Ye; Sun, Peng; Wu, Haiyan; Bai, Na; Wang, Ruixue; Zhu, Weidong; Zhang, Jue; Liu, Fuxiang

    2010-07-01

    A direct-current, cold atmospheric-pressure air plasma microjet (PMJ) was performed to inactivate Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) in air. The process of sterilization and morphology of bacteria was observed. We wish to know the possible inactivation mechanisms of PMJ and explore a potential application in dental and other temperature sensitive treatment. In this study, we employed a direct current, atmospheric pressure, cold air PMJ to inactivate bacterias. Scanning electron microscopy was employed to evaluate the morphology of S. aureus and showed rupture of cell walls after the plasma treatment and Optical emission spectrum (OES) were used to understand the possible inactivation mechanisms of PMJ. The inactivation rates could reach 100% in 5 min. When the distance between the exit nozzle of the PMJ device and Petri dish was extended from 1 cm to 3 cm, effective inactivation was also observed with a similar inactivation curve. The inactivation of bacteria is attributed to the abundant reactive oxygen and nitrogen species, as well as ultroviolet radiation in the plasma. Different life spans and defensibilities of these killing agents may hold the key to understanding the different inactivation curves at different treatment distances.

  3. Cell inactivation and chromosomal aberrations induced by X-rays and fast neutrons in cells of the Chinese hamster. 1

    International Nuclear Information System (INIS)

    Tolkendorf, E.

    1979-01-01

    Asynchronously grown cultures of Chinese hamster cells V79-4 were irradiated in suspension with 180 kV X-rays and fast neutrons (average energy of 6.2 MeV). The damage was assessed by measuring cell survival and frequencies of chromosome aberrations in the first post-irradiation metaphases. The experimental data for survival and chromosome aberrations were fitted by computer programmes. From the fitted curves the relative biological effectiveness (RBE) of fast neutrons was calculated. The RBE shows a similar dose dependence for killed and aberrant cells. The RBE decreases with increasing dose and amounts to approximately 5 for both effects for small neutron doses. The highest RBE is found for asymmetrical chromosomal exchanges and is dependent on the neutron dose, too. However, for isochromatid deletions the RBE is dose independent with a value of 3.6. (author)

  4. Effect of adrenalectomy on recipients of allogeneic lymphocytes on inactivation of endogenous colony-forming cells in mice

    International Nuclear Information System (INIS)

    Semenkov, V.F.

    1985-01-01

    This paper presents a study of the killer functions of lymph node cells directed against endogenous colony-forming cells in adrenalectomized recipients in a genetic system with one-way incompatibility: parental line - F 1 hybrid. Mice were irradiated with Co 60 gamma rays on the EGO-2 apparatus with dose rate from 200 to 250 R/min. The results were subjected to statistical analysis by Student's test. It can be tentatively suggested that the killer action of T lymphocytes on endogenous colonies was intensified in adrenal-ectomized recipients with endogenous hypocorticism, as a result of cooperation with the cortisol-sensitive subpopulation of T helper cells, of a change in the properties of the antigen-recognizing receptors, or an increase in the sensitivity of target cells to the killer action of T lymphocytes

  5. Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

    Science.gov (United States)

    Geens, M; Seriola, A; Barbé, L; Santalo, J; Veiga, A; Dée, K; Van Haute, L; Sermon, K; Spits, C

    2016-04-01

    Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture

  6. How reduction of theta rhythm by medial septum inactivation may covary with disruption of entorhinal grid cell responses due to reduced cholinergic transmission

    Directory of Open Access Journals (Sweden)

    Praveen K. Pilly

    2013-10-01

    Full Text Available Oscillations in the coordinated firing of brain neurons have been proposed to play important roles in perception, cognition, attention, learning, navigation, and sensory-motor control. The network theta rhythm has been associated with properties of spatial navigation, as has the firing of entorhinal grid cells and hippocampal place cells. Two recent studies reduced the theta rhythm by inactivating the medial septum (MS and demonstrated a correlated reduction in the characteristic hexagonal spatial firing patterns of grid cells. These results, along with properties of intrinsic membrane potential oscillations (MPOs in slice preparations of entorhinal cells, have been interpreted to support oscillatory interference models of grid cell firing. The current article shows that an alternative self-organizing map model of grid cells can explain these data about intrinsic and network oscillations without invoking oscillatory interference. In particular, the adverse effects of MS inactivation on grid cells can be understood in terms of how the concomitant reduction in cholinergic inputs may increase the conductances of leak potassium (K+ and slow and medium after-hyperpolarization (sAHP and mAHP channels. This alternative model can also explain data that are problematic for oscillatory interference models, including how knockout of the HCN1 gene in mice, which flattens the dorsoventral gradient in MPO frequency and resonance frequency, does not affect the development of the grid cell dorsoventral gradient of spatial scales, and how hexagonal grid firing fields in bats can occur even in the absence of theta band modulation. These results demonstrate how models of grid cell self-organization can provide new insights into the relationship between brain learning, oscillatory dynamics, and navigational behaviors.

  7. RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

    International Nuclear Information System (INIS)

    Poch, Enric; Minambres, Rebeca; Mocholi, Enric; Ivorra, Carmen; Perez-Arago, Amparo; Guerri, Consuelo; Perez-Roger, Ignacio; Guasch, Rosa M.

    2007-01-01

    Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines

  8. Anti-cancer effect of bee venom in prostate cancer cells through activation of caspase pathway via inactivation of NF-κB.

    Science.gov (United States)

    Park, Mi Hee; Choi, Myoung Suk; Kwak, Dong Hoon; Oh, Ki-Wan; Yoon, Do Young; Han, Sang Bae; Song, Ho Sueb; Song, Min Jong; Hong, Jin Tae

    2011-06-01

    Bee venom has been used as a traditional medicine to treat arthritis, rheumatism, back pain, cancerous tumors, and skin diseases. However, the effects of bee venom on the prostate cancer and their action mechanisms have not been reported yet. To determine the effect of bee venom and its major component, melittin on the prostate cancer cells, apoptosis is analyzed by tunnel assay and apoptotic gene expression. For xenograft studies, bee venom was administrated intraperitoneally twice per week for 4 weeks, and the tumor growth was measured and the tumor were analyzed by immunohistochemistry. To investigate whether bee venom and melittin can inactivate nuclear factor kappa B (NF-κB), we assessed NF-κB activity in vitro and in vivo. Bee venom (1-10 µg/ml) and melittin (0.5-2.5 µg/ml) inhibited cancer cell growth through induction of apoptotic cell death in LNCaP, DU145, and PC-3 human prostate cancer cells. These effects were mediated by the suppression of constitutively activated NF-κB. Bee venom and melittin decreased anti-apoptotic proteins but induced pro-apoptotic proteins. However, pan caspase inhibitor abolished bee venom and melittin-induced apoptotic cell death and NF-κB inactivation. Bee venom (3-6 mg/kg) administration to nude mice implanted with PC-3 cells resulted in inhibition of tumor growth and activity of NF-κB accompanied with apoptotic cell death. Therefore, these results indicated that bee venom and melittin could inhibit prostate cancer in in vitro and in vivo, and these effects may be related to NF-κB/caspase signal mediated induction of apoptotic cell death. Copyright © 2010 Wiley-Liss, Inc.

  9. Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Liu, Maoxi; Fu, Zhongxue; Wu, Xingye; Du, Kunli; Zhang, Shouru; Zeng, Li

    2016-05-01

    Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

  10. Inactivation Data.xlsx

    Data.gov (United States)

    U.S. Environmental Protection Agency — The data set is a spreadsheet that contains results of inactivation experiments that were conducted to to determine the effectiveness of chlorine in inactivating B....

  11. Inactivation of the Lateral Entorhinal Area Increases the Influence of Visual Cues on Hippocampal Place Cell Activity

    Directory of Open Access Journals (Sweden)

    Kristin M. Scaplen

    2017-05-01

    Full Text Available The hippocampus is important for both navigation and associative learning. We previously showed that the hippocampus processes two-dimensional (2D landmarks and objects differently. Our findings suggested that landmarks are more likely to be used for orientation and navigation, whereas objects are more likely to be used for associative learning. The process by which cues are recognized as relevant for navigation or associative learning, however, is an open question. Presumably both spatial and nonspatial information are necessary for classifying cues as landmarks or objects. The lateral entorhinal area (LEA is a good candidate for participating in this process as it is implicated in the processing of three-dimensional (3D objects and object location. Because the LEA is one synapse upstream of the hippocampus and processes both spatial and nonspatial information, it is reasonable to hypothesize that the LEA modulates how the hippocampus uses 2D landmarks and objects. To test this hypothesis, we temporarily inactivated the LEA ipsilateral to the dorsal hippocampal recording site using fluorophore-conjugated muscimol (FCM 30 min prior to three foraging sessions in which either the 2D landmark or the 2D object was back-projected to the floor of an open field. Prior to the second session we rotated the 2D cue by 90°. Cues were returned to the original configuration for the third session. Compared to the Saline treatment, FCM inactivation increased the percentage of rotation responses to manipulations of the landmark cue, but had no effect on information content of place fields. In contrast, FCM inactivation increased information content of place fields in the presence of the object cue, but had no effect on rotation responses to the object cue. Thus, LEA inactivation increased the influence of visual cues on hippocampal activity, but the impact was qualitatively different for cues that are useful for navigation vs. cues that may not be useful for

  12. Radiobiological inactivation of Epstein-Barr virus

    International Nuclear Information System (INIS)

    Henderson, E.; Heston, L.; Grogan, E.; Miller, G.

    1978-01-01

    Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either uv or x irradiation. No dose of irradiation increases the transforming capacity of EBV. The x-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to uv irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of uv damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of uv and x irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P 3 HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction

  13. Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection▿

    Science.gov (United States)

    Whiteside, Theresa L.; Piazza, Paolo; Reiter, Amanda; Stanson, Joanna; Connolly, Nancy C.; Rinaldo, Charles R.; Riddler, Sharon A.

    2009-01-01

    In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8+ and CD4+ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4+ T cells with the virus; (iii) inactivation of the virus in CD4+ T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4+ T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4+ T cells. CD4+ T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID50; which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4+ T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID50 of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 μg/ml) and UVB irradiation (312 nm) reduced the TCID50 of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4+ T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137). PMID:19038780

  14. Production of a dendritic cell-based vaccine containing inactivated autologous virus for therapy of patients with chronic human immunodeficiency virus type 1 infection.

    Science.gov (United States)

    Whiteside, Theresa L; Piazza, Paolo; Reiter, Amanda; Stanson, Joanna; Connolly, Nancy C; Rinaldo, Charles R; Riddler, Sharon A

    2009-02-01

    In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8(+) and CD4(+) T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4(+) T cells with the virus; (iii) inactivation of the virus in CD4(+) T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4(+) T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4(+) T cells. CD4(+) T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID(50); which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4(+) T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID(50) of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 microg/ml) and UVB irradiation (312 nm) reduced the TCID(50) of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4(+) T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).

  15. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation.

    Science.gov (United States)

    Harms, Paul W; Collie, Angela M B; Hovelson, Daniel H; Cani, Andi K; Verhaegen, Monique E; Patel, Rajiv M; Fullen, Douglas R; Omata, Kei; Dlugosz, Andrzej A; Tomlins, Scott A; Billings, Steven D

    2016-03-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes

  16. Next Generation Sequencing of Cytokeratin 20-Negative Merkel Cell Carcinoma Reveals Ultraviolet Signature Mutations and Recurrent TP53 and RB1 Inactivation

    Science.gov (United States)

    Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.

    2016-01-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping

  17. Pathogen inactivation techniques.

    Science.gov (United States)

    Pelletier, J P R; Transue, S; Snyder, E L

    2006-01-01

    The desire to rid the blood supply of pathogens of all types has led to the development of many technologies aimed at the same goal--eradication of the pathogen(s) without harming the blood cells or generating toxic chemical agents. This is a very ambitious goal, and one that has yet to be achieved. One approach is to shun the 'one size fits all' concept and to target pathogen-reduction agents at the Individual component types. This permits the development of technologies that might be compatible with, for example, plasma products but that would be cytocidal and thus incompatible with platelet concentrates or red blood cell units. The technologies to be discussed include solvent detergent and methylene blue treatments--designed to inactivate plasma components and derivatives; psoralens (S-59--amotosalen) designed to pathogen-reduce units of platelets; and two products aimed at red blood cells, S-303 (a Frale--frangible anchor-linker effector compound) and Inactine (a binary ethyleneimine). A final pathogen-reduction material that might actually allow one material to inactivate all three blood components--riboflavin (vitamin B2)--is also under development. The sites of action of the amotosalen (S-59), the S-303 Frale, Inactine, and riboflavin are all localized in the nucleic acid part of the pathogen. Solvent detergent materials act by dissolving the plasma envelope, thus compromising the integrity of the pathogen membrane and rendering it non-infectious. By disrupting the pathogen's ability to replicate or survive, its infectivity is removed. The degree to which bacteria and viruses are affected by a particular pathogen-reducing technology relates to its Gram-positive or Gram-negative status, to the sporulation characteristics for bacteria, and the presence of lipid or protein envelopes for viruses. Concerns related to photoproducts and other breakdown products of these technologies remain, and the toxicology of pathogen-reduction treatments is a major ongoing area

  18. Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion.

    Science.gov (United States)

    Li, Yangyang; Wang, Cheng; Liu, Xinye; Song, Jian; Li, Hongjian; Sui, Zhipeng; Zhang, Ming; Fang, Shuang; Chu, Jinfang; Xin, Mingming; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2016-04-01

    Heterosis has been widely used in agriculture, but the underlying molecular principles are still largely unknown. During seed germination, we observed that maize (Zea mays) hybrid B73/Mo17 was less sensitive than its parental inbred lines to exogenous abscisic acid (ABA), and endogenous ABA content in hybrid embryos decreased more rapidly than in the parental inbred lines. ZmABA8ox1b, an ABA inactivation gene, was consistently more highly up-regulated in hybrid B73/Mo17 than in its parental inbred lines at early stages of seed germination. Moreover, ectopic expression of ZmABA8ox1b obviously promoted seed germination in Arabidopsis Remarkably, microscopic observation revealed that cell expansion played a major role in the ABA-mediated maize seed germination heterosis, which could be attributed to the altered expression of cell wall-related genes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  19. Antiproliferative Action of Conjugated Linoleic Acid on Human MCF-7 Breast Cancer Cells Mediated by Enhancement of Gap Junctional Intercellular Communication through Inactivation of NF-κB

    Directory of Open Access Journals (Sweden)

    Md. Abdur Rakib

    2013-01-01

    Full Text Available The major conjugated linoleic acid (CLA isomers, c9,t11-CLA and t10,c12-CLA, have anticancer effects; however, the exact mechanisms underlying these effects are unknown. Evidence suggests that reversal of reduced gap junctional intercellular communication (GJIC in cancer cells inhibits cell growth and induces cell death. Hence, we determined that CLA isomers enhance GJIC in human MCF-7 breast cancer cells and investigated the underlying molecular mechanisms. The CLA isomers significantly enhanced GJIC of MCF-7 cells at 40 μM concentration, whereas CLA inhibited cell growth and induced caspase-dependent apoptosis. CLA increased connexin43 (Cx43 expression both at the transcriptional and translational levels. CLA inhibited nuclear factor-κB (NF-κB activity and enhanced reactive oxygen species (ROS generation. No significant difference was observed in the efficacy of c9,t11-CLA and t10,c12-CLA. These results suggest that the anticancer effect of CLA is associated with upregulation of GJIC mediated by enhanced Cx43 expression through inactivation of NF-κB and generation of ROS in MCF-7 cells.

  20. UV-inactivation of Epstein-Barr virus: differences in early antigen expression in two different non-productive cell lines and influence of caffeine

    International Nuclear Information System (INIS)

    Suchankova, A.; Vonka, V.

    1978-01-01

    Two non-productive Epstein-Barr (EB) virus genome-carrying lymphoblastoid cell lines, namely Raji and NC37, were used for studying the effect of UV irradiation on the ability of P3HR-1 EB virus to induce early antigen (EA) formation. In NC37 cells infected with UV-irradiated virus the formation of EA was delayed; thus the slope of inactivation curve based on the early (24 hr) reading was steeper than that based on the late (72 hr) reading. This was not observed in Raji cells. Caffeine did not influence the percentage of EA positive cells in cultures infected with untreated virus; however, the drug exhibited a marked inhibitory effect on EA production after infection with UV-irradiated virus. The sensitivity to caffeine decreased more rapidly with time after infection of Raji than of NC37 cells, suggesting a higher degree of readiness of the host cell repair system in the former than in the latter cells. The caffeine effect was merely directed against the synthesis of R (restricted) component of EA; its influence on the D (diffuse) component formation was negligible. (author)

  1. Combined treatments of enterocin AS-48 with biocides to improve the inactivation of methicillin-sensitive and methicillin-resistant Staphylococcus aureus planktonic and sessile cells.

    Science.gov (United States)

    Caballero Gómez, Natacha; Abriouel, Hikmate; Grande, M José; Pérez Pulido, Rubén; Gálvez, Antonio

    2013-05-15

    Control of staphylococci during cleaning and disinfection is important to the food industry. Broad-spectrum bacteriocins with proved anti-staphylococcal activity, such as enterocin AS-48, could open new possibilities for disinfection in combination with biocides. In the present study, enterocin AS-48 was tested singly or in combination with biocides against a cocktail of six Staphylococcus aureus strains (including three methicillin-resistant strains) in planktonic state as well as in biofilms formed on polystyrene microtiter plates. Cells were challenged with enterocin, biocides or enterocin/biocide combinations. Inactivation of planktonic cells increased significantly (penterocin AS-48 (25mg/l) was tested in combination with benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and non-significantly in combination with didecyldimethylammonium bromide (AB), triclosan (TC), hexachlorophene (CF), polyhexamethylen guanidinium chloride (PHMG), chlorhexidine (CH) or P3-oxonia (OX). In the sessile state (24h biofilms), staphylococci required higher biocide concentrations in most cases, except for OX. Inactivation of sessile staphylococci increased remarkably when biocides were applied in combination with enterocin AS-48, especially when the bacteriocin was added at 50mg/l. During storage, the concentrations of sessile as well as planktonic cells in the treated samples decreased remarkably for BC, TC and PHMG, but OX failed to inhibit proliferation of the treated biofilms as well as growth of planktonic cells. The observed inhibitory effects during storage were potentiated when the biocides were combined with 50 mg/l enterocin AS-48. Results from this study suggest that selected combinations of enterocin AS-48 and biocides offer potential use against planktonic and sessile, methicillin-sensitive and methicillin-resistant S. aureus. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Inactivation of human osteosarcoma cells in vitro by 211At-TP-3 monoclonal antibody: Comparison with astatine-211 and external-beam X rays

    International Nuclear Information System (INIS)

    Larsen, R.H.; Bruland, O.S.; Hoff, P.; Alstad, J.; Lindmo, T.; Rofstad, E.K.

    1994-01-01

    The potential usefulness of α-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) 211 At-TP-3 of different specific activities, (b) 211 At-labeled bovine serum albumin (BSA), (c) free 211 At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, 211 At-BSA or free 211 At. The D o 's were lower for free 211 At than for 211 At-BSA. The survival curves for 211 At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to 211 At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to 211 At-TP-3 treatment was the antigen expression. Cell inactivation after 211 At-TP-3 treatment increased substantially with increasing specific activity of the 211 At-TP-3. At high specific activities, the cytotoxic effect of 211 At-TP-3 was significantly higher than that of 211 At-BSA. In conclusion, 211 At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma. 39 refs., 5 figs., 2 tabs

  3. Comprehensive genomic profiling reveals inactivating SMARCA4 mutations and low tumor mutational burden in small cell carcinoma of the ovary, hypercalcemic-type.

    Science.gov (United States)

    Lin, Douglas I; Chudnovsky, Yakov; Duggan, Bridget; Zajchowski, Deborah; Greenbowe, Joel; Ross, Jeffrey S; Gay, Laurie M; Ali, Siraj M; Elvin, Julia A

    2017-12-01

    Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

    Science.gov (United States)

    2013-01-01

    Introduction Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). Methods To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis. Results Surprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration. Conclusions Although WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs. PMID:23295128

  5. Inactivated Tianjin strain, a novel genotype of Sendai virus, induces apoptosis in HeLa, NCI-H446 and Hep3B cells.

    Science.gov (United States)

    Chen, Jun; Han, Han; Wang, Bin; Shi, Liying

    2016-07-01

    The Sendai virus strain Tianjin is a novel genotype of the Sendai virus. In previous studies, ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) demonstrated antitumor effects on human breast cancer cells. The aim of the present study was to investigate the in vitro antitumor effects of UV-Tianjin on the human cervical carcinoma HeLa, human small cell lung cancer NCI-H446 and human hepatocellular carcinoma Hep 3B cell lines, and the possible underlying mechanisms of these antitumor effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that UV-Tianjin treatment inhibited the proliferation of HeLa, NCI-H446 and Hep 3B cells in a dose- and time-dependent manner. Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide double staining indicated that UV-Tianjin induced dose-dependent apoptosis in all three cell lines with the most significant effect observed in the HeLa cell line. In the HeLa cell line, UV-Tianjin-induced apoptosis was further confirmed by the disruption of the mitochondria membrane potential and the activation of caspases, as demonstrated by fluorescent cationic dye and colorimetric assays, respectively. In addition, western blot analysis revealed that UV-Tianjin treatment resulted in significant upregulation of cytochrome c , apoptosis protease activating factor-1, Fas, Fas ligand and Fas-associated protein with death domain, and activated caspase-9, -8 and -3 in HeLa cells. Based on these results, it is hypothesized that UV-Tianjin exhibits anticancer activity in HeLa, NCI-H446 and Hep 3B cell lines via the induction of apoptosis. In conclusion, the results of the present study indicate that in the HeLa cell line, intrinsic and extrinsic apoptotic pathways may be involved in UV-Tianjin-induced apoptosis.

  6. Skewed X-inactivation in cloned mice

    International Nuclear Information System (INIS)

    Senda, Sho; Wakayama, Teruhiko; Yamazaki, Yukiko; Ohgane, Jun; Hattori, Naka; Tanaka, Satoshi; Yanagimachi, Ryuzo; Shiota, Kunio

    2004-01-01

    In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P < 0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders

  7. Dihydroartemisinin Accentuates the Anti-Tumor Effects of Photodynamic Therapy via Inactivation of NF-κB in Eca109 and Ec9706 Esophageal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yan Jing Li

    2014-05-01

    Full Text Available Background: Photodynamic therapy (PDT is a new treatment for esophageal cancer which has been shown to be effective in the elimination of tumor. However, PDT could induce the activation of nuclear factor-kappa B (NF-κB in many photosensitizers based PDT, which plays a negative role in PDT. In addition, our previous results have shown that dihydroartemisinin (DHA, which was the most potent one of artemisinin derivatives, has anticancer activity in esophageal cancer cells. Methods: Cell viability was determined by MTT analysis, and apoptosis was evaluated by flow cytometry. Nuclear extract was obtained for determining NF-κB DNA-binding activity, while total protein extract obtained for downstream gene expression by western blot. Results: We demonstrated DHA enhanced PDT-induced growth inhibition and apoptosis in both human esophageal cancer cell lines Eca109 and Ec9706 in vitro. The mechanism was at least partially due to DHA deactivated PDT-induced NF-κB activation, so as to decrease tremendously the expression of its target gene Bcl-2. Conclusion: Our results demonstrate that DHA augments PDT-induced growth inhibition and apoptosis in esophageal cancer cells, and that inactivation of NF-κB activity is a potential mechanism by which DHA sensitizes esophageal cancer cells to PDT-induced growth inhibition and apoptosis.

  8. Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

    Science.gov (United States)

    Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

    2016-08-01

    Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Guizhi Fuling Wan, a Traditional Chinese Herbal Formula, Sensitizes Cisplatin-Resistant Human Ovarian Cancer Cells through Inactivation of the PI3K/AKT/mTOR Pathway

    Directory of Open Access Journals (Sweden)

    Li Han

    2016-01-01

    Full Text Available The aim of the study was to explore the possible mechanisms that Guizhi Fuling Wan (GFW enhances the sensitivity of the SKOV3/DDP ovarian cancer cells and the resistant xenograft tumours to cisplatin. Rat medicated sera containing GFW were prepared by administering GFW to rats, and the primary bioactive constituents of the sera were gallic acid, paeonol, and paeoniflorin analysed by HPLC/QqQ MS. Cell counting kit-8 analysis was shown that coincubation of the sera with cisplatin/paclitaxel enhanced significantly the cytotoxic effect of cisplatin or paclitaxel in SKOV3/DDP cells. The presence of the rat medicated sera containing GFW resulted in an increase in rhodamine 123 accumulation by flow cytometric assays and a decrease in the protein levels of P-gp, phosphorylation of AKT at Ser473, and mTOR in a dose-dependent manner in SKOV3/DDP cells by western blot analysis, but the sera had no effect on the protein levels of PI3K p110α and total AKT. The low dose of GFW enhanced the anticancer efficacy of cisplatin and paclitaxel treatment in resistant SKOV3/DDP xenograft tumours. GFW could sensitize cisplatin-resistant SKOV3/DDP cells by inhibiting the protein level and function of P-gp, which may be medicated through inactivation of the PI3K/AKT/mTOR pathway.

  10. Cell membrane temperature rate sensitivity predicted from the Nernst equation.

    Science.gov (United States)

    Barnes, F S

    1984-01-01

    A hyperpolarized current is predicted from the Nernst equation for conditions of positive temperature derivatives with respect to time. This ion current, coupled with changes in membrane channel conductivities, is expected to contribute to a transient potential shift across the cell membrane for silent cells and to a change in firing rate for pacemaker cells.

  11. Tendon cell outgrowth rates and morphology associated with kevlar-49.

    Science.gov (United States)

    Zimmerman, M; Gordon, K E

    1988-12-01

    A rat tendon cell model was used to evaluate the in vitro biocompatibility of kevlar-49. The cell response to kevlar was compared to carbon AS-4 and nylon sutures. Three trials were run and cell growth rates were statistically similar for all the materials tested. A separate experiment was conducted in which the same fiber materials were placed in the same Petri dish. Again, the rates were similar for each material. Finally, the cells were observed with a scanning electron microscope, and the three classic cell morphologies associated with this tendon cell model were observed. Also, cellular attachment to the fiber and cellular encapsulation of the fiber were identical for the three materials tested. Kevlar-49 proved to be comparable to carbon AS4 and nylon sutures in terms of cellular response and cell outgrowth rates.

  12. Zurampic Protects Pancreatic β-Cells from High Uric Acid Induced-Damage by Inhibiting URAT1 and Inactivating the ROS/AMPK/ERK Pathways

    Directory of Open Access Journals (Sweden)

    Ying Xin

    2018-05-01

    Full Text Available Background/Aims: Zurampic is a US FDA approved drug for treatment of gout. However, the influence of Zurampic on pancreatic β-cells remains unclear. The study aimed to evaluate the effects of Zurampic on high uric acid-induced damage of pancreatic β-cells and the possible underlying mechanisms. Methods: INS-1 cells and primary rat islets were stimulated with Zurampic and the mRNA expression of urate transporter 1 (URAT1 was assessed by qRT-PCR. Cells were stimulated with uric acid or uric acid plus Zurampic, and cell viability, apoptosis and ROS release were measured by MTT and flow cytometry assays. Western blot analysis was performed to evaluate the expressions of active Caspase-3 and phosphorylation of AMPK and ERK. Finally, cells were stimulated with uric acid or uric acid plus Zurampic at low/high level of glucose (2.8/16.7 mM glucose, and the insulin release was assessed by ELISA. Results: mRNA expression of URAT1 was decreased by Zurampic in a dose-dependent manner. Uric acid decreased cell viability, promoted cell apoptosis and induced ROS release. Uric acid-induced alterations could be reversed by Zurampic. Activation of Caspase-3 and phosphorylation of AMPK and ERK were enhanced by uric acid, and the enhancements were reversed by Zurampic. Decreased phosphorylation of AMPK and ERK, induced by Zurampic, was further reduced by adding inhibitor of AMPK or ERK. Besides, uric acid inhibited high glucose-induced insulin secretion and the inhibition was rescued by Zurampic. Conclusions: Zurampic has a protective effect on pancreatic β-cells against uric acid induced-damage by inhibiting URAT1 and inactivating the ROS/AMPK/ERK pathway.

  13. Comparison of two different methods for inactivation of viruses in serum

    DEFF Research Database (Denmark)

    Preuss, T.; Kamstrup, Søren; Kyvsgaard, N.C.

    1997-01-01

    enterovirus (PEV) was inactivated within 3 h, The inactivation with electron-beam irradiation resulted in almost linear curves in a semilogarithmic plot of virus titer versus irradiation dose, reflecting a first-order inactivation, The rate of inactivation was almost twice as fast in the liquid samples...

  14. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

    Science.gov (United States)

    Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

    2016-11-18

    The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

  15. Inactivation of Src-to-Ezrin Pathway: A Possible Mechanism in the Ouabain-Mediated Inhibition of A549 Cell Migration

    Directory of Open Access Journals (Sweden)

    Hye Kyoung Shin

    2015-01-01

    Full Text Available Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na+/K+-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416 was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925, p-paxillin (Y118, p130CAS, and Na+/K+-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

  16. Inactivation by oxidation and recruitment into stress granules of hOGG1 but not APE1 in human cells exposed to sub-lethal concentrations of cadmium

    International Nuclear Information System (INIS)

    Bravard, Anne; Campalans, Anna; Vacher, Monique; Gouget, Barbara; Levalois, Celine; Chevillard, Sylvie; Radicella, J. Pablo

    2010-01-01

    The induction of mutations in mammalian cells exposed to cadmium has been associated with the oxidative stress triggered by the metal. There is increasing evidence that the mutagenic potential of Cd is not restricted to the induction of DNA lesions. Cd has been shown to inactivate several DNA repair enzymes. Here we show that exposure of human cells to sub-lethal concentrations of Cd leads to a time- and concentration-dependent decrease in hOGG1 activity, the major DNA glycosylase activity responsible for the initiation of the base excision repair (BER) of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. Although there is a slight effect on the level of hOGG1 transcripts, we show that the inhibition of the 8-oxoguanine DNA glycosylase activity is mainly associated with an oxidation of the hOGG1 protein and its disappearance from the soluble fraction of total cell extracts. Confocal microscopy analyses show that in cells exposed to Cd hOGG1-GFP is recruited to discrete structures in the cytoplasm. These structures were identified as stress granules. Removal of Cd from the medium allows the recovery of the DNA glycosylase activity and the presence of hOGG1 in a soluble form. In contrast to hOGG1, we show here that exposure to Cd does not affect the activity of the second enzyme of the pathway, the major AP endonuclease APE1.

  17. Dendritic cells pulsed with Pythium insidiosum (1,3)(1,6)-β-glucan, Heat-inactivated zoospores and immunotherapy prime naïve T cells to Th1 differentiation in vitro.

    Science.gov (United States)

    Ledur, Pauline C; Tondolo, Juliana S M; Jesus, Francielli P K; Verdi, Camila M; Loreto, Érico S; Alves, Sydney H; Santurio, Janio M

    2018-03-01

    Pythiosis is a life-threatening disease caused by the fungus-like microorganism Pythium insidiosum that can lead to death if not treated. Since P. insidiosum has particular cell wall characteristics, pythiosis is difficult to treat, as it does not respond well to traditional antifungal drugs. In our study, we investigated a new immunotherapeutic approach with potential use in treatment and in the acquisition of immunity against pythiosis. Dendritic cells from both human and mouse, pulsed with P. insidiosum heat-inactivated zoospore, (1,3)(1,6)-β-glucan and the immunotherapeutic PitiumVac ® efficiently induced naïve T cell differentiation in a Th1 phenotype by the activation of specific Th1 cytokine production in vitro. Heat-inactivated zoospores showed the greatest Th1 response among the tested groups, with a significant increase in IL-6 and IFN-γ production in human cells. In mice cells, we also observed a Th17 pathway induction, with an increase on the IL-17A levels in lymphocytes cultured with β-glucan pulsed DCs. These results suggest a potential use of DCs pulsed with P. insidiosum antigens as a new therapeutic strategy in the treatment and acquisition of immunity against pythiosis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Fisetin induces G2/M phase cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation in human endometrial cancer cells

    Directory of Open Access Journals (Sweden)

    Zhan-Ying Wang

    2015-06-01

    Full Text Available Endometrial cancer is one of the most prevalent gynaecological malignancies where, currently available therapeutic options remain limited. Recently phytochemicals are exploited for their efficiency in cancer therapy. The present study investigates the anti-proliferative effect of fisetin, a flavonoid on human endometrial cancer cells (KLE and Hec1 A. Fisetin (20-100 µM effectively reduced the viability of Hec1 A and KLE cells and potentially altered the cell population at G2/M stage. Expression levels of the cell cycle proteins (cyclin B1, p-Cdc2, p-Cdc25C, p-Chk1, Chk2, p-ATM, cyclin B1, H2AX, p21 and p27 were analyzed. Fisetin suppressed cyclin B1 expression and caused inactiva-tion of Cdc25C and Cdc2 by increasing their phosphorylation levels and further activated ATM, Chk1 and Chk2. Increased levels of p21 and p27 were observed as well. These results suggest that fisetin induced G2/M cell cycle arrest via inactivating Cdc25c and Cdc2 through activation of ATM, Chk1 and Chk2.

  19. Patulin reduction in apple juice by inactivated Alicyclobacillus spp.

    Science.gov (United States)

    Yuan, Y; Wang, X; Hatab, S; Wang, Z; Wang, Y; Luo, Y; Yue, T

    2014-12-01

    This study aimed to investigate the reduction of patulin (PAT) in apple juice by 12 inactivated Alicyclobacillus strains. The reduction rate of PAT by each strain was determined by high-performance liquid chromatography (HPLC). The results indicated that the removal of PAT was strain specific. Alicyclobacillus acidoterrestris 92 and A. acidoterrestris 96 were the most effective ones among the 12 tested strains in the removal of PAT. Therefore, these two strains were selected to study the effects of incubation time, initial PAT concentration and bacteria powder amount on PAT removal abilities of Alicyclobacillus. The highest PAT reduction rates of 88·8 and 81·6% were achieved after 24-h incubation with initial PAT concentration of 100 μg l(-1) and bacteria powder amount of 40 g l(-1) , respectively. Moreover, it was found that the treatment by these 12 inactivated Alicyclobacillus strains had no negative effect on the quality parameters of apple juice. Similar assays were performed in supermarket apple juice, where inactivated Alicyclobacillus cells could efficiently reduce PAT content. Taken together, these data suggest the possible application of this strategy as a means to detoxify PAT-contaminated juices. Inactivated Alicyclobacillus cells can efficiently reduce patulin concentration in apple juice. It provides a theoretical foundation for recycling of Alicyclobacillus cells from spoiled apple juice to reduce the source of pollution and the cost of juice industry. This is the first report on the use of Alicyclobacillus to remove patulin from apple juice. © 2014 The Society for Applied Microbiology.

  20. Turnover rate of hypoxic cells in solid tumors

    International Nuclear Information System (INIS)

    Ljungkvist, A.S.E.; Bussink, J.; Rijken, P.F.J.W.; Van Der Kogel, A.J.

    2003-01-01

    Most solid tumors contain hypoxic cells, and both the amount and duration of tumor hypoxia has been shown to influence the effect of radiation treatment negatively. It is important to understand the dynamic processes within the hypoxic cell population in non-treated tumors, and the effect of different treatment modalities on the kinetics of hypoxic cells to be able to design optimal combined modality treatments. The turnover rate of hypoxic cells was analyzed in three different solid tumor models with a double bio-reductive hypoxic marker assay with sequential injection of the two hypoxic markers. Previously it was shown that this assay could be used to detect both a decrease and an increase of tumor hypoxia in relation to the tumor vasculature with high spatial resolution. In this study the first hypoxic marker, pimonidazole, was administered at variable times relative to tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. The hypoxic cell turnover rate was calculated as the loss of pimonidazole positive cells relative to CCI-103F. The murine C38 line had the fastest hypoxic turnover rate of 60% /24h and the human xenograft line SCCNij3 had the slowest hypoxic turnover rate of 30% /24 h. The hypoxic turnover rate was most heterogeneous in the SCCNij3 line that even contained viable groups of cells that had been hypoxic for at least 5 days. The human xenograft line MEC82 fell in between with a hypoxic turnover rate of 50% /24 h. The hypoxic cell turnover was related to the potential tumor volume doubling time (Tpot) with a Tpot of 26h in C38 and 103h in SCCNij3. The dynamics of hypoxic cells, quantified with a double hypoxic marker method, showed large differences in hypoxic cell turnover rate and were related to Tpot

  1. Microtubules Growth Rate Alteration in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Irina B. Alieva

    2010-01-01

    Full Text Available To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC and “fast” (three times as much growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  2. Inactivation of certain insect pathogens by ultraviolet radiation

    International Nuclear Information System (INIS)

    Krieg, A.; Groener, A.; Huber, J.; Zimmermann, G.

    1981-01-01

    The UV-sensitivity of two baculoviruses (granulosis virus, nuclear polyhedrosis virus) and two entomopathogenic microorganisms (Bacillus thuringiensis, Beauveria bassiana) was determined by radiation tests. In the far UV (254 nm) the stability, measured at an inactivation rate of 99%, was in declining order: nuclear polyhedra >= conidia of B. bassiana > granula > spores of B. thuringiensis >= vegetative cells of B. thuringiensis. In the near UV (285-380 nm) the following order could be found: conidia of B. bassiana >= nuclear polyhedra > spores of B. thuringiensis >= granula > vegetative cells of B. thuringiensis. Far UV had a much higher germicidal effect for all pathogens tested than near UV. (orig.) [de

  3. Inactivation of certain insect pathogens by ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Krieg, A.; Groener, A.; Huber, J.; Zimmermann, G.

    1981-01-01

    The UV-sensitivity of two baculoviruses (granulosis virus, nuclear polyhedrosis virus) and two entomopathogenic microorganisms (Bacillus thuringiensis, Beauveria bassiana) was determined by radiation tests. In the far UV (254 nm) the stability, measured at an inactivation rate of 99%, was in declining order: nuclear polyhedra >= conidia of B. bassiana > granula > spores of B. thuringiensis >= vegetative cells of B. thuringiensis. In the near UV (285-380 nm) the following order could be found: conidia of B. bassiana >= nuclear polyhedra > spores of B. thuringiensis >= granula > vegetative cells of B. thuringiensis. Far UV had a much higher germicidal effect for all pathogens tested than near UV.

  4. p53 inactivation decreases dependence on estrogen/ERK signalling for proliferation but promotes EMT and susceptility to 3-bromopyruvate in ERα+ breast cancer MCF-7 cells.

    Science.gov (United States)

    Rieber, Manuel; Strasberg-Rieber, Mary

    2014-03-15

    Most breast cancers express the estrogen receptor alpha (ERα(+)), harbor wt TP53, depend on estrogen/ERK signalling for proliferation, and respond to anti-estrogens. However, concomittant activation of the epidermal growth factor receptor (EGFR)/MEK pathway promotes resistance by decreasing estrogen dependence. Previously, we showed that retroviral transduction of mutant p53 R175H into wt TP53 ERα(+) MCF-7 cells induces epidermal growth factor (EGF)-independent proliferation, activation of the EGF receptor (p-EGFR) and some characteristics of epithelial-mesenchymal transition (EMT). To investigate whether p53 inactivation augments ERα(+) cell proliferation in response to restrictive estradiol, chemical MEK inhibition or metabolic inhibitors. Introduction of mutant p53 R175H lowered expression of p53-dependent PUMA and p21WAF1, decreased E-cadherin and cytokeratin 18 associated with EMT, but increased the % of proliferating ERα(+)/Ki67 cells, diminishing estrogen dependence. These cells also exhibited higher proliferation in the presence of MEK-inhibitor UO126, reciprocally correlating with preferential susceptibility to the pyruvate analog 3-bromopyruvate (3-BrPA) without a comparable response to 2-deoxyglucose. p53 siRNA silencing by electroporation in wt TP53 MCF-7 cells also decreased estrogen dependence and response to MEK inhibition, while also conferring susceptibility to 3-BrPA. (a) ERα(+) breast cancer cells dysfunctional for TP53 which proliferate irrespective of low estrogen and chemical MEK inhibition are likely to increase metabolic consumption becoming increasingly susceptible to 3-BrPA; (b) targeting the pyruvate pathway may improve response to endocrine therapy in ERα(+) breast cancer with p53 dysfunction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  5. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

    DEFF Research Database (Denmark)

    Harada, H; Andersen, Jens S.; Mann, M

    2001-01-01

    Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site-specific phospho...

  6. Metabolic rate determines haematopoietic stem cell self-renewal.

    Science.gov (United States)

    Sastry, P S R K

    2004-01-01

    The number of haematopoietic stem cells (HSCs) per animal is conserved across species. This means the HSCs need to maintain hematopoiesis over a longer period in larger animals. This would result in the requirement of stem cell self-renewal. At present the three existing models are the stochastic model, instructive model and the third more recently proposed is the chiaro-scuro model. It is a well known allometric law that metabolic rate scales to the three quarter power. Larger animals have a lower metabolic rate, compared to smaller animals. Here it is being hypothesized that metabolic rate determines haematopoietic stem cell self-renewal. At lower metabolic rate the stem cells commit for self-renewal, where as at higher metabolic rate they become committed to different lineages. The present hypothesis can explain the salient features of the different models. Recent findings regarding stem cell self-renewal suggest an important role for Wnt proteins and their receptors known as frizzleds, which are an important component of cell signaling pathway. The role of cGMP in the Wnts action provides further justification for the present hypothesis as cGMP is intricately linked to metabolic rate. One can also explain the telomere homeostasis by the present hypothesis. One prediction of the present hypothesis is with reference to the limit of cell divisions known as Hayflick limit, here it is being suggested that this is the result of metabolic rate in laboratory conditions and there can be higher number of cell divisions in vivo if the metabolic rate is lower. Copyright 2004 Elsevier Ltd.

  7. ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

    Science.gov (United States)

    Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S; Allen, Joshua E; Oster, Wolfgang; Duvic, Madeleine

    2017-09-22

    Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4 + malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V + cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4 + malignant T cells, not in normal CD4 + T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

  8. Mycobacteria inactivation using Engineered Water Nanostructures (EWNS).

    Science.gov (United States)

    Pyrgiotakis, Georgios; McDevitt, James; Gao, Ya; Branco, Alan; Eleftheriadou, Mary; Lemos, Bernardo; Nardell, Edward; Demokritou, Philip

    2014-08-01

    Airborne transmitted pathogens such as Mycobacterium tuberculosis (Mtb) cause serious, often fatal infectious disease with enormous global health implications. Due to their unique cell wall and slow growth, mycobacteria are among the most resilient microbial forms. Herein we evaluate the ability of an emerging, chemical-free, nanotechnology-based method to inactivate M. parafortuitum (Mtb surrogate). This method is based on the transformation of atmospheric water vapor into engineered water nano-structures (EWNS) via electrospray. We demonstrate that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. Additionally, EWNS can inactivate M. parafortuitum on surfaces eight times faster than the control. The mechanism of mycobacteria inactivation was also investigated in this study. It was demonstrated that the EWNS effectively deliver the reactive oxygen species, encapsulated during the electrospray process, to the bacteria oxidizing their cell membrane resulting into inactivation. Overall, this is a method with the potential to become an effective intervention technology in the battle against airborne infections. This study demonstrates the feasibility of mycobacterium inactivation in airborne form or on contact surfaces using electrospray activated water nano-structures. Given that the method is free of toxic chemicals, this might become an important tool in the prevention of mycobacterial infections, which are notoriously hard to treat. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer.

    Science.gov (United States)

    Lee, Jeong-Yeon; Jang, Ki-Seok; Shin, Dong-Hui; Oh, Mi-Yun; Kim, Hyun-Jun; Kim, Yongseok; Kong, Gu

    2008-06-01

    Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

  10. Prevention of allergic rhinitis by ginger and the molecular basis of immunosuppression by 6-gingerol through T cell inactivation.

    Science.gov (United States)

    Kawamoto, Yoshiyuki; Ueno, Yuki; Nakahashi, Emiko; Obayashi, Momoko; Sugihara, Kento; Qiao, Shanlou; Iida, Machiko; Kumasaka, Mayuko Y; Yajima, Ichiro; Goto, Yuji; Ohgami, Nobutaka; Kato, Masashi; Takeda, Kozue

    2016-01-01

    The incidence of allergies has recently been increasing worldwide. Immunoglobulin E (IgE)-mediated hypersensitivity is central to the pathogenesis of asthma, hay fever and other allergic diseases. Ginger (Zingiber officinale Roscoe) and its extracts have been valued for their medical properties including antinausea, antiinflammation, antipyresis and analgesia properties. In this study, we investigated the antiallergic effects of ginger and 6-gingerol, a major compound of ginger, using a mouse allergy model and primary/cell line culture system. In mice with ovalbumin (OVA)-induced allergic rhinitis, oral administration of 2% ginger diet reduced the severity of sneezing and nasal rubbing by nasal sensitization of OVA and suppressed infiltration of mast cells in nasal mucosa and secretion of OVA-specific IgE in serum. 6-Gingerol inhibited the expression of not only Th2 cytokines but also Th1 cytokines in OVA-sensitized spleen cells. Accordingly, 6-gingerol suppressed in vitro differentiation of both Th1 cells and Th2 cells from naïve T cells. In addition, 6-gingerol suppressed both superantigen staphylococcal enterotoxin B (SEB)- and anti-CD3-induced T cell proliferation. 6-Gingerol also abrogated PMA plus ionomycin- and SEB-induced IL-2 production in T cells, suggesting that 6-gingerol affected T cell receptor-mediated signal transduction rather than the antigen-presentation process. Indeed, 6-gingerol inhibited the phosphorylation of MAP kinases, calcium release and nuclear localization of c-fos and NF-κB by PMA and ionomycin stimulation. Thus, our results demonstrate that 6-gingerol suppresses cytokine production for T cell activation and proliferation, thereby not causing B cell and mast cell activation and resulting in prevention or alleviation of allergic rhinitis symptoms. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

    Science.gov (United States)

    Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

    2015-10-13

    Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Bending spring rate investigation of nanopipette for cell injection

    Science.gov (United States)

    Shen, Yajing; Zhang, Zhenhai; Fukuda, Toshio

    2015-04-01

    Bending of nanopipette tips during cell penetration is a major cause of cell injection failure. However, the flexural rigidity of nanopipettes is little known due to their irregular structure. In this paper, we report a quantitative method to estimate the flexural rigidity of a nanopipette by investigating its bending spring rate. First nanopipettes with a tip size of 300 nm are fabricated from various glass tubes by laser pulling followed by focused ion beam (FIB) milling. Then the bending spring rate of the nanopipettes is investigated inside a scanning electron microscope (SEM). Finally, a yeast cell penetration test is performed on these nanopipettes, which have different bending spring rates. The results show that nanopipettes with a higher bending spring rate have better cell penetration capability, which confirms that the bending spring rate may well reflect the flexural rigidity of a nanopipette. This method provides a quantitative parameter for characterizing the mechanical property of a nanopipette that can be potentially taken as a standard specification in the future. This general method can also be used to estimate other one-dimensional structures for cell injection, which will greatly benefit basic cell biology research and clinical applications.

  13. Bending spring rate investigation of nanopipette for cell injection

    International Nuclear Information System (INIS)

    Shen, Yajing; Zhang, Zhenhai; Fukuda, Toshio

    2015-01-01

    Bending of nanopipette tips during cell penetration is a major cause of cell injection failure. However, the flexural rigidity of nanopipettes is little known due to their irregular structure. In this paper, we report a quantitative method to estimate the flexural rigidity of a nanopipette by investigating its bending spring rate. First nanopipettes with a tip size of 300 nm are fabricated from various glass tubes by laser pulling followed by focused ion beam (FIB) milling. Then the bending spring rate of the nanopipettes is investigated inside a scanning electron microscope (SEM). Finally, a yeast cell penetration test is performed on these nanopipettes, which have different bending spring rates. The results show that nanopipettes with a higher bending spring rate have better cell penetration capability, which confirms that the bending spring rate may well reflect the flexural rigidity of a nanopipette. This method provides a quantitative parameter for characterizing the mechanical property of a nanopipette that can be potentially taken as a standard specification in the future. This general method can also be used to estimate other one-dimensional structures for cell injection, which will greatly benefit basic cell biology research and clinical applications. (paper)

  14. Clonal variation in proliferation rate of cultures of GPK cells.

    Science.gov (United States)

    Riley, P A; Hola, M

    1981-09-01

    Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. The mean interdivision time (IDT) for the culture was 1143 +/- 215 (SD) min. The mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93--10.2 x 10(-4)/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. The data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. The mean generation rate of quadrats was 8.73 x 10(-4)/min (SD = 4.9%). The spatial distribution showed some clustering of cells. The mean local density of the clones (2.25 +/- 0.62 cells/10(-4) cm2) was significantly higher than the quadrat density (1.76 +/- 0.8 cells/10(-4) cm2). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P = 2.7%). The results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.

  15. Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck.

    Science.gov (United States)

    Poi, M J; Yen, T; Li, J; Song, H; Lang, J C; Schuller, D E; Pearl, D K; Casto, B C; Tsai, M D; Weghorst, C M

    2001-01-01

    The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or

  16. Investigation of high-rate lithium-thionyl chloride cells

    Science.gov (United States)

    Hayes, Catherine A.; Gust, Steven; Farrington, Michael D.; Lockwood, Judith A.; Donaldson, George J.

    Chemical analysis of a commercially produced high-rate D-size lithium-thionyl cell was carried out, as a function of rate of discharge (1 ohm and 5 ohms), depth of discharge, and temperature (25 C and -40 C), using specially developed methods for identifying suspected minor cell products or impurities which may effect cell performance. These methods include a product-retrieval system which involves solvent extraction to enhance the recovery of suspected semivolatile minor chemicals, and methods of quantitative GC analysis of volatile and semivolatile products. The nonvolatile products were analyzed by wet chemical methods. The results of the analyses indicate that the predominant discharge reaction in this cell is 4Li + 2SOCl2 going to 4LiCl + S + SO2, with SO2 formation decreasing towards the end of cell life (7 to 12 Ah). The rate of discharge had no effect on the product distribution. Upon discharge of the high-rate cell at -40 C, one cell exploded, and all others exhibited overheating and rapid internal pressure rise when allowed to warm up to room temperature.

  17. The action of microsecond-pulsed plasma-activated media on the inactivation of human lung cancer cells

    International Nuclear Information System (INIS)

    Kumar, Naresh; Park, Ji Hoon; Jeon, Su Nam; Park, Bong Sang; Choi, Eun Ha; Attri, Pankaj

    2016-01-01

    In the present work, we have generated reactive species (RS) through microsecond-pulsed plasma (MPP) in the cell culture media using a Marx generator with point–point electrodes of approximately 0.06 J discharge energy/pulse. RS generated in culture media through MPP have a selective action between growth of the H460 lung cancer cells and L132 normal lung cells. We observed that MPP-activated media (MPP-AM) induced apoptosis on H460 lung cancer cells through an oxidative DNA damage cascade. Additionally, we studied the apoptosis-related mRNA expression, DNA oxidation and polymerase-1 (PARP-1) cleaved analysis from treated cancer cells. The result proves that radicals generated through MPP play a pivotal role in the activation of media that induces the selective killing effect. (paper)

  18. Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

    International Nuclear Information System (INIS)

    Do, Minh Truong; Kim, Hyung Gyun; Khanal, Tilak; Choi, Jae Ho; Kim, Dong Hee; Jeong, Tae Cheon; Jeong, Hye Gwang

    2013-01-01

    Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancer A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells. The inactivation of AMPK by siRNA, DN-AMPK or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner. These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anticancer therapy in response to metformin treatment. - Highlights: • Metformin inhibits HO-1 expression in cancer cells. • Metformin attenuates Raf-ERK-Nrf2 signaling. • Suppression of HO-1 by metformin is independent of AMPK. • HO-1 inhibition contributes to anti-proliferative effects of metformin

  19. Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

    International Nuclear Information System (INIS)

    Chen, Zhihong; Forman, Lora W; Williams, Robert M; Faller, Douglas V

    2014-01-01

    A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach. shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA. We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human

  20. Abuse resistant high rate lithium/thionyl chloride cells

    Energy Technology Data Exchange (ETDEWEB)

    Surprenant, J.; Snuggerud, D.

    1982-08-01

    A compact, disc shaped lithium/thionyl chloride cell has been developed by Altus Corporation. The cell has a 6 Amphr capacity and is capable of high rate discharge at high voltage. Discharge data is presented over the range of 0.07 to 1.1 Amperes. The cell is operable over the temperature range of -40/sup 0/C to +70/sup 0/C, and has a 10 year shelf life at 20/sup 0/C. Safety features allow the cells to withstand fire, puncture, shock, spin, forced discharge or forced charge without dangerous reactions.

  1. Abuse resistant high rate lithium/thionyl chloride cells

    Science.gov (United States)

    Surprenant, J.; Snuggerud, D.

    A compact, disk shaped lithium/thionyl chloride cell has been developed. The cell has a 6 Amphr capacity and is capable of high rate discharge at high voltage. Discharge data are presented over the range of 0.07 to 1.1 amperes. The cell is operable over the temperature range of -40 C to +70 C, and has a 10 year shelf life at 20 C. Safety features allow the cells to withstand fire, puncture, shock, spin, forced discharge or forced charge without dangerous reactions.

  2. BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    Directory of Open Access Journals (Sweden)

    Keisuke Taniuchi

    2012-05-01

    Full Text Available We report that Binder of Arl Two (BART plays a role in inhibiting cell invasion by regulating the activity of the Rho small guanosine triphosphatase protein Rac1 in pancreatic cancer cells. BART was originally identified as a binding partner of ADP-ribosylation factor-like 2, a small G protein implicated as a regulator of microtubule dynamics and folding. BART interacts with active forms of Rac1, and the BART-Rac1 complex localizes at the leading edges of migrating cancer cells. Suppression of BART increases active Rac1, thereby increasing cell invasion. Treatment of pancreatic cancer cells in which BART is stably knocked down with a Rac1 inhibitor decreases invasiveness. Thus, BART-dependent inhibition of cell invasion is likely associated with decreased active Rac1. Suppression of BART induces membrane ruffling and lamellipodial protrusion and increases peripheral actin structures in membrane ruffles at the edges of lamellipodia. The Rac1 inhibitor inhibits the lamellipodia formation that is stimulated by suppression of BART. Our results imply that BART regulates actin-cytoskeleton rearrangements at membrane ruffles through modulation of the activity of Rac1, which, in turn, inhibits pancreatic cancer cell invasion.

  3. Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

    Science.gov (United States)

    Misawa, Yuki; Misawa, Kiyoshi; Kawasaki, Hideya; Imai, Atsushi; Mochizuki, Daiki; Ishikawa, Ryuji; Endo, Shiori; Mima, Masato; Kanazawa, Takeharu; Iwashita, Toshihide; Mineta, Hiroyuki

    2017-07-01

    The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

  4. Virally inactivated human platelet concentrate lysate induces regulatory T cells and immunosuppressive effect in a murine asthma model.

    Science.gov (United States)

    Lee, Yueh-Lun; Lee, Lin-Wen; Su, Chen-Yao; Hsiao, George; Yang, Yi-Yuan; Leu, Sy-Jye; Shieh, Ying-Hua; Burnouf, Thierry

    2013-09-01

    Platelet concentrate lysates (PCLs) are increasingly used in regenerative medicine. We have developed a solvent/detergent (S/D)-treated PCL. The functional properties of this preparation should be unveiled. We hypothesized that, due to transforming growth factor-β1 (TGF-β1) content, PCLs may exert immunosuppressive and anti-inflammatory functions. PCL was prepared by S/D treatment, oil extraction, and hydrophobic interaction chromatography. The content of TGF-β in PCL was determined by enzyme-linked immunosorbent assay. Cultured CD4+ T cells were used to investigate the effects of PCL on expression of transcription factor forkhead box P3 (Foxp3), the inhibition of T-cell proliferation, and cytokine production. The regulatory function of PCL-converted CD4+ T cells was analyzed by suppressive assay. The BALB/c mice were given PCL-converted CD4+ T cells before ovalbumin (OVA) sensitization and challenge using an asthma model. Inflammatory parameters, such as the level of immunoglobulin E (IgE), airway hyperresponsiveness (AHR), bronchial lavage fluid eosinophils, and cytokines were assayed. Recombinant human (rHu) TGF-β1 was used as control. PCL significantly enhanced the development of CD4+Foxp3+-induced regulatory T cells (iTregs). Converted iTregs produced neither Th1 nor Th2 cytokines and inhibited normal T-cell proliferation. PCL- and rHuTGF-β-converted CD4+ T cells prevented OVA-induced asthma. PCL- and rHuTGF-β-modified T cells both significantly reduced expression levels of OVA-specific IgE and significantly inhibited the development of AHR, airway eosinophilia, and Th2 responses in mice. S/D-treated PCL promotes Foxp3+ iTregs and exerts immunosuppressive and anti-inflammatory properties. This finding may help to understand the clinical properties of platelet lysates. © 2013 American Association of Blood Banks.

  5. Turnover rates of B cells, T cells, and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques

    NARCIS (Netherlands)

    Boer, R.J. de; Mohri, H.; Ho, D.D.; Perelson, A.S.

    2003-01-01

    We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques

  6. Self-discharge rate of lithium thionyl-chloride cells

    Energy Technology Data Exchange (ETDEWEB)

    Cieslak, W.R.

    1993-12-31

    Our low-rate lithium/thionyl-chloride ``D`` cell is required to provide power continuously for up to 10 years. The cell was designed at Sandia National Laboratories and manufactured at Eagle-Picher Industries, Joplin, Missouri. We have conducted accelerated aging studies at elevated temperatures to predict long-term performance of cells fabricated in 1992. Cells using 1.0M LiAlCl{sub 4} electrolyte follow Arrhenius kinetics with an activation energy of 14.6 Kcal/mol. This results in an annual capacity loss to self-discharge of 0.13 Ah at 25 C. Cells using a 1.0M LiAlCl{sub 4}{sm_bullet}SO{sub 2} electrolyte do not follow Arrhenius behavior. The performance of aged cells from an earlier fabrication lot is variable.

  7. Inactivation of enteroviruses in sewage with ozone

    Energy Technology Data Exchange (ETDEWEB)

    Ivanova, O.E.; Bogdanov, M.V.; Kazantseva, V.A.; Gabrilevskaia, L.N.; Kodkind, G.K.H.

    The study of ozone inactivation of enteroviruses in sewage showed the presence in sewage of suspensions of organic origin and bacterial flora to influence the rate of inactivation. The inactivation rate of poliomyelitis virus in sewage free from organic suspension and bacterial flora was significantly higher than that in sewage containing such suspension and bacterial flora. The inactivation rate of enteroviruses was found not to depend upon the protein and salt composition and pH of sewage or strain appurtenance of viruses. The inactivation rate of enteroviruses directly depended upon the dose of ozone and time of contact with it. Differences in the resistance of different types of poliomyelitis virus, ECHO and Coxsackie viruses to the effect of ozone are likely exist. These differences are manifested within the range of relatively small doses of ozone. E. coli is more resistant to ozone than entero-viruses. The results of laboratory studies were used to choose the regimen of sanitation of urban sewage to be used in technological cycles of industrial enterprises.

  8. Use of In Situ-Generated Dimethyldioxirane for Inactivation of Biological Agents

    National Research Council Canada - National Science Library

    Wallace, William H; Bushway, Karen E; Miller, Susan D; Delcomyn, Carrie A; Renard, Jean J; Henley, Michael V

    2005-01-01

    ...) at neutral pH, was investigated for inactivation of biological warfare agent simulants. The DMDO solution inactivated bacterial spores, fungal spores, vegetative bacterial cells, viruses, and protein by 7 orders of magnitude in less than 10 min...

  9. Yeast cell inactivation related to local heating induced by low-intensity electric fields with long-duration pulses.

    Science.gov (United States)

    Guyot, Stéphane; Ferret, Eric; Boehm, Jean-Baptiste; Gervais, Patrick

    2007-01-25

    The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investigated using a PG200 electroporator (Hoefer Scientific Instrument, San Fransisco, CA, USA) with specific attention to induced thermal effects on cell death. Lethal electric fields (1.5 kV cm(-1) for 5 s) were shown to cause heat variations in the cell suspension medium (water+glycerol), while corresponding classical thermal treatments at equivalent temperatures had no effect on the cells viability. Variations of the electrical conductivity of the intra- and extracellular matrix caused by ions and solutes transfer across the membrane were shown to be involved in the observed heating. The results permitted to build a theoretical model for the temperature variations induced by electric fields. Using this model and the electrical conductivity of the different media, a plausible explanation of the cell death induced by low-intensity electric fields with long-duration pulses has been proposed. Indeed, cell mortality could in part be caused by direct and indirect effects of electric fields. Direct effects are related to well known electromechanical phenomena, whereas indirect effects are related to secondary thermal stress caused by plasma membrane thermoporation. This thermoporation was attributed to electrical conductivity variations and the corresponding intracellular heating.

  10. Ecto-ATPase CD39 Inactivates Isoprenoid-Derived Vγ9Vδ2 T Cell Phosphoantigens

    Directory of Open Access Journals (Sweden)

    Georg Gruenbacher

    2016-07-01

    Full Text Available In humans, Vγ9Vδ2 T cells respond to self and pathogen-associated, diphosphate-containing isoprenoids, also known as phosphoantigens (pAgs. However, activation and homeostasis of Vγ9Vδ2 T cells remain incompletely understood. Here, we show that pAgs induced expression of the ecto-ATPase CD39, which, however, not only hydrolyzed ATP but also abrogated the γδ T cell receptor (TCR agonistic activity of self and microbial pAgs (C5 to C15. Only mevalonate-derived geranylgeranyl diphosphate (GGPP, C20 resisted CD39-mediated hydrolysis and acted as a regulator of CD39 expression and activity. GGPP enhanced macrophage differentiation in response to the tissue stress cytokine interleukin-15. In addition, GGPP-imprinted macrophage-like cells displayed increased capacity to produce IL-1β as well as the chemokine CCL2 and preferentially activated CD161-expressing CD4+ T cells in an innate-like manner. Our studies reveal a previously unrecognized immunoregulatory function of CD39 and highlight a particular role of GGPP among pAgs.

  11. Astragaloside IV Inhibits Oxidative Stress-Induced Mitochondrial Permeability Transition Pore Opening by Inactivating GSK-3β via Nitric Oxide in H9c2 Cardiac Cells

    Directory of Open Access Journals (Sweden)

    Yonggui He

    2012-01-01

    Full Text Available Objective. This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP opening through glycogen synthase kinase 3β (GSK-3β in H9c2 cells. Methods. H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3β (Ser9, Akt (Ser473, and VASP (Ser239 activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨm. Nitric oxide (NO generation was measured by 4-amino-5-methylamino-2′, 7′-difluorofluorescein (DAF-FM diacetate. Fluorescence images were obtained with confocal microscopy. Results. Astragaloside IV significantly enhanced GSK-3β phosphorylation and prevented H2O2-induced loss of ΔΨm. These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury. Conclusions. These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3β through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.

  12. Berberine and a Berberis lycium extract inactivate Cdc25A and induce α-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis

    International Nuclear Information System (INIS)

    Khan, Musa; Giessrigl, Benedikt; Vonach, Caroline; Madlener, Sibylle; Prinz, Sonja; Herbaceck, Irene; Hoelzl, Christine; Bauer, Sabine; Viola, Katharina; Mikulits, Wolfgang; Quereshi, Rizwana Aleem; Knasmueller, Siegfried; Grusch, Michael; Kopp, Brigitte; Krupitza, Georg

    2010-01-01

    Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H 2 O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 μg BuOH extract (that was gained from 1 mg dried root) contained 2.0 μg berberine and 0.3 μg/ml palmatine. 1.2 μg/ml berberine inhibited cell proliferation significantly, while 0.5 μg/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of α-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.

  13. Retinol uptake and esterification in the rate sertoli cell

    International Nuclear Information System (INIS)

    Shingleton, J.L.

    1989-01-01

    The mechanism by which Sertoli cells accumulate retinol from retinol-binding protein (RBP) and the cellular metabolism of the accumulated retinol were investigated here using primary cultures of Sertoli cells isolated from 20 day-old rats. Cells incubated with [ 3 H]retinol-RBP accumulated [ 3 H]retinol in a time- and temperature dependent manner. The rate of [ 3 H] retinol accumulation declined when cellular [ 3 H] retinol concentrations reached approximately 0.53 pmol of retinol per μg of cellular DNA, equivalent to the cellular content of cellular retinol-binding protein (CRBP). Excess unlabeled retinol-RBP competed with [ 3 H] retinol-RBP for [ 3 H] retinol delivery to the cells but free retinol did not. Furthermore, free [ 3 H] retinol associated with Sertoli cells in a non-saturable manner. The transport constant for specific retinol accumulation from RBP was 1.9 μM, suggesting that any change in the normal circulating retinol-RBP level would directly affect the rate of retinol accumulation. Competition studies and studies using labeled RBP, cellular energy inhibitors, and lysosomal poisons indicated that the specific retinol accumulation by Sertoli cells occurs by interaction with a cell-surface receptor that internalizes retinol without concomitant internalization of RBP. Extraction and HPLC analysis of the radioactivity associated with Sertoli cells after incubation with [ 3 H] retinol-RBP yielded retinol and retinyl esters

  14. Tumor cell proliferation kinetics and tumor growth rate

    Energy Technology Data Exchange (ETDEWEB)

    Tubiana, M

    1989-01-01

    The present knowledge on the growth rate and the proliferation kinetics of human tumor is based on the measurement of the tumor doubling times (DT) in several hundred patients and on the determination of the proportion of proliferating cells with radioactive thymidine or by flow cytometry in large numbers of patients. The results show that the DT of human tumor varies widely, from less than one week to over one year with a median value of approximately 2 months. The DTs are significantly correlated with the histological type. They depend upon (1) the duration of the cell cycle whose mean duration is 2 days with small variations from tumor to tumor, (2) the proportion of proliferating cells and consequently the cell birth rate which varies widely among tumors and which is significantly correlated to the DT, (3) the cell loss factors which also vary widely and which are the greatest when proliferation is most intensive. These studies have several clinical implications: (a) they have further increased our understanding of the natural history of human tumor, (b) they have therapeutic implications since tumor responsiveness and curability by radiation and drugs are strongly influenced by the cell kinetic parameters of the tumor, (c) the proportion of proliferating cells is of great prognostic value in several types of human cancers. The investigation of the molecular defects, which are correlated with the perturbation of control of cell proliferation, should lead to significant fundamental and therapeutic advances. (orig.).

  15. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  16. Inactivation of a single copy of Crebbp selectively alters pre-mRNA processing in mouse hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Madeleine E Lemieux

    Full Text Available Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/- FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/- FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs.

  17. Epigenetic inactivation of CHFR in human tumors.

    Science.gov (United States)

    Toyota, Minoru; Sasaki, Yasushi; Satoh, Ayumi; Ogi, Kazuhiro; Kikuchi, Takefumi; Suzuki, Hiromu; Mita, Hiroaki; Tanaka, Nobuyuki; Itoh, Fumio; Issa, Jean-Pierre J; Jair, Kam-Wing; Schuebel, Kornel E; Imai, Kohzoh; Tokino, Takashi

    2003-06-24

    Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

  18. Human T-cell leukemia virus type 1 (HTLV-1 tax requires CADM1/TSLC1 for inactivation of the NF-κB inhibitor A20 and constitutive NF-κB signaling.

    Directory of Open Access Journals (Sweden)

    Rajeshree Pujari

    2015-03-01

    Full Text Available Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1 oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP. K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1 recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

  19. Inactivation of Mycobacterium avium with free chlorine.

    Science.gov (United States)

    Luh, Jeanne; Mariñas, Benito J

    2007-07-15

    The inactivation kinetics of Mycobacterium avium with free chlorine was characterized by two stages: an initial phase at a relatively fast rate followed by a slower second stage of pseudo first-order kinetics. The inactivation rate of each stage was approximately the same for all experiments performed at a certain condition of pH and temperature; however, variability was observed for the disinfectant exposure at which the transition between the two stages occurred. This variability was not a function of the initial disinfectant concentration, the initial bacterial density, or the bacterial stock. However, the transition to the second stage varied more significantly at high temperatures (30 degrees C), while lower variability was observed at lower temperatures (5 and 20 degrees C). Experiments conducted at pH values in the range of 6-9 revealed that the inactivation of M. avium was primarily due to hypochlorous acid, with little contribution from hypochlorite ion within this pH range. The inactivation kinetics was represented with a two-population model. The activation energies for the resulting pseudo first-order rate constants for the populations with fast and slow kinetics were 100.3 and 96.5 kJ/mol, respectively. The magnitude of these values suggested that for waters of relatively high pH and low temperatures, little inactivation of M. avium would be achieved within treatment plants, providing a seeding source for distribution systems.

  20. Survival rate of eukaryotic cells following electrophoretic nanoinjection.

    Science.gov (United States)

    Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

    2017-01-25

    Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.

  1. Survival rate of eukaryotic cells following electrophoretic nanoinjection

    Science.gov (United States)

    Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

    2017-01-01

    Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells. PMID:28120926

  2. Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Cho, O Yeon; Hwang, Hye Sook; Lee, Bok Soon; Oh, Young Taek; Kim, Chul Ho; Chun, Mi Son [Ajou University School of Medicine, Suwon (Korea, Republic of)

    2015-12-15

    Past studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation. Wound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF). This study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling. These results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy.

  3. Inactivation of Orientia tsutsugamushi in red blood cells, plasma, and platelets with riboflavin and light, as demonstrated in an animal model.

    Science.gov (United States)

    Rentas, Francisco; Harman, Ronald; Gomez, Charlotte; Salata, Jeanne; Childs, Joseph; Silva, Tonya; Lippert, Lloyd; Montgomery, Joshua; Richards, Allen; Chan, Chye; Jiang, Ju; Reddy, Heather; Li, John; Goodrich, Raymond

    2007-02-01

    Treatment of blood products with riboflavin and light has been used to reduce the number of certain pathogens. Orientia (formerly Rickettsia) tsutsugamushi, the scrub typhus agent, is an obligate intracellular bacterium that grows free in the cytoplasm of infected cells. This study evaluated the capability of riboflavin and light to inactivate O. tsutsugamushi in red blood cells (RBCs), platelets (PLTs), and plasma, as measured by mouse infectivity. A total of 108 mice, equally divided into groups receiving RBCs, plasma, and PLTs, received untreated products infected with 10(0) to 10(5) organisms. Eighteen mice received products infected with 10(5) organisms and were subsequently treated with riboflavin and light. Mice were monitored daily for up to 17 days for signs and symptoms of infection (e.g., lethargy, labored breathing, rough coat) and killed upon appearance of symptoms or on Day 17 after infection. Real-time polymerase chain reaction (PCR) on blood and Giemsa stains from peritoneal exudates were performed. A total of 102 of 108 mice receiving the untreated products developed signs and symptoms of infection and had positive PCR and Giemsa stain results. None of the 18 animals receiving riboflavin and light-treated blood products exhibited signs or symptoms of infection, nor was infection observed by PCR testing or Giemsa staining. Riboflavin and light are effective in reducing O. tsutsugamushi. Mice injected with blood products inoculated with 10(5) organisms and treated with riboflavin and light did not experience any signs or symptoms of infection, 17 days after inoculation. A 5-log reduction of this organism in blood was achieved as assayed in an animal model.

  4. Numerical evaluation of lactoperoxidase inactivation during continuous pulsed electric field processing.

    Science.gov (United States)

    Buckow, Roman; Semrau, Julius; Sui, Qian; Wan, Jason; Knoerzer, Kai

    2012-01-01

    A computational fluid dynamics (CFD) model describing the flow, electric field and temperature distribution of a laboratory-scale pulsed electric field (PEF) treatment chamber with co-field electrode configuration was developed. The predicted temperature increase was validated by means of integral temperature studies using thermocouples at the outlet of each flow cell for grape juice and salt solutions. Simulations of PEF treatments revealed intensity peaks of the electric field and laminar flow conditions in the treatment chamber causing local temperature hot spots near the chamber walls. Furthermore, thermal inactivation kinetics of lactoperoxidase (LPO) dissolved in simulated milk ultrafiltrate were determined with a glass capillary method at temperatures ranging from 65 to 80 °C. Temperature dependence of first order inactivation rate constants was accurately described by the Arrhenius equation yielding an activation energy of 597.1 kJ mol(-1). The thermal impact of different PEF processes on LPO activity was estimated by coupling the derived Arrhenius model with the CFD model and the predicted enzyme inactivation was compared to experimental measurements. Results indicated that LPO inactivation during combined PEF/thermal treatments was largely due to thermal effects, but 5-12% enzyme inactivation may be related to other electro-chemical effects occurring during PEF treatments. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  5. Selection of inactivation medium for fungal spores in clinical wastes by supercritical carbon dioxide.

    Science.gov (United States)

    Noman, Efaq; Norulaini Nik Ab Rahman, Nik; Al-Gheethi, Adel; Nagao, Hideyuki; Talip, Balkis A; Ab Kadir, Omar

    2018-05-21

    The present study aimed to select the best medium for inactivation of Aspergillus fumigatus, Aspergillus spp. in section Nigri, A. niger, A. terreus var. terreus, A. tubingensis, Penicillium waksmanii, P. simplicissimum, and Aspergillus sp. strain no. 145 spores in clinical wastes by using supercritical carbon dioxide (SC-CO 2 ). There were three types of solutions used including normal saline, seawater, distilled water, and physiological saline with 1% of methanol; each solution was tested at 5, 10, and 20 mL of the water contents. The experiments were conducted at the optimum operating parameters of supercritical carbon dioxide (30 MPa, 75 °C, 90 min). The results showed that the inactivation rate was more effective in distilled water with the presence of 1% methanol (6 log reductions). Meanwhile, the seawater decreases inactivation rate more than normal saline (4.5 vs. 5.1 log reduction). On the other hand, the experiments performed with different volumes of distilled water (5, 10, and 20 mL) indicated that A. niger spores were completely inactivated with 10 mL of distilled water. The inactivation rate of fungal spores decreased from 6 to 4.5 log as the amount of distilled water increased from 10 to 20 mL. The analysis for the spore morphology of A. fumigatus and Aspergillus spp. in section Nigri using scanning electron microscopy (SEM) has revealed the role of temperature and pressure in the SC-CO 2 in the destruction of the cell walls of the spores. It can be concluded that the distilled water represent the best medium for inactivation of fungal spores in the clinical solid wastes by SC-CO 2 .

  6. Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

    Science.gov (United States)

    Selmin, Ornella I; Fang, Changming; Lyon, Adam M; Doetschman, Tom C; Thompson, Patricia A; Martinez, Jesse D; Smith, Jeffrey W; Lance, Peter M; Romagnolo, Donato F

    2016-02-01

    The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells

  7. Independent rate and temporal coding in hippocampal pyramidal cells.

    Science.gov (United States)

    Huxter, John; Burgess, Neil; O'Keefe, John

    2003-10-23

    In the brain, hippocampal pyramidal cells use temporal as well as rate coding to signal spatial aspects of the animal's environment or behaviour. The temporal code takes the form of a phase relationship to the concurrent cycle of the hippocampal electroencephalogram theta rhythm. These two codes could each represent a different variable. However, this requires the rate and phase to vary independently, in contrast to recent suggestions that they are tightly coupled, both reflecting the amplitude of the cell's input. Here we show that the time of firing and firing rate are dissociable, and can represent two independent variables: respectively the animal's location within the place field, and its speed of movement through the field. Independent encoding of location together with actions and stimuli occurring there may help to explain the dual roles of the hippocampus in spatial and episodic memory, or may indicate a more general role of the hippocampus in relational/declarative memory.

  8. Inferring time derivatives including cell growth rates using Gaussian processes

    Science.gov (United States)

    Swain, Peter S.; Stevenson, Keiran; Leary, Allen; Montano-Gutierrez, Luis F.; Clark, Ivan B. N.; Vogel, Jackie; Pilizota, Teuta

    2016-12-01

    Often the time derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the population's growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time derivatives as a function of time from time-series data. Our approach is based on Gaussian processes and applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, and allows interpolation with the corresponding error estimation. As illustrations, we infer growth rates of microbial cells, the rate of assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies. Our algorithm should thus be broadly applicable.

  9. Ultraviolet inactivation of papain

    International Nuclear Information System (INIS)

    Baugher, J.F.; Grossweiner, L.I.

    1975-01-01

    Flash photolysis transient spectra (lambda > 250 nm) of aqueous papain showed that the initial products are the neutral tryptophan radical Trp (lambdasub(max) 510 nm), the tryptophan triplet state 3 Trp (lambdasub(max) 460 nm), the disulfide bridge electron adduct -SS - - (lambdasub(max) 420 nm) and the hydrated electron esub(aq) - . The -SS - - yield was not altered by nitrous oxide or air, indicating that the formation of this product does not involve electrons in the external medium. The original papain preparation was activated by irradiating under nitrogen. The action spectrum supports previous work attributing the low initial activity to blocking of cysteinyl site 25 with a mixed disulfide. Flask lamp irradiation in nitrogen led to activation at low starting activities and inactivation at higher starting activities, while only inactivation at the same quantum yield was observed with air saturation. The results are consistent with photoionization of an essential tryptophyl residue as the key inactivating step. (author)

  10. Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of beta-Lactamase-Producing Escherichia coli

    DEFF Research Database (Denmark)

    Baker, Kristin R.; Sigurdardottir, Helga Høeg; Jana, Bimal

    2017-01-01

    Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid...... was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dap...... for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness...

  11. High rate capability of lithium/silver vanadium oxide cells

    International Nuclear Information System (INIS)

    Takeuchi, E.S.; Zelinsky, M.A.; Keister, P.

    1986-01-01

    High rate characteristics of the lithium/silver vanadium oxide system were investigated in test cells providing four different limiting surface areas. The cells were tested by constant current and constant resistance discharge with current densities ranging from 0.04 to 6.4 mA/cm/sup 2/. The maximum current density under constant resistance and constant current discharges which would deliver 50% of theoretical capacity was determined. The ability of the cells to deliver high current pulses was evaluated by application of 10 second pulses with current densities ranging from 3 to 30 mA/cm/sup 2/. The voltage delay characteristics of the cells were determined after 1 to 3 months of storage at open circuit voltage or under low level background currents. The volumetric and gravimetric energy density of the SVO system is compared to other cathode materials

  12. Photosensitized inactivation of DNA by monochromatic 334-nm radiation in the presence of 2-thiouracil: genetic activity and backbone breaks

    International Nuclear Information System (INIS)

    Peak, M.J.; Ito, A.; Peak, J.G.; Foote, C.S.

    1988-01-01

    Monochromatic 334-nm radiation delivered under aerobic conditions inactivates the genetic activity (ability to transform auxotrophic recipient cells to nutritional prototrophy) of isolated transforming Bacillus subtilis DNA. The presence of superoxide dismutase (SOD), catalase, and mannitol reduces the 334-nm inactivation. The rate of inactivation of the genetic activity by 334-nm radiation is enhanced fivefold by the sensitizer 2-thiouracil (s 2 Ura). This enhancement is substantially reversed when the irradiations are performed in the presence of mannitol, and, to a lesser extent, SOD. Catalase slightly reduces the s 2 Ura enhancement of 334-nm inactivation of transforming activity. Backbone breaks induced in the same DNA by aerobic 334-nm radiation were also enhanced markedly by the presence of s 2 Ura; this enhancement was reversed by the presence of mannitol and, to a lesser extent, SOD during irradiation. Catalase had no effect upon s 2 Ura-enhanced, 334-nm-induced SSBs. Whereas DNA breakage may be responsible for a portion of the inactivation of the DNA by the photosensitized reaction between s 2 Ura and 334-nm radiation, it is not the only inactivating lesion, because the yield of SSBs per lethal hit per unit length of DNA is not constant for all the irradiation conditions studied. (author)

  13. Live vaccinia-rabies virus recombinants, but not an inactivated rabies virus cell culture vaccine, protect B-lymphocyte-deficient A/WySnJ mice against rabies: considerations of recombinant defective poxviruses for rabies immunization of immunocompromised individuals.

    Science.gov (United States)

    Lodmell, Donald L; Esposito, Joseph J; Ewalt, Larry C

    2004-09-03

    Presently, commercially available cell culture rabies vaccines for humans and animals consist of the five inactivated rabies virus proteins. The vaccines elicit a CD4+ helper T-cell response and a humoral B-cell response against the viral glycoprotein (G) resulting in the production of virus neutralizing antibody. Antibody against the viral nucleoprotein (N) is also present, but the mechanism(s) of its protection is unclear. HIV-infected individuals with low CD4+ T-lymphocyte counts and individuals undergoing treatment with immunosuppressive drugs have an impaired neutralizing antibody response after pre- and post-exposure immunization with rabies cell culture vaccines. Here we show the efficacy of live vaccinia-rabies virus recombinants, but not a cell culture vaccine consisting of inactivated rabies virus, to elicit elevated levels of neutralizing antibody in B-lymphocyte deficient A/WySnJ mice. The cell culture vaccine also failed to protect the mice, whereas a single immunization of a vaccinia recombinant expressing the rabies virus G or co-expressing G and N equally protected the mice up to 18 months after vaccination. The data suggest that recombinant poxviruses expressing the rabies virus G, in particular replication defective poxviruses such as canarypox or MVA vaccinia virus that undergo abortive replication in non-avian cells, or the attenuated vaccinia virus NYVAC, should be evaluated as rabies vaccines in immunocompromised individuals.

  14. Method of inactivating reproducible forms of mycoplasma in biological preparations

    International Nuclear Information System (INIS)

    Veber, P.; Jurmanova, K.; Lesko, J.; Hana, L.; Veber, V.

    1978-01-01

    Inactivation of mycoplasms in biological materials was achieved using gamma radiation with a dose rate of 1x10 4 to 5x10 6 rads/h for 1 to 250 hours. The technique is advantageous for allowing the inactivation of the final form of products (tablets, vaccines, etc.). (J.P.)

  15. Inactivation of pathogenic bacteria in food matrices: high pressure processing, photodynamic inactivation and pressure-assisted photodynamic inactivation

    Science.gov (United States)

    Cunha, A.; Couceiro, J.; Bonifácio, D.; Martins, C.; Almeida, A.; Neves, M. G. P. M. S.; Faustino, M. A. F.; Saraiva, J. A.

    2017-09-01

    Traditional food processing methods frequently depend on the application of high temperature. However, heat may cause undesirable changes in food properties and often has a negative impact on nutritional value and organoleptic characteristics. Therefore, reducing the microbial load without compromising the desirable properties of food products is still a technological challenge. High-pressure processing (HPP) can be classified as a cold pasteurization technique, since it is a non-thermal food preservation method that uses hydrostatic pressure to inactivate spoilage microorganisms. At the same time, it increases shelf life and retains the original features of food. Photodynamic inactivation (PDI) is also regarded as promising approach for the decontamination of food matrices. In this case, the inactivation of bacterial cells is achieved by the cytotoxic effects of reactive oxygens species (ROS) produced from the combined interaction of a photosensitizer molecule, light and oxygen. This short review examines some recent developments on the application of HPP and PDI with food-grade photosensitizers for the inactivation of listeriae, taken as a food pathogen model. The results of a proof-of-concept trial of the use of high-pressure as a coadjutant to increase the efficiency of photodynamic inactivation of bacterial endospores is also addressed.

  16. Relationship of milking rate to somatic cell count.

    Science.gov (United States)

    Brown, C A; Rischette, S J; Schultz, L H

    1986-03-01

    Information on milking rate, monthly bucket somatic cell counts, mastitis treatment, and milk production was obtained from 284 lactations of Holstein cows separated into three lactation groups. Significant correlations between somatic cell count (linear score) and other parameters included production in lactation 1 (-.185), production in lactation 2 (-.267), and percent 2-min milk in lactation 2 (.251). Somatic cell count tended to increase with maximum milking rate in all lactations, but correlations were not statistically significant. Twenty-nine percent of cows with milking rate measurements were treated for clinical mastitis. Treated cows in each lactation group produced less milk than untreated cows. In the second and third lactation groups, treated cows had a shorter total milking time and a higher percent 2-min milk than untreated cows, but differences were not statistically significant. Overall, the data support the concept that faster milking cows tend to have higher cell counts and more mastitis treatments, particularly beyond first lactation. However, the magnitude of the relationship was small.

  17. Mutual inactivation of Notch receptors and ligands facilitates developmental patterning.

    Directory of Open Access Journals (Sweden)

    David Sprinzak

    2011-06-01

    Full Text Available Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

  18. Stretching of red blood cells at high strain rates

    Science.gov (United States)

    Mancuso, J. E.; Ristenpart, W. D.

    2017-10-01

    Most work on the mechanical behavior of red blood cells (RBCs) in flow has focused on simple shear flows. Relatively little work has examined RBC deformations in the physiologically important extensional flow that occurs at the entrance to a constriction. In particular, previous work suggests that RBCs rapidly stretch out and then retract upon entering the constriction, but to date no model predicts this behavior for the extremely high strain rates typically experienced there. In this Rapid Communication, we use high speed video to perform systematic measurements of the dynamic stretching behavior of RBCs as they enter a microfluidic constriction. We demonstrate that both the Kelvin-Voigt and Skalak viscoelastic models capture the observed stretching dynamics, up to strain rates as high as 2000 s-1. The results indicate that the effective elastic modulus of the RBC membrane at these strain rates is an order of magnitude larger than moduli measured by micropipette aspiration or other low strain rate techniques.

  19. Safety and tolerability of a cell culture derived trivalent subunit inactivated influenza vaccine administered to healthy children and adolescents: A Phase III, randomized, multicenter, observer-blind study.

    Science.gov (United States)

    Nolan, Terry; Chotpitayasunondh, Tawee; Capeding, Maria Rosario; Carson, Simon; Senders, Shelly David; Jaehnig, Peter; de Rooij, Richard; Chandra, Richa

    2016-01-04

    Cell culture-derived inactivated influenza vaccines (TIVc) are necessary for scale and predictability of production to meet global demand. This study compared the safety and tolerability of TIVc with an egg-derived trivalent influenza vaccine (TIVf) in 4-17 yearolds. A Phase 3 observer blind, multicenter study enrolled 2055 healthy participants randomized 2:1 to receive either TIVc or TIVf, respectively (1372 TIVc and 683 TIVf evaluable subjects). Participants received one dose each on Days 1 and 28 (4-8 year-olds not previously vaccinated [NPV]) or one dose on Day 1 (4-8 and 9-17 yearolds previously vaccinated [PV]). Solicited adverse events (AEs) occurring within 7 days after each vaccination were assessed; participants were followed up for 6 months after their last dose for safety. Most solicited and unsolicited AEs were mild to moderate with vaccine-related SAEs were reported. TIVc and TIVf were similar in percentages of participants reporting solicited reactions in 4-8 years NPV group after the 1st dose: local reactions, TIVc: 48%, TIVf: 43%; systemic reactions, TIVc: 34%, TIVf: 32%; percentages were lower following the 2nd dose in TIVc; local reactions: TIVc: 40%; TIVf: 43%; systemic reactions: TIVc: 21%; TIVf: 22%. In 4-17 years PV group, solicited reactions were lower following TIVf, local reactions: TIVc: 53%; TIVf: 43%; systemic reactions: TIVc: 37%, TIVf: 30%. Injection-site pain was the most common solicited reaction, and was similar following TIVc and TIVf in 4-8 yearolds (TIVc: 56%; TIVf: 55%), and lower following TIVf in 9-17 years group (TIVc: 52%; TIVf: 42%). Reporting of unsolicited AEs was similar for TIVc and TIVf across the two age groups. TIVc was well tolerated and had a safety and reactogenicity profile similar to that of TIVf in healthy 4-17 yearolds (NCT01857206). Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Continuous Production of Isomalto-oligosaccharides by Thermo-inactivated Cells of Aspergillus niger J2 with Coarse Perlite as an Immobilizing Material.

    Science.gov (United States)

    Huang, Zhihua; Li, Zhihong; Su, Yongjian; Zhu, Yongfeng; Zeng, Wei; Chen, Guiguang; Liang, Zhiqun

    2018-02-13

    The coarse perlite 40-80 mesh was selected as an immobilizing material and put into a packed bed reactor (PBR) to continuously convert maltose to isomalto-oligosaccharides (IMOs). The PBR was prepared by mixing the thermo-inactivated cells (TIC) from Aspergillus niger J2 strain with the coarse perlite, then the mixture was put into an overpressure-resistant column. Compared with diatomite 40-80 mesh and thin perlite 80-120 mesh in PBR, coarse perlite was chosen as the best filtration aid, when the ratio of coarse perlite versus TIC was 1:1. The thermal and pH stability of the free and immobilized TIC and the optimum conditions for the transglycosylation reactions were determined. The results show that approximately 75 and 82% and 87 and 91% of α-glucosidase activity were reserved for free and immobilized TIC at temperatures from 30 to 60 °C and pH from 3.00 to 7.00 for 12 h, respectively. With 30% malt syrup under the conditions of 50 °C and pH 4.00, a mini-scale packed bed reactor (Mi-PBR) and medium-scale packed bed reactor (Me-PBR) could continuously produce IMO over 25 and 34 days with the yield of effective IMO (eIMO) ≥ 35% and total IMO (tIMO) ≥ 50%, respectively. The strategy of mixing the coarse perlite with TIC in PBR is a novel approach to continuously produce IMO and has great application potential in industry.

  1. Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

    Science.gov (United States)

    Castiglia, Sara; Mareschi, Katia; Labanca, Luciana; Lucania, Graziella; Leone, Marco; Sanavio, Fiorella; Castello, Laura; Rustichelli, Deborah; Signorino, Elena; Gunetti, Monica; Bergallo, Massimiliano; Bordiga, Anna Maria; Ferrero, Ivana; Fagioli, Franca

    2014-06-01

    Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Validation of γ-radiation and ultraviolet as a new inactivators for foot and mouth disease virus in comparison with the traditional methods

    Science.gov (United States)

    Mahdy, Safy El din; Hassanin, Amr Ismail; Gamal El-Din, Wael Mossad; Ibrahim, Ehab El-Sayed; Fakhry, Hiam Mohamed

    2015-01-01

    Aim: The present work deals with different methods for foot and mouth disease virus (FMDV) inactivation for serotypes O/pan Asia, A/Iran05, and SAT-2/2012 by heat, gamma radiation, and ultraviolet (UV) in comparison with the traditional methods and their effects on the antigenicity of viruses for production of inactivated vaccines. Materials and Methods: FMDV types O/pan Asia, A/Iran05, and SAT-2/2012 were propagated in baby hamster kidney 21 (BHK21) and titrated then divided into five parts; the first part inactivated with heat, the second part inactivated with gamma radiation, the third part inactivated with UV light, the fourth part inactivated with binary ethylamine, and the last part inactivated with combination of binary ethylamine and formaldehyde (BEI+FA). Evaluate the method of inactivation via inoculation in BHK21, inoculation in suckling baby mice and complement fixation test then formulate vaccine using different methods of inactivation then applying the quality control tests to evaluate each formulated vaccine. Results: The effect of heat, gamma radiation, and UV on the ability of replication of FMDV “O/pan Asia, A/Iran05, and SAT-2/2012” was determined through BHK cell line passage. Each of the 9 virus aliquots titer 108 TCID50 (3 for each strain) were exposed to 37, 57, and 77°C for 15, 30, and 45 min. Similarly, another 15 aliquots (5 for each strain) contain 1 mm depth of the exposed samples in petri-dish was exposed to UV light (252.7 nm wavelength: One foot distance) for 15, 30, 45, 60, and 65 min. Different doses of gamma radiation (10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 KGy) were applied in a dose rate 0.551 Gy/s for each strain and repeated 6 times for each dose. FMDV (O/pan Asia, A/Iran05, and SAT-2/2012) were inactivated when exposed to heat ≥57°C for 15 min. The UV inactivation of FMDV (O/pan Asia and SAT-2) was obtained within 60 min and 65 min for type A/Iran05. The ideal dose for inactivation of FMDV (O/pan Asia, A/Iran05

  3. Validation of γ-radiation and ultraviolet as a new inactivators for foot and mouth disease virus in comparison with the traditional methods

    Directory of Open Access Journals (Sweden)

    Safy El din Mahdy

    2015-09-01

    Full Text Available Aim: The present work deals with different methods for foot and mouth disease virus (FMDV inactivation for serotypes O/pan Asia, A/Iran05, and SAT-2/2012 by heat, gamma radiation, and ultraviolet (UV in comparison with the traditional methods and their effects on the antigenicity of viruses for production of inactivated vaccines. Materials and Methods: FMDV types O/pan Asia, A/Iran05, and SAT-2/2012 were propagated in baby hamster kidney 21 (BHK21 and titrated then divided into five parts; the first part inactivated with heat, the second part inactivated with gamma radiation, the third part inactivated with UV light, the fourth part inactivated with binary ethylamine, and the last part inactivated with combination of binary ethylamine and formaldehyde (BEI+FA. Evaluate the method of inactivation via inoculation in BHK21, inoculation in suckling baby mice and complement fixation test then formulate vaccine using different methods of inactivation then applying the quality control tests to evaluate each formulated vaccine. Results: The effect of heat, gamma radiation, and UV on the ability of replication of FMDV "O/pan Asia, A/Iran05, and SAT-2/2012" was determined through BHK cell line passage. Each of the 9 virus aliquots titer 108 TCID50 (3 for each strain were exposed to 37, 57, and 77°C for 15, 30, and 45 min. Similarly, another 15 aliquots (5 for each strain contain 1 mm depth of the exposed samples in petri-dish was exposed to UV light (252.7 nm wavelength: One foot distance for 15, 30, 45, 60, and 65 min. Different doses of gamma radiation (10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 KGy were applied in a dose rate 0.551 Gy/s for each strain and repeated 6 times for each dose. FMDV (O/pan Asia, A/Iran05, and SAT-2/2012 were inactivated when exposed to heat ≥57°C for 15 min. The UV inactivation of FMDV (O/pan Asia and SAT-2 was obtained within 60 min and 65 min for type A/Iran05. The ideal dose for inactivation of FMDV (O/pan Asia, A

  4. SMARCA4 inactivating mutations cause concomitant Coffin–Siris syndrome, microphthalmia and small‐cell carcinoma of the ovary hypercalcaemic type

    Science.gov (United States)

    Mustafa, Noor; Vetro, Annalisa; Notarangelo, Lucia Dora; de Jonge, Hugo; Rinaldi, Berardo; Vergani, Debora; Giglio, Sabrina Rita; Morbini, Patrizia; Zuffardi, Orsetta

    2017-01-01

    Abstract SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin–Siris syndrome (CSS) patients and in almost all small‐cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain‐of‐function or dominant‐negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole‐exome sequencing to study a 15‐year‐old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild‐type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense‐mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non‐truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under‐recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations

  5. SMARCA4 inactivating mutations cause concomitant Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary hypercalcaemic type.

    Science.gov (United States)

    Errichiello, Edoardo; Mustafa, Noor; Vetro, Annalisa; Notarangelo, Lucia Dora; de Jonge, Hugo; Rinaldi, Berardo; Vergani, Debora; Giglio, Sabrina Rita; Morbini, Patrizia; Zuffardi, Orsetta

    2017-09-01

    SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations. © 2017 The Authors. The

  6. Inactivation of Vegetative Cells, but Not Spores, of Bacillus anthracis, B. cereus, and B. subtilis on Stainless Steel Surfaces Coated with an Antimicrobial Silver- and Zinc-Containing Zeolite Formulation

    Science.gov (United States)

    Galeano, Belinda; Korff, Emily; Nicholson, Wayne L.

    2003-01-01

    Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25°C and 80% relative humidity), the zeolite coating produced approximately 3 log10 inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected. PMID:12839825

  7. Inactivation of Bacterial Spores and Vegetative Bacterial Cells by Interaction with ZnO-Fe2O3 Nanoparticles and UV Radiation

    Directory of Open Access Journals (Sweden)

    José Luis Sánchez-Salas

    2017-09-01

    Full Text Available ZnO-Fe2O3 nanoparticles (ZnO-Fe NPs were synthesized and characterized by scanning electron microscopy (SEM, energy dispersive X-ray spectroscopy (EDS and dynamic light scattering (DLS. The generation of chemical reactive hydroxyl radicals (•OH was measured spectrophotometrically (UV-Vis by monitoring of p-nitrosodimethylaniline (pNDA bleaching. Inactivation of E. coli and B. subtilis spores in the presence of different concentrations of ZnO-Fe NPs, under UV365nm or visible radiation, was evaluated. We observed the best results under visible light, of which inactivation of E. coli of about 90% was accomplished in 30 minutes, while B. subtilis inactivation close to 90% was achieved in 120 minutes. These results indicate that the prepared photocatalytic systems are promising for improving water quality by reducing the viability of water-borne microorganisms, including bacterial spores.

  8. Determination of in vitro oxygen consumption rates for tumor cells

    International Nuclear Information System (INIS)

    Cardenas-Navia, L.I.; Moeller, B.J.; Kirkpatrick, J.P.; Laursen, T.A.; Dewhirst, M.W.

    2003-01-01

    To determine pO 2 at the surface of a monolayer of confluent HCT 116 cells, and to then determine consumption rate in vitro by examining the pO 2 profile in media above the cells. Materials and Methods: A recessed-tip polarographic oxygen microelectrode (diameter ∼10μm) was used to measure pO 2 profiles of media above a confluent monolayer of HCT 116 human colon adenocarcinoma cells in a T25 flask exposed to a 95% air, 5% CO 2 mixture. A two-dimensional finite element analysis of the diffusion equation was used to fit the data, thereby extracting a steady-state O 2 consumption rate. The diffusion equation was solved for zeroth and first-order expressions. No-flux boundary conditions were imposed on its bottom and side boundaries and experimental data was used for boundary conditions at the gas-media boundary. All flasks show an O 2 gradient in the media, with a mean (SE) media layer of 1677 (147) μm and a mean pO 2 at the cell layer/media interface of 44 (8) mm Hg (n=9). pO 2 gradient over the entire media layer is 630 (90) mm Hg/cm, equivalent to a consumption rate of 6.3 x 10 -4 (9.0 x 10 -5 ) mm Hg/s. The mean values for the zeroth and first order rate constants are 8.1 x 10 -9 (1.3 x 10 -9 ) g mol O 2 /cm 3 s and 1.0 x 10 3 (0.46 x 10 3 ) /s, respectively. Control experiments in flasks containing no cells show slight gradients in pO 2 of 38 (12) mm Hg/cm, resulting from some O 2 diffusion through the flask into the surrounding water bath. An addition of 10 -3 M NaCN to the media results in a dramatic increase in pO 2 at the cell layer, consistent with a shut-down in respiration. Under normal cell culture conditions there is an O 2 gradient present in the media of cull culture systems, resulting in physiologic O 2 concentrations at the cell layer, despite the non-physiologic O 2 concentration of the gas mixture to which the cell culture system is exposed. This significant (p -6 ) O 2 gradient in the media of cell culture systems is a result of cell O 2

  9. Covariation of metabolic rates and cell size in coccolithophores

    Science.gov (United States)

    Aloisi, G.

    2015-08-01

    Coccolithophores are sensitive recorders of environmental change. The size of their coccosphere varies in the ocean along gradients of environmental conditions and provides a key for understanding the fate of this important phytoplankton group in the future ocean. But interpreting field changes in coccosphere size in terms of laboratory observations is hard, mainly because the marine signal reflects the response of multiple morphotypes to changes in a combination of environmental variables. In this paper I examine the large corpus of published laboratory experiments with coccolithophores looking for relations between environmental conditions, metabolic rates and cell size (a proxy for coccosphere size). I show that growth, photosynthesis and, to a lesser extent, calcification covary with cell size when pCO2, irradiance, temperature, nitrate, phosphate and iron conditions change. With the exception of phosphate and temperature, a change from limiting to non-limiting conditions always results in an increase in cell size. An increase in phosphate or temperature (below the optimum temperature for growth) produces the opposite effect. The magnitude of the coccosphere-size changes observed in the laboratory is comparable to that observed in the ocean. If the biological reasons behind the environment-metabolism-size link are understood, it will be possible to use coccosphere-size changes in the modern ocean and in marine sediments to investigate the fate of coccolithophores in the future ocean. This reasoning can be extended to the size of coccoliths if, as recent experiments are starting to show, coccolith size reacts to environmental change proportionally to coccosphere size. The coccolithophore database is strongly biased in favour of experiments with the coccolithophore Emiliania huxleyi (E. huxleyi; 82 % of database entries), and more experiments with other species are needed to understand whether these observations can be extended to coccolithophores in general. I

  10. Cancer Cell Growth Inhibitory Effect of Bee Venom via Increase of Death Receptor 3 Expression and Inactivation of NF-kappa B in NSCLC Cells

    Directory of Open Access Journals (Sweden)

    Kyung Eun Choi

    2014-07-01

    Full Text Available Our previous findings have demonstrated that bee venom (BV has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well as DAPI and TUNEL assay. Cell death related protein expression was determined with Western blotting. An EMSA was used for nuclear factor kappaB (NF-κB activity assay. BV (1–5 μg/mL inhibited growth of lung cancer cells by induction of apoptosis in a dose dependent manner in lung cancer cell lines A549 and NCI-H460. Consistent with apoptotic cell death, expression of DR3 and DR6 was significantly increased. However, deletion of DRs by small interfering RNA significantly reversed BV induced cell growth inhibitory effects. Expression of pro-apoptotic proteins (caspase-3 and Bax was concomitantly increased, but the NF-κB activity and expression of Bcl-2 were inhibited. A combination treatment of tumor necrosis factor (TNF-like weak inducer of apoptosis, TNF-related apoptosis-inducing ligand, docetaxel and cisplatin, with BV synergistically inhibited both A549 and NCI-H460 lung cancer cell growth with further down regulation of NF-κB activity. These results show that BV induces apoptotic cell death in lung cancer cells through the enhancement of DR3 expression and inhibition of NF-κB pathway.

  11. Inactivation of poliovirus in wastewater sludge with radiation and thermoradiation

    International Nuclear Information System (INIS)

    Ward, R.L.

    1977-01-01

    The effect of sludge on the rate of viral inactivation by radiation and thermoradiation was determined. The virus used for the experiments was the poliovirus type 1 strain CHAT, which was grown in HeLa cells. Radiation, heat, and thermoradiation treatments were carried out in a chamber specifically designed to permit rapid heating and cooling of the samples at the beginning and completion of treatment, respectively. The treated samples were then assayed for plaque-forming units on HeLa cells after sonication in 0.1% sodium dodecylsulfate (SDS). For the radiation treatment virus was diluted 10-fold into PBS containing new sludge, irradiated at 20 0 C with 137 Cs at a dose rate of 30 krads/min, and assayed for infectious virus. The results show that raw sludge is protective of poliovirus against ionizing radiation but that small concentrations of sludge are nearly as protective as large concentrations. When heat and radiation are given simultaneously, however, the amount of protection afforded by sludge is less than the additive effects of the individual treatments. This result is especially evident at low concentrations of sludge. It appears, therefore, that thermoradiation treatment may be an effective way of inactivation viruses in waters containing low concentrations of suspended solids

  12. Co-culture with NK-92MI cells enhanced the anti-cancer effect of bee venom on NSCLC cells by inactivation of NF-κB.

    Science.gov (United States)

    Kollipara, Pushpa Saranya; Kim, Jung Hyun; Won, Dohee; Lee, Sang Min; Sung, Ha Chang; Chang, Hyun Sok; Lee, Kang Tae; Lee, Kang Sik; Park, Mi Hee; Song, Min Jong; Song, Ho Sueb; Hong, Jin Tae

    2014-03-01

    In the present study we experimented on a multimodal therapeutic approach, such as combining chemotherapy agent (Bee venom) with cellular (NK-92MI) immunotherapy. Previously bee venom has been found to show anti-cancer effect in various cancer cell lines. In lung cancer cells bee venom showed an IC(50) value of 3 μg/ml in both cell lines. The co-culture of NK-92MI cell lines with lung cancer cells also show a decrease in viability upto 50 % at 48 h time point. Hence we used bee venom treated NK-92MI cells to co-culture with NSCLC cells and found that there is a further decrease in cell viability upto 70 and 75 % in A549 and NCI-H460 cell lines respectively. We further investigated the expression of various apoptotic and anti-apoptotic proteins and found that Bax, cleaved caspase-3 and -8 were increasing where as Bcl-2 and cIAP-2 was decreasing. The expression of various death receptor proteins like DR3, DR6 and Fas was also increasing. Concomitantly the expression of various death receptor ligands (TNFalpha, Apo3L and FasL) was also increasing of NK-92MI cells after co-culture. Further the DNA binding activity and luciferase activity of NF-κB was also inhibited after co-culture with bee venom treated NK-92MI cell lines. The knock down of death receptors with si-RNA has reversed the decrease in cell viability and NF-κB activity after co-culture with bee venom treated NK-92MI cells. Thus this new approach can enhance the anti-cancer effect of bee venom at a much lower concentration.

  13. Mechanisms of poliovirus inactivation by the direct and indirect effects of ionizing radiation

    International Nuclear Information System (INIS)

    Ward, R.L.

    1980-01-01

    This study was designed to measure the effects of ionizing radiation on poliovirus particles when given under conditions where either direct (in broth) or indirect (in water) effects were predominant. Under direct conditions, inactivation of poliovirus was found to be due primarily to RNA damage, although capsid damage could account for about one-third of the viral inactivation. RNA damage did not appear to be due to strand breakage and therefore was probably caused primarily by base damage or crosslink formation. Capsid damage under direct irradiation conditions did not result in significant alterations of either the sedimentation coefficients or the isoelectric points of the poliovirus particles or detectable modification of the sizes of the viral proteins. It did, however, cause loss of availability to bind to host cells. Under indirect conditions no more than 25% of viral inactivation appeared to be due to RNA damage. However, the sedimentation coefficients and isoelectric points of the viral particles were greatly altered, and their abilities to bind to cells were lost at about three-fourths the rate of loss of infectivity. Capsid damage in this case did result in changes in the sizes of capsid proteins. Therefore, the majority of the radiation inactivation under indirect conditions appeared to be due to protein damage

  14. Multi-Layered TiO2 Films towards Enhancement of Escherichia coli Inactivation

    Directory of Open Access Journals (Sweden)

    Sorachon Yoriya

    2016-09-01

    Full Text Available Crystalline TiO2 has shown its great photocatalytic properties in bacterial inactivation. This work presents a design fabrication of low-cost, layered TiO2 films assembled reactors and a study of their performance for a better understanding to elucidate the photocatalytic effect on inactivation of E. coli in water. The ability to reduce the number of bacteria in water samples for the layered TiO2 composing reactors has been investigated as a function of time, while varying the parameters of light sources, initial concentration of bacteria, and ratios of TiO2 film area and volume of water. Herein, the layered TiO2 films have been fabricated on the glass plates by thermal spray coating prior to screen printing, allowing a good adhesion of the films. Surface topology and crystallographic phase of TiO2 for the screen-printed active layer have been characterized, resulting in the ratio of anatase:rutile being 80:20. Under exposure to sunlight and a given condition employed in this study, the optimized film area:water volume of 1:2.62 has shown a significant ability to reduce the E. coli cells in water samples. The ratio of surface area of photocatalytic active base to volume of water medium is believed to play a predominant role facilitating the cells inactivation. The kinetic rate of inactivation and its behavior are also described in terms of adsorption of reaction species at different contact times.

  15. CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

    Science.gov (United States)

    This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

  16. Use of genetic algorithms for high hydrostatic pressure inactivation ...

    African Journals Online (AJOL)

    ) for high hydrostatic pressure (HHP) inactivation of Bacillus cereus spores, Bacillus subtilis spores and cells, Staphylococcus aureus and Listeria monocytogenes, all in milk buffer, were used to demonstrate the utility of genetic algorithms ...

  17. Place cell rate remapping by CA3 recurrent collaterals.

    Directory of Open Access Journals (Sweden)

    Trygve Solstad

    2014-06-01

    Full Text Available Episodic-like memory is thought to be supported by attractor dynamics in the hippocampus. A possible neural substrate for this memory mechanism is rate remapping, in which the spatial map of place cells encodes contextual information through firing rate variability. To test whether memories are stored as multimodal attractors in populations of place cells, recent experiments morphed one familiar context into another while observing the responses of CA3 cell ensembles. Average population activity in CA3 was reported to transition gradually rather than abruptly from one familiar context to the next, suggesting a lack of attractive forces associated with the two stored representations. On the other hand, individual CA3 cells showed a mix of gradual and abrupt transitions at different points along the morph sequence, and some displayed hysteresis which is a signature of attractor dynamics. To understand whether these seemingly conflicting results are commensurate with attractor network theory, we developed a neural network model of the CA3 with attractors for both position and discrete contexts. We found that for memories stored in overlapping neural ensembles within a single spatial map, position-dependent context attractors made transitions at different points along the morph sequence. Smooth transition curves arose from averaging across the population, while a heterogeneous set of responses was observed on the single unit level. In contrast, orthogonal memories led to abrupt and coherent transitions on both population and single unit levels as experimentally observed when remapping between two independent spatial maps. Strong recurrent feedback entailed a hysteretic effect on the network which diminished with the amount of overlap in the stored memories. These results suggest that context-dependent memory can be supported by overlapping local attractors within a spatial map of CA3 place cells. Similar mechanisms for context-dependent memory may

  18. The rate of spontaneous mutations in human myeloid cells

    International Nuclear Information System (INIS)

    Araten, David J.; Krejci, Ondrej; DiTata, Kimberly; Wunderlich, Mark; Sanders, Katie J.; Zamechek, Leah; Mulloy, James C.

    2013-01-01

    Highlights: • We provide the first measurement of the mutation rate (μ) in human myeloid cells. • μ is measured to be 3.6–23 × 10 −7 per cell division. • The AML-ETO and MLL-AF9 fusions do not seem to increase μ. • Cooperating mutations in NRAS, FLT3 and p53 not seem to increase μ. • Hypermutability may be required to explain leukemogenesis. - Abstract: The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼9.4 × 10 −7 (range ∼3.6–23 × 10 −7 ) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis

  19. Oxygen transfer rate identifies priming compounds in parsley cells.

    Science.gov (United States)

    Schilling, Jana Viola; Schillheim, Britta; Mahr, Stefan; Reufer, Yannik; Sanjoyo, Sandi; Conrath, Uwe; Büchs, Jochen

    2015-11-25

    In modern agriculture, the call for an alternative crop protection strategy increases because of the desired reduction of fungicide and pesticide use and the continuously evolving resistance of pathogens and pests to agrochemicals. The direct activation of the plant immune system does not provide a promising plant protection measure because of high fitness costs. However, upon treatment with certain natural or synthetic compounds, plant cells can promote to a fitness cost-saving, primed state of enhanced defense. In the primed state, plants respond to biotic and abiotic stress with faster and stronger activation of defense, and this is often associated with immunity and abiotic stress tolerance. Until now, the identification of chemical compounds with priming-inducing activity (so-called plant activators) relied on tedious and invasive approaches, or required the late detection of secreted furanocoumarin phytoalexins in parsley cell cultures. Thus, simple, fast, straightforward, and noninvasive techniques for identifying priming-inducing compounds for plant protection are very welcome. This report demonstrates that a respiration activity-monitoring system (RAMOS) can identify compounds with defense priming-inducing activity in parsley cell suspension in culture. RAMOS relies on the quasi-continuous, noninvasive online determination of the oxygen transfer rate (OTR). Treatment of parsley culture cells with the known plant activator salicylic acid (SA), a natural plant defense signal, resulted in an OTR increase. Addition of the defense elicitor Pep13, a cell wall peptide of Phythophthora sojae, induced two distinctive OTR peaks that were higher in SA-primed cells than in unprimed cells upon Pep13 challenge. Both, the OTR increase after priming with SA and the Pep13 challenge were dose-dependent. Furthermore, there was a close correlation of a compound's activity to enhance the oxygen consumption in parsley cells and its capacity to prime Pep13-induced furanocoumarin

  20. Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima

    2018-01-01

    The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

    Science.gov (United States)

    Reeve, C A; Baldwin, T O

    1981-06-01

    Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.

  2. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Wogonin induced G1 cell cycle arrest by regulating Wnt/β-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells

    International Nuclear Information System (INIS)

    He, Licheng; Lu, Na; Dai, Qinsheng; Zhao, Yue; Zhao, Li; Wang, Hu; Li, Zhiyu; You, Qidong; Guo, Qinglong

    2013-01-01

    Highlights: • Wogonin inhibited HCT116 cells growth and arrested at G1 phase of the cell cycle. • Wogonin down-regulated the canonical Wnt/β-catenin signaling pathway. • Wogonin interfered in the combination of β-catenin and TCF/Lef. • Wogonin limited the kinase activity of CDK8. - Abstract: Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/β-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted β-catenin for proteasomal degradation. Wogonin inhibited β-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/β-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer

  4. Measurement of blowdown flow rates using load cells

    International Nuclear Information System (INIS)

    Dolas, P.K.; Venkat Raj, V.; Ghosh, A.K.; Murty, L.G.K.; Muralidhar Rao, S.

    1980-01-01

    To establish a reliable method for measuring two-phase flow, experiments were planned for measurement of transient single phase flow rates from vessels using load cells. Suitability of lead-zirconate-titanate piezoelectric ceramic discs was examined. Discharge time constant of the disc used was low, leading to large measurement errors. Subsequently, experiments were carried out using strain gauge load cells and these were found satisfactory. The unsteady flow equation has been derived for the system under investigation. The equation has been solved numerically using the fourth order Runge-Kutta method and also by integrating it analytically. The experimental results are compared with the theoretical results and presented in this report. (auth.)

  5. Elevated O-GlcNAcylation stabilizes FOXM1 by its reduced degradation through GSK-3β inactivation in a human gastric carcinoma cell line, MKN45 cells.

    Science.gov (United States)

    Inoue, Yosuke; Moriwaki, Kazumasa; Ueda, Yasuhiro; Takeuchi, Toshihisa; Higuchi, Kazuhide; Asahi, Michio

    2018-01-08

    O-GlcNAcylation is a dynamic post-translational modification of cytonuclear proteins for intracellular signaling. Elevated O-GlcNAcylation is a general feature of cancer and contributes to cancer progression, and recent studies indicate the contribution to increasing incidence of various types of cancer in diabetic patients. However, the role of O-GlcNAcylation in tumor progression is not fully elucidated. Forkhead box M1 (FOXM1), a master mitotic transcription factor, has been implicated in all major hallmarks of cancer, and is wildly expressed in solid tumors. Given that FOXM1 expression was reported to be elevated in gastric cancer, we examined the effect of high glucose or an inhibitor of O-GlcNAc hydrolase, Thiamet G (TMG), on FOXM1 protein expression in a human gastric cancer cell line, MKN45 cells, and confirmed that FOXM1 protein level and the cell proliferation were upregulated. To investigate the molecular mechanisms by which FOXM1 protein expression is regulated by O-GlcNAcylation, the effect of high glucose and TMG on FOXM1 ubiquitination was examined in MKN45 cells. As a result, the ubiquitination and degradation of FOXM1 protein were both suppressed by high glucose and TMG treatment. However, the O-GlcNAcylation was not detected on FOXM1 but not on GSK-3β. High glucose and TMG treatment increased phospho-serine 9 GSK-3β, an inactive form, and the degradation of FOXM1 protein was suppressed by treatment of GSK-3β inhibitors in MKN45 cells. Taken together, we suggest that high glucose and elevated O-GlcNAcylation stabilize FOXM1 protein by its reduced degradation via GSK-3β inactivation in MKN45 cells, suggesting that the higher risk of gastric cancer in diabetic patients could be partially due to O-GlcNAcylation-mediated FOXM1 stabilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Chemotherapeutic drugs sensitize human renal cell carcinoma cells to ABT-737 by a mechanism involving the Noxa-dependent inactivation of Mcl-1 or A1

    Directory of Open Access Journals (Sweden)

    Zantl Niko

    2010-06-01

    Full Text Available Abstract Background Human renal cell carcinoma (RCC is very resistant to chemotherapy. ABT-737 is a novel inhibitor of anti-apoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. Results We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposide-treatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. Conclusions Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells.

  7. Cortical inactivation by cooling in small animals

    Directory of Open Access Journals (Sweden)

    Ben eCoomber

    2011-06-01

    Full Text Available Reversible inactivation of the cortex by surface cooling is a powerful method for studying the function of a particular area. Implanted cooling cryoloops have been used to study the role of individual cortical areas in auditory processing of awake-behaving cats. Cryoloops have also been used in rodents for reversible inactivation of the cortex, but recently there has been a concern that the cryoloop may also cool non-cortical structures either directly or via the perfusion of blood, cooled as it passed close to the cooling loop. In this study we have confirmed that the loop can inactivate most of the auditory cortex without causing a significant reduction in temperature of the auditory thalamus or other sub-cortical structures. We placed a cryoloop on the surface of the guinea pig cortex, cooled it to 2°C and measured thermal gradients across the neocortical surface. We found that the temperature dropped to 20-24°C among cells within a radius of about 2.5mm away from the loop. This temperature drop was sufficient to reduce activity of most cortical cells and led to the inactivation of almost the entire auditory region. When the temperature of thalamus, midbrain, and middle ear were measured directly during cortical cooling, there was a small drop in temperature (about 4°C but this was not sufficient to directly reduce neural activity. In an effort to visualise the extent of neural inactivation we measured the uptake of thallium ions following an intravenous injection. This confirmed that there was a large reduction of activity across much of the ipsilateral cortex and only a small reduction in subcortical structures.

  8. The quantitative description of radiation-unduced cell inactivation. 9. Some remarks on the relative biological effect of ionizing radiation upon reproductive death of diploid and polyploid cells

    International Nuclear Information System (INIS)

    Barsukov, V.S.; Malinovskij, O.V.

    1978-01-01

    A model of repproductive death of cells is suggested and tested on the basis of data on relative biological efficiency (RBE) with provision for microdosimetry approaches. The model considers irradiation of cells with charged particles under conditions of linear energy transfers (LET) to the cell nucleus by a particle. This energy is subject to great fluctuations, and irradiation of a cell should be characterized not by the absorbed dose but rather by its stochastic analogue - specific energy. It appears then that RBE should be determined from the specific energy-response relationshjp rather than from dose-response relationship. The followjng main results were obtained: experimental relationship between the yield of sublesions, one-track and two-track lethals and LET correspond to that expected from the literature data; under the conditions considered, RBE can be determined from absorbed dose-pesponse dependency; RBE for sublesions, one- and two-track lethals taken separatly does not depend on the radiation dose and the relationshjp between the resulting RBE and the response is governed by redistribution of contributions of one- and two-track lethals at different doses. The plausibility of the model suggested is thus confirmed

  9. Influence of Cell-Cell Interactions on the Population Growth Rate in a Tumor

    Science.gov (United States)

    Chen, Yong

    2017-12-01

    The understanding of the macroscopic phenomenological models of the population growth at a microscopic level is important to predict the population behaviors emerged from the interactions between the individuals. In this work, we consider the influence of the population growth rate R on the cell-cell interaction in a tumor system and show that, in most cases especially small proliferative probabilities, the regulative role of the interaction will be strengthened with the decline of the intrinsic proliferative probabilities. For the high replication rates of an individual and the cooperative interactions, the proliferative probability almost has no effect. We compute the dependences of R on the interactions between the cells under the approximation of the nearest neighbor in the rim of an avascular tumor. Our results are helpful to qualitatively understand the influence of the interactions between the individuals on the growth rate in population systems. Supported by the National Natural Science Foundation of China under Grant Nos. 11675008 and 21434001

  10. Inactivated yellow fever 17D vaccine: development and nonclinical safety, immunogenicity and protective activity.

    Science.gov (United States)

    Monath, Thomas P; Lee, Cynthia K; Julander, Justin G; Brown, Alicja; Beasley, David W; Watts, Douglas M; Hayman, Edward; Guertin, Patrick; Makowiecki, Joseph; Crowell, Joseph; Levesque, Philip; Bowick, Gavin C; Morin, Merribeth; Fowler, Elizabeth; Trent, Dennis W

    2010-05-14

    In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age 60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with beta-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX [geometric mean titer (GMT) 20,480 vs. 1940, respectively (Pvaccine or a single dose of YF-VAX were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Influence of cell detachment on the respiration rate of tumor and endothelial cells.

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

  12. Influence of Cell Detachment on the Respiration Rate of Tumor and Endothelial Cells

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F.; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study. PMID:23382841

  13. Maintenance of mesenchymal stem cells culture due to the cells with reduced attachment rate

    Directory of Open Access Journals (Sweden)

    Shuvalova N. S.

    2013-01-01

    Full Text Available Aim. The classic detachment techniques lead to changes in cells properties. We offer a simple method of cultivating the population of cells that avoided an influence on the surface structures. Methods. Mesenchymal stem cells (MSC from human umbilical cord matrix were obtained and cultivated in standard conditions. While substituting the culture media by a fresh portion, the conditioned culture medium, where the cells were maintained for three days, was transferred to other culture flacks with addition of serum and growth factors. Results. In the flacks, one day after medium transfer, we observed attached cells with typical MSC morphology. The cultures originated from these cells had the same rate of surface markers expression and clonogenic potential as those replated by standard methods. Conclusions. MSC culture, derived by preserving the cells with reduced attachment ability, actually has the properties of «parent» passage. Using this method with accepted techniques of cells reseeding would allow maintaining the cells that avoided an impact on the cell surface proteins.

  14. Dengue virus inactivation by minipool TnBP/Triton X-45 treatment of plasma and cryoprecipitate.

    Science.gov (United States)

    Burnouf, T; Chou, M-L; Cheng, L-H; Li, Z-R; Wu, Y-W; El-Ekiaby, M; Tsai, K-H

    2013-01-01

    A minipool solvent/detergent (S/D; 1% TnBP/1% Triton X-45; 31°C) process was developed for viral inactivation of plasma and cryoprecipitate used for transfusion. The goal of this study was to determine the rate and extent of inactivation of dengue virus (DENV) during this process. DENV-1 was propagated using C6/36 mosquito cells to an infectivity titre close to 9 log and spiked (10% v/v) into individual plasma and cryoprecipitate samples from two distinct donors. Samples were taken right after spiking and during viral inactivation treatment by 1% TnBP-1% Triton X-45 at 31°C. DENV-1 infectivity was assessed on Vero E6 cells by a focus-forming assay (FFA). Culture medium and complement-inactivated plasma were used as experimental controls. Experiments were done in duplicate. DENV-1 infectivity was 7·5 log in spiked plasma and 7·1 and 7·3 log in spiked cryoprecipitate. There was no loss of DENV-1 infectivity in the spiked materials, nor in the controls not subjected to S/D treatment. No infectivity was found in plasma and cryoprecipitate subjected to S/D treatment at the first time-point evaluated (10 min). DENV-1 was strongly inactivated in plasma and cryoprecipitate, respectively, within 10 min of 1% TnBP/1% Triton X-45 treatment at 31°C. These data provide a reassurance of the safety of such S/D-treated plasma and cryoprecipitate with regard to the risk of transmission of all DENV serotypes and other flaviviruses. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

  15. Epigenetic inactivation of CHFR in human tumors

    OpenAIRE

    Toyota, Minoru; Sasaki, Yasushi; Satoh, Ayumi; Ogi, Kazuhiro; Kikuchi, Takefumi; Suzuki, Hiromu; Mita, Hiroaki; Tanaka, Nobuyuki; Itoh, Fumio; Issa, Jean-Pierre J.; Jair, Kam-Wing; Schuebel, Kornel E.; Imai, Kohzoh; Tokino, Takashi

    2003-01-01

    Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, w...

  16. Inactivation of viruses in municipal effluent by chlorine.

    OpenAIRE

    Hajenian, H. G.; Butler, M.

    1980-01-01

    The influence of pH and temperature on the efficiency of chlorine inactivation of two unrelated picornaviruses in a typical urban wastewater effluent was examined. Temperature, unlike pH, had relatively little effect on the rate of inactivation. The pH effect was complex and the two viruses differed. The f2 coliphage was more sensitive to chlorine at low pH, but at all values there was a threshold above which additional chlorine resulted in very rapid inactivation. The amount of chlorine requ...

  17. Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

    Science.gov (United States)

    Schenk-Meuser, K; Pawlowsky, K; Kiefer, J

    1992-07-15

    The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.

  18. Effect of different saccharides on growth, sporulation rate and d ...

    African Journals Online (AJOL)

    MFCS

    2012-05-17

    May 17, 2012 ... general, high sporulation rate was related with high growth rate and high viable cell count (>1.5 x 1012 cfu/ml). .... The sterile culture medium (180 ml) in a 1000 ml Erlenmeyer flask was ... The column temperature was set at 85°C. A series of ..... inactivation of certain sugar-metabolizing operons, such as lac ...

  19. A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

    Science.gov (United States)

    Filatova, Lyubov Y; Donovan, David M; Ishnazarova, Nadiya T; Foster-Frey, Juli A; Becker, Stephen C; Pugachev, Vladimir G; Balabushevich, Nadezda G; Dmitrieva, Natalia F; Klyachko, Natalia L

    2016-10-01

    A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal ( opt ) conditions: pH opt 6.0-10.0, t opt 20-30 °C, NaCl opt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

  20. Inactivation of the Radiation-Resistant Spoilage Bacterium Micrococcus radiodurans

    Science.gov (United States)

    Duggan, D. E.; Anderson, A. W.; Elliker, P. R.

    1963-01-01

    A simplified technique permitting the pipetting of raw puréed meats for quantitative bacteriological study is described for use in determining survival of these non-sporing bacteria, which are exceptionally resistant to radiation. Survival curves, using gamma radiation as the sterilizing agent, were determined in raw beef with four strains of Micrococcus radiodurans. Survival curves of the R1 strain in other meat substrates showed that survival was significantly greater in raw beef and raw chicken than in raw fish or in cooked beef. Resistance was lowest in the buffer. Cells grown in broth (an artificial growth medium) and resuspended in beef did not differ in resistance from cells that had been grown and irradiated in beef. Survival rate was statistically independent of the initial cell concentration, even though there appeared to be a correlation between lower death rate and lower initial cell concentrations. The initial viable count of this culture of the domesticated R1 strain in beef was reduced by a factor of about 10-5 by 3.0 megarad, and 4.0 megarad reduced the initial count by a factor of more than 10-9. Data suggest that M. radiodurans R1 is more resistant to radiation than spore-forming spoilage bacteria for which inactivation rates have been published. PMID:14063780

  1. Performance and Abuse Testing of 5 Year Old Low Rate and Medium Rate Lithium Thionyl Chloride Cells

    Science.gov (United States)

    Frerker, Rick; Zhang, Wenlin; Jeevarajan, Judith; Bragg, Bobby J.

    2001-01-01

    Most cells survived the 3 amp (A) over-discharge at room temperature for 2 hours. The cell that failed was the LTC-114 after high rate discharge of 500 mA similar to the results of the 1 A over-discharge test. Most cells opened during 0.05 Ohm short circuit test without incident but three LTC-111 cells exploded apparently due to a lack of a thermal cutoff switch. The LTC-114 cells exposed to a hard short of 0.05 Ohms recovered but the LTC-114 cells exposed to a soft short of 1 Ohm did not. This is probably due to the activation of a resetable fuse during a hard short. Fresh cells tend to survive exposure to higher temperatures than cells previously discharged at high rate (1 Amp). LTC-111 cells tend to vent at lower temperatures than the all LTC-114 cells and the LTC-115 cells that were previously discharged at rates exceeding 1 Amp.

  2. Dose-rate effects on the cell cycle and survival of S3 HeLa and V79 cells

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Bedford, J.S.; Bailey, S.M.

    1979-01-01

    The effects of continuous irradiation at different dose rates on the cell cycle and on cell survival were studied using synchronized S3 HeLa and V79 cells. The minimum dose rate necessary to stop cell division was found to be approximately 23 rad/hr for HeLa cells and 270 rad/hr for V79 cells. For dose rates that stop cell division, cells progress through G 1 and S, with a small delay in the S phase, and are blocked in G 2 . Appreciable mitotic accumulation was observed for HeLa cells at dose rates which stopped cell division. By comparison, much less mitotic accumulation was observed for V79 cells over a range of dose rates from 37 to 270 rad/hr. Minimum mitotic delays for a variety of dose rates were determined for both cell lines. S3 HeLa cells are much more sensitive in this respect than V79 cells; however, it appeared that for higher dose rates the minimum mitotic delay in HeLa cells asymptotically approached a value of about 35 hr. In addition to the qualitative differences observed for the two cell lines in regard to mitotic accumulation, HeLa cells accumulated for prolonged periods in the presence of colcemid while V79 cells were blocked for only a few hours, HeLa cells show a dramatic effect of redistribution of cells into sensitive phases of the cell cycle during exposure, which was reflected in the survival curves at low dose rate. More cell killing per unit dose was observed at 37 than at 74 rad/hr

  3. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation

    International Nuclear Information System (INIS)

    Elliott, L.H.; McCormick, J.B.; Johnson, K.M.

    1982-01-01

    Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with 60 CO gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of 60 CO radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose per rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. The authors found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents

  4. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation

    International Nuclear Information System (INIS)

    Elliott, L.H.; McCormick, J.B.; Johnson, K.M.

    1982-01-01

    Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with 60 Co gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of 60 Co radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose per rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. We found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents

  5. Role of dopant concentration, crystal phase and particle size on microbial inactivation of Cu-doped TiO{sub 2} nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Sahu, Manoranjan; Wu Bing; Zhu Liying; Jacobson, Craig; Wang Weining; Jones, Kristen; Goyal, Yogesh; Tang, Yinjie J; Biswas, Pratim, E-mail: pbiswas@wustl.edu [Department of Energy, Environmental and Chemical Engineering, Washington University in St Louis, St Louis, MO 63130 (United States)

    2011-10-14

    The properties of Cu-doped TiO{sub 2} nanoparticles (NPs) were independently controlled in a flame aerosol reactor by varying the molar feed ratios of the precursors, and by optimizing temperature and time history in the flame. The effect of the physico-chemical properties (dopant concentration, crystal phase and particle size) of Cu-doped TiO{sub 2} nanoparticles on inactivation of Mycobacterium smegmatis (a model pathogenic bacterium) was investigated under three light conditions (complete dark, fluorescent light and UV light). The survival rate of M. smegmatis (in a minimal salt medium for 2 h) exposed to the NPs varied depending on the light irradiation conditions as well as the dopant concentrations. In dark conditions, pristine TiO{sub 2} showed insignificant microbial inactivation, but inactivation increased with increasing dopant concentration. Under fluorescent light illumination, no significant effect was observed for TiO{sub 2}. However, when TiO{sub 2} was doped with copper, inactivation increased with dopant concentration, reaching more than 90% (>3 wt% dopant). Enhanced microbial inactivation by TiO{sub 2} NPs was observed only under UV light. When TiO{sub 2} NPs were doped with copper, their inactivation potential was promoted and the UV-resistant cells were reduced by over 99%. In addition, the microbial inactivation potential of NPs was also crystal-phase-and size-dependent under all three light conditions. A lower ratio of anatase phase and smaller sizes of Cu-doped TiO{sub 2} NPs resulted in decreased bacterial survival. The increased inactivation potential of doped TiO{sub 2} NPs is possibly due to both enhanced photocatalytic reactions and leached copper ions.

  6. In vitro and in vivo anti-microbial activity evaluation of inactivated cells of Lactobacillus salivarius CECT 5713 against Streptococcus mutans.

    Science.gov (United States)

    Sañudo, Ana I; Luque, Roberto; Díaz-Ropero, Mª Paz; Fonollá, Juristo; Bañuelos, Óscar

    2017-12-01

    Defining the etiology of dental caries is a complex problem. The microbiological approach has included Streptococcus mutans as one of the bacterial species involved in this disease. This research investigates the inhibitory effects of heat-inactivated Lactobacillus salivarius CECT 5713 against S. mutans using in vitro and in vivo assays. On the one hand, the effect of non-viable L. salivarius CECT 5713 on the in vitro adhesion of S. mutans to hydroxyapatite discs was evaluated. On the other hand, levels of Streptococcus mutans, amount of salivary flow and salivary pH before and after taking the rinse with the non-viable L. salivarius CECT 5713 in healthy volunteers were assessed (self-controlled open-label pilot study). The levels of S. mutans seemed to decrease in the in vitro and in vivo assays (p<0.05). The in vitro effect of non-viable L. salivarius was maintained until 36 months of storage. In addition, the reduction of S. mutans salivary concentration in the volunteers was statistically significant from the third day until two weeks of treatment. Heat-inactivated L. salivarius CECT 5713 prevents S. mutans adhesion to hydroxyapatite and could be used as a strategy to reduce the salivary concentration of this oral pathogen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

    Science.gov (United States)

    Grant, I R; Ball, H J; Neill, S D; Rowe, M T

    1996-01-01

    The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions. PMID:8593064

  8. Inactivation of Microorganisms

    Science.gov (United States)

    Alzamora, Stella Maris; Guerrero, Sandra N.; Schenk, Marcela; Raffellini, Silvia; López-Malo, Aurelio

    Minimal processing techniques for food preservation allow better retention of product flavor, texture, color, and nutrient content than comparable conventional treatments. A wide range of novel alternative physical factors have been intensely investigated in the last two decades. These physical factors can cause inactivation of microorganisms at ambient or sublethal temperatures (e.g., high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, and ultraviolet light). These technologies have been reported to reduce microorganism population in foods while avoiding the deleterious effects of severe heating on quality. Among technologies, high-energy ultrasound (i.e., intensities higher than 1 W/cm2, frequencies between 18 and 100 kHz) has attracted considerable interest for food preservation applications (Mason et al., 1996; Povey and Mason, 1998).

  9. Mean inactivation dose (D)

    International Nuclear Information System (INIS)

    Vijayakumar, S.; Ng, T.C.; Raudkivi, U.; Meaney, T.J.

    1990-01-01

    By predicting treatment outcome to radiotherapy from in vitro radiobiological parameters, not only individual patient treatments can be tailored, but also new promising treatment protocols can be tried in patients in whom unfavorable outcome is predicted. In this respect, choosing the right parameter can be very important. Unlike D 0 and N which provide information of the distal part of the survival curve, mean inactivation dose (D) estimates overall radiosensitivity. However, the parameters reflecting the response at the clinically relevant low-dose region are neglected in the literature. In a literature survey of 98 papers in which survival curves or D 0 /N were used, only in 2 D was used. In 21 papers the D 0 /n values were important in drawing conclusions. By calculating D in 3 of these 21 papers, we show that the conclusion drawn may be altered with the use of D. The importance of ''low-dose-region-parameters'' is reviewed. (orig.)

  10. γ-ray dose rate effect in DNA double-strand break repair deficient murine cells

    International Nuclear Information System (INIS)

    Li Liya; Li Peiwen

    2002-01-01

    Objective: To analyze the dose rate effect and potentially lethal damage repair in DNA double-strand break repair deficient murine cells (SCID) irradiated by γ-ray. Methods: The wild type (CB.17+/+) and SCID cells were exposed to γ-ray at high and low dose rates. The high dose rate exposure was fractionated into two equal doses at 24 h intervals. The survival rates of irradiated cells were calculated by clone-forming analysis. Results: When γ-ray was given to wild type (CB.17+/+) cells in two fractions at 24 h intervals, the survival rate was significantly higher than that when the same total dose was given singly. In contrast, there was no difference in the survival rates between the single and fractionated exposure in SCID cells. SCID cells were more sensitive than CB.17+/+ cells to both low and high dose rates γ-ray exposure for cell killing. The survival rate by low dose rate exposure was significantly higher than that by high dose rate exposure, not only in CB.17+/+ cells but also in SCID cells. Conclusions: SCID cells are deficient in repairing γ-ray induced double-strand breaks. There is dose rate effect in both SCID and CB.17+/+ cells

  11. PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence.

    Science.gov (United States)

    Byun, H-O; Jung, H-J; Kim, M-J; Yoon, G

    2014-09-01

    Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

  12. Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine.

    Science.gov (United States)

    Dima, V F; Ionescu, M D; Palade, R; Balotescu, C; Becheanu, G; Dima, S V

    2001-01-01

    In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.

  13. Survival rate of eukaryotic cells following electrophoretic nanoinjection

    OpenAIRE

    Simonis, Matthias; H?bner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

    2017-01-01

    Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for...

  14. Evaluation of Hematopoietic Stem Cell Mobilization Rates with Early Plerixafor Administration for Adult Stem Cell Transplantation.

    Science.gov (United States)

    Stover, Jessica T; Shaw, J Ryan; Kuchibhatla, Maragatha; Horwitz, Mitchell E; Engemann, Ashley M

    2017-08-01

    The addition of plerixafor to high-dose colony-stimulating growth factor has been shown to improve stem cell mobilization rates in autologous transplant patients with multiple myeloma and non-Hodgkin lymphoma. This study evaluates the change in administration time of plerixafor to determine if cell mobilization rates are similar between the US Food and Drug Administration-approved administration time of 11 hours before apheresis and an earlier administration time of 16 hours before apheresis. Medical records of patients age ≥ 18 years undergoing autologous stem cell transplantation requiring the use of plerixafor after at least 4 days of granulocyte colony-stimulating factor therapy to complete stem cell mobilization from January 1, 2010 through September 30, 2014 were retrospectively reviewed. The primary outcome was CD34 + cell mobilization success rates when plerixafor was administered 11 ± 2 hours (standard administration group) compared with 16 ± 2 hours before cell apheresis (early administration group), as defined as collection of  ≥2 × 10 6 CD34 + cells/kg. Secondary outcomes included the number of plerixafor therapy days required to collect a total of ≥2 × 10 6 CD34 + cells/kg, the number of apheresis cycles required to achieve ≥2 × 10 6 CD34 + cells/kg, the median CD34 + cells/kg collected in each apheresis session, and the rates of reported adverse events that occurred in the standard administration time group compared with the early administration time group. Of the 197 patients included, 114 patients received plerixafor 11 ± 2 hours before apheresis and 83 patients received plerixafor 16 hours ± 2 hours before apheresis. Ninety-four percent of patients in the early administration group achieved successful stem cell mobilization compared with 81.6% in the standard administration group (P = .0111). The median number of plerixafor days to reach the collection goal of  ≥2 × 10 6 CD34 + cells/kg was 1 day for

  15. Some factors affecting urokinase inactivation. [Gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Hiroo; Iketa, Yoshito

    1985-10-01

    The enzymatic activity of urokinase adsorbed on various polymer surfaces was measured to study the interaction between protein and polymers. The polymer films on which urokinase was adsorbed were exposed to either a high temperature or ..gamma..-radiation. The thermal inactivation rates were higher on hydrophobic polymers such as poly(ethylene terephthalate), nylon 6, and poly(vinylidene fluoride) than hydrophilic polymers like cellulose and ethylene-vinyl alcohol copolymer, indicating their substantial dependence on the interfacial free energy between the polymer and water. A similar dependence was also seen for the ..gamma..-radiation inactivation. Urokinase adsorbed on the hydrophobic polymers lost more easily its enzymatic activity by exposure to ..gamma..-radiation. The interfacial free energy seems to be one of the driving forces to denaturate proteins on polymers.

  16. Dose-rate effects on mammalian cells exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Mitchell, J.B.

    1978-01-01

    The effect of irradiation on the life cycle and on cell survival was studied for a range of different dose rates. Log phase, plateau phase and synchronized cultures of different mammalian cells were used. Cell cycle redistribution during the radiation exposure was found to be a very important factor in determining the overall dose-rate effect for log phase and synchronized cells. In fact, cell cycle redistribution during the exposure, in some instances, resulted in a lower dose rate being more effective in cell killing per unit dose than a higher dose rate. For plateau phase cultures, where cell cycle times are greatly lengthened, the effects of redistribution in regard to cell killing was virtually eliminated. Both fed and unfed plateau phase cultures exhibited a dose-rate effect, but it was found that below dose rates of 154 rad/h there is no further loss in effectiveness

  17. Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Tzu-Li Lu

    2011-01-01

    Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

  18. Inactivation of E. Coli in Water Using Photocatalytic, Nanostructured Films Synthesized by Aerosol Routes

    Directory of Open Access Journals (Sweden)

    Pratim Biswas

    2013-03-01

    Full Text Available TiO2 nanostructured films were synthesized by an aerosol chemical vapor deposition (ACVD method with different controlled morphologies: columnar, granular, and branched structures for the photocatalytic inactivation of Escherichia coli (E. coli in water. Effects of film morphology and external applied voltage on inactivation rate were investigated. As-prepared films were characterized using scanning electron microscopy (SEM, transmission electron microscopy (TEM, X-ray diffractometry (XRD, and UV-VIS. Photocatalytic and photoelectrochemical inactivation of E. coli using as-prepared TiO2 films were performed under irradiation of UVA light (note: UVA has a low efficiency to inactivate E. coli. Inactivation rate constants for each case were obtained from their respective inactivation curve through a 2 h incubation period. Photocatalytic inactivation rate constants of E. coli are 0.02/min (using columnar films, and 0.08/min (using branched films. The inactivation rate constant for the columnar film was enhanced by 330% by applied voltage on the film while that for the branched film was increased only by 30%. Photocatalytic microbial inactivation rate of the columnar and the branched films were also compared taking into account their different surface areas. Since the majority of the UV radiation that reaches the Earth’s surface is UVA, this study provides an opportunity to use sunlight to efficiently decontaminate drinking water.

  19. Rates of incorporation of radioactive molecules during the cell cycle

    International Nuclear Information System (INIS)

    Gray, J.W.; Pallavicini, M.G.; George, Y.S.; Groppi, V.; Look, M.; Dean, P.N.

    1981-01-01

    We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine ( 3 H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3 H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) 3 H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing 3 H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of 3 H-TdR and 35 S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3 H-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3 H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of 35 S-methionine increased continuously with cell size and DNA content. Incorporation of 3 H-TdR in CHO cells was proportional to DNA synthesis

  20. Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-κBα degradation in RAW 264.7 cells

    International Nuclear Information System (INIS)

    Lee, Hwa Jin; Lim, Hyo Jin; Lee, Da Yeon; Jung, Hyeyoun; Kim, Mi-Ran; Moon, Dong-Cheul; Kim, Keun Il; Lee, Myeong-Sok; Ryu, Jae-Ha

    2010-01-01

    Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-κB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.

  1. Survival of bacteria of laboratory animal origin on cage bedding and inactivation by hydrogen peroxide vapour.

    Science.gov (United States)

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Schulze-Röbbecke, Roland; Sager, Martin

    2017-08-01

    This study aims to determine the ability of laboratory animal bacteria to resist desiccation and inactivation by hydrogen peroxide vapour (HPV) on paper bedding pieces. Bedding pieces were saturated with bacterial suspensions in water or 2% (w/v) bovine serum albumin (BSA) in water, and held in a mouse facility. Viable counts showed variable survival rates over time for the bacterial species used ([ Pasteurella] pneumotropica, Muribacter muris, Pseudomonas aeruginosa, Acinetobacter redioresistens, Escherichia coli, Klebsiella oxytoca, Bordetella bronchiseptica, Bordetella hinzii, Enterococcus faecalis, β-haemolytic Streptococcus spp., Staphylococcus aureus and Staphylococcus xylosus). Overall, BSA increased bacterial survival in the bedding pieces. The survival rates of Bacillus safensis were not influenced by BSA but depended on sporulation. When bedding pieces and Petri dishes inoculated with E. coli, P. aeruginosa and S. aureus were subjected to HPV disinfection, all bacterial species on the bedding pieces inoculated with bacterial suspensions in water were readily inactivated. By contrast, S. aureus and P. aeruginosa, but not E. coli cells survived HPV treatment in high numbers when inoculated on bedding pieces as a BSA suspension. Notably, all three bacterial species were readily inactivated by HPV even in the presence of BSA when smeared on smooth surfaces. In conclusion, the suspension medium and the carrier can influence the environmental survival and susceptibility of bacterial species to HPV. Our results may help to develop standard protocols that can be used to ensure the microbiological quality of experimental rodent housing.

  2. Inactivation of the P16INK4/MTS1 gene by a chromosome translocation t(9;14)(p21-22;q11) in an acute lymphoblastic leukemia of B-cell type.

    Science.gov (United States)

    Duro, D; Bernard, O; Della Valle, V; Leblanc, T; Berger, R; Larsen, C J

    1996-02-15

    We have reported previously a preliminary study of a t(9;14)(p21-22; q11) in B-cell acute lymphoblastic leukemia. This translocation had rearranged the TCRA/D locus on chromosome band 14q11 and the locus encoding the tumor suppressor gene P16INK4/MTS1 (P16) on band 9p21 (D. Duro et al., Oncogene, 11: 21-29, 1995). In the present report, the breakpoints were precisely localized on each chromosome partner. On the 14q- derivative, the sequence derived from chromosome 9 was interrupted at 1.0 kb upstream of the first exon of P16, close to a consensus recombination heptamer, CACTGTG. In addition, the chromosome 14 breakpoint was localized at the end of the TCRD2 (delta 2) segment, and 22 residues with unknown origin were present at the translocation junction. On the 9p+ derivative, chromosome 9 sequences were in continuity with those displaced onto chromosome 14, and the 14q11 breakpoint was located within TCRJA29 segment. These features are consistent with aberrant activity of the TCR gene recombinase complex. Although all three coding exons of P16 were displaced onto the chromosome 14q-derivative, no P16 transcript was detected in the leukemic cells. Because the region spanning the P16 exon 1 was not inactivated by methylation and because the other P16 allele was deleted, the implication is that the chromosome breakpoint was likely to disrupt regulatory elements involved in the normal expression of the gene. As a whole, then, our results show that translocations affecting band 9p21 can participate to the inactivation of P16, thus justifying a systematic survey of translocations of the 9p21 band in acute lymphoblastic leukemia.

  3. Effect of radiation doses rate on SOS response induction in irradiated Escherichia coli Cells

    International Nuclear Information System (INIS)

    Cuetara Lugo, Elizabeth B.; Fuentes Lorenzo, Jorge L.; Almeida Varela, Eliseo; Prieto Miranda, Enrique F.; Sanchez Lamar, Angel; Llagostera Casal, Montserrat

    2005-01-01

    The present work is aimed to study the effect of radiation dose rate on the induction of SOS response in Escherichia coli cells. We measured the induction of sul A reporter gene in PQ-37 (SOS Chromotest) cells. Lead devises were built with different diameter and these were used for diminishing the dose rate of PX- -30M irradiator. Our results show that radiation doses rate significantly modifies the induction of SOS response. Induction factor increases proportionally to doses rate in Escherichia coli cells defective to nucleotide excision repair (uvrA), but not in wild type cells. We conclude that the dose rate affects the level of induction of SOS response

  4. Heat generation rates in lithium thionyl chloride cells

    Science.gov (United States)

    Frank, H.

    1982-03-01

    An empirical equation that is useful for good first approximation in thermal modeling is presented. Indications and measurements of electrochemical heat effects were investigated. The particular cells of interest are of the D size, with spiral wound configuration and were instrumented with a thermocouple. It is found that cathode limited cells can explode on reversal at moderate temperatures.

  5. Radiobiological responses for two cell lines following continuous low dose-rate (CLDR) and pulsed dose rate (PDR) brachytherapy

    International Nuclear Information System (INIS)

    Hanisch, Per Henrik; Furre, Torbjoern; Olsen, Dag Rune; Pettersen, Erik O.

    2007-01-01

    The iso-effective irradiation of continuous low-dose-rate (CLDR) irradiation was compared with that of various schedules of pulsed dose rate (PDR) irradiation for cells of two established human lines, T-47D and NHIK 3025. Complete single-dose response curves were obtained for determination of parameters α and β by fitting of the linear quadratic formula. Sublethal damage repair constants μ and T 1/2 were determined by split-dose recovery experiments. On basis of the acquired parameters of each cell type the relative effectiveness of the two regimens of irradiation (CLDR and PDR) was calculated by use of Fowler's radiobiological model for iso-effect irradiation for repeated fractions of dose delivered at medium dose rates. For both cell types the predicted and observed relative effectiveness was compared at low and high iso-effect levels. The results indicate that the effect of PDR irradiation predicted by Fowler's model is equal to that of CLDR irradiation for both small and large doses with T-47D cells. With NHIK 3025 cells PDR irradiation induces a larger effect than predicted by the model for small doses, while it induces the predicted effect for high doses. The underlying cause of this difference is unclear, but cell-cycle parameters, like G2-accumulation is tested and found to be the same for the two cell lines

  6. Yes-associated protein and WW-containing transcription regulator 1 regulate the expression of sex-determining genes in Sertoli cells, but their inactivation does not cause sex reversal.

    Science.gov (United States)

    Levasseur, Adrien; Paquet, Marilène; Boerboom, Derek; Boyer, Alexandre

    2017-07-01

    Yes-associated protein (YAP) and WW-containing transcription regulator 1 (WWTR1) are two functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway, and that act as major regulators of cell growth and differentiation. To elucidate their role in Sertoli cells, primary Sertoli cell culture from Yapflox/flox; Wwtr1flox/flox animals were infected with a Cre recombinase-expressing adenovirus. Concomitant inactivation of Yap and Wwtr1 resulted in a decrease in the mRNA levels of the male sex differentiation genes Dhh, Dmrt1, Sox9, and Wt1, whereas those of genes involved in female differentiation (Wnt4, Rspo1, and Foxl2) were induced. SOX9, FOXL2, and WNT4 proteins were regulated in the same manner as their mRNAs in response to loss of YAP and WWTR1. To further characterize the role of YAP and WWTR1 in Sertoli cells, we generated a mouse model (Yapflox/flox; Wwtr1flox/flox; Amhcre/+) in which Yap and Wwtr1 were conditionally deleted in Sertoli cells. An increase in the number of apoptotic cells was observed in the seminiferous tubules of 4 dpp mutant mice, leading to a reduction in testis weights and a decrease in the number of Sertoli cells in adult animals. Gene expression analyses of testes from 4 dpp Yapflox/flox; Wwtr1flox/flox; Amhcre/+ mice showed that Sertoli cell differentiation is initially altered, as Dhh, Dmrt1, and Sox9 mRNA levels were downregulated, whereas Wnt4 mRNA levels were increased. However, expression of these genes was not changed in older animals. Together, these results suggest a novel role of the Hippo signaling pathway in the mechanisms of sex differentiation. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Estimation of the rate of energy production of rat mast cells in vitro

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    Rat mast cells were treated with glycolytic and respiratory inhibitors. The rate of adenosine triphosphate depletion of cells incubated with both types of inhibitors and the rate of lactate produced in presence of antimycin A and glucose were used to estimate the rate of oxidative and glycolytic...

  8. Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels

    Science.gov (United States)

    Fineberg, Jeffrey D.; Ritter, David M.

    2012-01-01

    A-type voltage-gated K+ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na+ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously

  9. MCLR-induced PP2A inhibition and subsequent Rac1 inactivation and hyperphosphorylation of cytoskeleton-associated proteins are involved in cytoskeleton rearrangement in SMMC-7721 human liver cancer cell line.

    Science.gov (United States)

    Wang, Hao; Liu, Jinghui; Lin, Shuyan; Wang, Beilei; Xing, Mingluan; Guo, Zonglou; Xu, Lihong

    2014-10-01

    Cyanobacteria-derived toxin microcystin-LR (MCLR) has been widely investigated in its effects on normal cells, there is little information concerning its effects on cancer cells. In the present study, the SMMC-7721 human liver cancer cell line treated with MCLR was used to investigate the change of PP2A, cytoskeleton rearrangement, phosphorylation levels of PP2A substrates that related with cytoskeleton stability and explored underlying mechanisms. Here, we confirmed that MCLR entered into SMMC-7721 cells, bound to PP2A/C subunit and inhibited the activity of PP2A. The upregulation of phosphorylation of the PP2A/C subunit and PP2A regulation protein α4, as well as the change in the association of PP2A/C with α4, were responsible for the decrease in PP2A activity. Another novel finding is that the rearrangement of filamentous actin and microtubules led by MCLR may attribute to the increased phosphorylation of HSP27, VASP and cofilin due to PP2A inhibition. As a result of weakened interactions with PP2A and alterations in its subcellular localization, Rac1 may contribute to the cytoskeletal rearrangement induced by MCLR in SMMC-7721 cells. The current paper presents the first report demonstrating the characteristic of PP2A in MCLR exposed cancer cells, which were more susceptible to MCLR compared with the normal cell lines we previously found, which may be owing to the absence of some type of compensatory mechanisms. The hyperphosphorylation of cytoskeleton-associated proteins and Rac1 inactivation which were induced by inhibition of PP2A are shown to be involved in cytoskeleton rearrangement. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine

    NARCIS (Netherlands)

    Sanders, Barbara P.; Edo-Matas, Diana; Custers, Jerome H. H. V.; Koldijk, Martin H.; Klaren, Vincent; Turk, Marije; Luitjens, Alfred; Bakker, Wilfried A. M.; Uytdehaag, Fons; Goudsmit, Jaap; Lewis, John A.; Schuitemaker, Hanneke

    2013-01-01

    There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to

  11. Strain energy storage and dissipation rate in active cell mechanics

    Science.gov (United States)

    Agosti, A.; Ambrosi, D.; Turzi, S.

    2018-05-01

    When living cells are observed at rest on a flat substrate, they can typically exhibit a rounded (symmetric) or an elongated (polarized) shape. Although the cells are apparently at rest, the active stress generated by the molecular motors continuously stretches and drifts the actin network, the cytoskeleton of the cell. In this paper we theoretically compare the energy stored and dissipated in this active system in two geometric configurations of interest: symmetric and polarized. We find that the stored energy is larger for a radially symmetric cell at low activation regime, while the polar configuration has larger strain energy when the active stress is beyond a critical threshold. Conversely, the dissipation of energy in a symmetric cell is always larger than that of a nonsymmetric one. By a combination of symmetry arguments and competition between surface and bulk stress, we argue that radial symmetry is an energetically expensive metastable state that provides access to an infinite number of lower-energy states, the polarized configurations.

  12. The short term effects of Low-dose-rate Radiation on EL4 Lymphoma Cell

    International Nuclear Information System (INIS)

    Bong, Jin Jong; Kang, Yu Mi; Shin, Suk Chull; Choi, Moo Hyun; Choi, Seung Jin; Kim, Hee Sun; Lee, Kyung Mi

    2012-01-01

    To determine the biological effects of low-dose-rate radiation ( 137 Cs, 2.95 mGy/h) on EL4 lymphoma cells during 24 h, we investigated the expression of genes related to apoptosis, cell cycle arrest, DNA repair, iron transport, and ribonucleotide reductase. EL4 cells were continuously exposed to low-dose-rate radiation (total dose: 70.8 mGy) for 24 h. We analyzed cell proliferation and apoptosis by trypan blue exclusion and flow cytometry, gene expression by real-time PCR, and protein levels with the apoptosis ELISA kit. Apoptosis increased in the Low-dose-rate irradiated cells, but cell number did not differ between non- (Non-IR) and Low-dose-rate irradiated (LDR-IR) cells. In concordance with apoptotic rate, the transcriptional activity of ATM, p53, p21, and Parp was upregulated in the LDR-IR cells. Similarly, Phospho-p53 (Ser15), cleaved caspase 3 (Asp175), and cleaved Parp (Asp214) expression was upregulated in the LDR-IR cells. No difference was observed in the mRNA expression of DNA repair-related genes (Msh2, Msh3, Wrn, Lig4, Neil3, ERCC8, and ERCC6) between Non-IR and LDR-IR cells. Interestingly, the mRNA of Trfc was upregulated in the LDR-IR cells. Therefore, we suggest that short-term Low-dose-rate radiation activates apoptosis in EL4 lymphoma cells.

  13. The short term effects of Low-dose-rate Radiation on EL4 Lymphoma Cell

    Energy Technology Data Exchange (ETDEWEB)

    Bong, Jin Jong; Kang, Yu Mi; Shin, Suk Chull; Choi, Moo Hyun; Choi, Seung Jin; Kim, Hee Sun [Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd, Seoul (Korea, Republic of); Lee, Kyung Mi [Global Research Lab, BAERI Institute, Dept. of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2012-06-15

    To determine the biological effects of low-dose-rate radiation ({sup 137}Cs, 2.95 mGy/h) on EL4 lymphoma cells during 24 h, we investigated the expression of genes related to apoptosis, cell cycle arrest, DNA repair, iron transport, and ribonucleotide reductase. EL4 cells were continuously exposed to low-dose-rate radiation (total dose: 70.8 mGy) for 24 h. We analyzed cell proliferation and apoptosis by trypan blue exclusion and flow cytometry, gene expression by real-time PCR, and protein levels with the apoptosis ELISA kit. Apoptosis increased in the Low-dose-rate irradiated cells, but cell number did not differ between non- (Non-IR) and Low-dose-rate irradiated (LDR-IR) cells. In concordance with apoptotic rate, the transcriptional activity of ATM, p53, p21, and Parp was upregulated in the LDR-IR cells. Similarly, Phospho-p53 (Ser15), cleaved caspase 3 (Asp175), and cleaved Parp (Asp214) expression was upregulated in the LDR-IR cells. No difference was observed in the mRNA expression of DNA repair-related genes (Msh2, Msh3, Wrn, Lig4, Neil3, ERCC8, and ERCC6) between Non-IR and LDR-IR cells. Interestingly, the mRNA of Trfc was upregulated in the LDR-IR cells. Therefore, we suggest that short-term Low-dose-rate radiation activates apoptosis in EL4 lymphoma cells.

  14. Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway.

    Science.gov (United States)

    Wang, Yingjun; Zhang, Mingzhi; Xu, Huanan; Wang, Yifei; Li, Zhaoming; Chang, Yu; Wang, Xinhuan; Fu, Xiaorui; Zhou, Zhiyuan; Yang, Siyuan; Wang, Bei; Shang, Yufeng

    2017-09-22

    Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

  15. Thin-film fixed-bed reactor for solar photocatalytic inactivation of Aeromonas hydrophila: influence of water quality

    Directory of Open Access Journals (Sweden)

    Khan Sadia J

    2012-11-01

    Full Text Available Abstract Background Controlling fish disease is one of the major concerns in contemporary aquaculture. The use of antibiotics or chemical disinfection cannot provide a healthy aquaculture system without residual effects. Water quality is also important in determining the success or failure of fish production. Several solar photocatalytic reactors have been used to treat drinking water or waste water without leaving chemical residues. This study has investigated the impact of several key aspects of water quality on the inactivation of the pathogenic bacterium Aeromonas hydrophila using a pilot-scale thin-film fixed-bed reactor (TFFBR system. Results The level of inactivation of Aeromonas hydrophila ATCC 35654 was determined using a TFFBR with a photocatalytic area of 0.47 m2 under the influence of various water quality variables (pH, conductivity, turbidity and colour under high solar irradiance conditions (980–1100 W m-2, at a flow rate of 4.8 L h-1 through the reactor. Bacterial enumeration were obtained through conventional plate count using trypticase soy agar media, cultured in conventional aerobic conditions to detect healthy cells and under ROS-neutralised conditions to detect both healthy and sub-lethally injured (oxygen-sensitive cells. The results showed that turbidity has a major influence on solar photocatalytic inactivation of A. hydrophila. Humic acids appear to decrease TiO2 effectiveness under full sunlight and reduce microbial inactivation. pH in the range 7–9 and salinity both have no major effect on the extent of photoinactivation or sub-lethal injury. Conclusions This study demonstrates the effectiveness of the TFFBR in the inactivation of Aeromonas hydrophila under the influence of several water quality variables at high solar irradiance, providing an opportunity for the application of solar photocatalysis in aquaculture systems, as long as turbidity remains low.

  16. The mechanism of photosystem-II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Nagy, Valéria; Vidal-Meireles, André; Podmaniczki, Anna; Szentmihályi, Klára; Rákhely, Gábor; Zsigmond, Laura; Kovács, László; Tóth, Szilvia Z

    2018-05-01

    Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H 2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  17. DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Inoue, Masao; Takebe, Hiraku.

    1978-01-01

    Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

  18. Sequential and Simultaneous Applications of UV and Chlorine for Adenovirus Inactivation.

    Science.gov (United States)

    Rattanakul, Surapong; Oguma, Kumiko; Takizawa, Satoshi

    2015-09-01

    Adenoviruses are water-borne human pathogens with high resistance to UV disinfection. Combination of UV treatment and chlorination could be an effective approach to deal with adenoviruses. In this study, human adenovirus 5 (HAdV-5) was challenged in a bench-scale experiment by separate applications of UV or chlorine and by combined applications of UV and chlorine in either a sequential or simultaneous manner. The treated samples were then propagated in human lung carcinoma epithelial cells to quantify the log inactivation of HAdV-5. When the processes were separate, a fluence of 100 mJ/cm(2) and a CT value of 0.02 mg min/L were required to achieve 2 log inactivation of HAdV-5 by UV disinfection and chlorination, respectively. Interestingly, synergistic effects on the HAdV-5 inactivation rates were found in the sequential process of chlorine followed by UV (Cl2-UV) (p simultaneous application of UV/Cl2. This implies that a pretreatment with chlorine may increase the sensitivity of the virus to the subsequent UV disinfection. In conclusion, this study suggests that the combined application of UV and chlorine could be an effective measure against adenoviruses as a multi-barrier approach in water disinfection.

  19. Influences of physicochemical stresses on injury and inactivation behaviour of Listeria innocua

    Directory of Open Access Journals (Sweden)

    Fátima A. Miller

    2017-10-01

    Full Text Available Many minimally processed foods depend on a combination of inhibitory factors to reduce the hazard of foodborne illness. Therefore, inactivation of Listeria innocua was studied according to a 24 factorial experiment designed to draw conclusions about temperature (52.5 °C and 65.0 °C, pH (4.5 and 7.5, water activity (aw=0.95 and 0.99 and solute type (NaCl and glycerol effects. Three different recovery media were used to assess injured cells. Survival data were fitted with a Gompertz-based model and kinetic parameters (shoulder, maximum inactivation rate – kmax, and tail were estimated. Results showed that shoulder was affected by temperature, pH and combined effects; kmax was influenced by all factors and their combinations; and tail was affected by aw, temperature and aw/pH combination. Results demonstrated the potential occurrence of microbial cross-protection survival techniques between the various stresses, e.g. heat and osmolarity. Indeed, this work clearly established that, to avoid hazards, Listeria inactivation must be evaluated with a maximum of environmental factors that undergo alterations. Only thus, appropriate food preservation treatments can be developed and consequently, the safety of food products can be assured.

  20. Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

    International Nuclear Information System (INIS)

    Foster, James S; Fish, Lindsay M; Phipps, Jonathan E; Bruker, Charles T; Lewis, James M; Bell, John L; Solomon, Alan; Kestler, Daniel P

    2013-01-01

    The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior

  1. Rating PV Power and Energy: Cell, Module, and System Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Emery, Keith

    2016-06-02

    A summary of key points related to research-level measurements of current vs. voltage measurement theory including basic PV operation, equivalent circuit, and concept of spectral error; PV power performance including PV irradiance sensors, simulators and commercial and generic I-V systems; PV measurement artifacts, intercomparisons, and alternative rating methods.

  2. Sunlight inactivation of Escherichia coli in waste stabilization microcosms in a sahelian region (Ouagadougou, Burkina Faso).

    Science.gov (United States)

    Maïga, Ynoussa; Denyigba, Kokou; Wethe, Joseph; Ouattara, Aboubakar Sidiki

    2009-02-09

    Experiments on sunlight inactivation of Escherichia coli were conducted from November 2006 to June 2007 in eight outdoors microcosms with different depths filled with maturation pond wastewater in order to determine pond depth influence on sunlight inactivation of E. coli. The long-term aim was to maximize sunlight inactivation of waterborne pathogens in waste stabilization ponds (WSPs) in sahelian regions where number of sunny days enable longer exposure of wastewater to sunlight. The inactivation was followed during daylight from 8.00 h to 17.00 h and during the night. Sunlight inactivation rates (K(S)), as a function of cumulative global solar radiation (insolation), were 16 and 24 times higher than the corresponding dark inactivation (K(D)) rates, respectively in cold and warm season. In warm season, E. coli was inactivated far more rapidly. Inactivation of E. coli follows the evolution of radiation during the day. In shallow depth microcosms, E. coli was inactivated far more rapidly than in high depth microcosms. The physical chemical parameters [pH, dissolved oxygen (DO)] of microcosms water were higher in shallow depth microcosms than in high depth microcosms suggesting a synergistic effect of sunlight and these parameters to damage E. coli. To increase the efficiency of the elimination of waterborne bacteria, the use of maturation ponds with intermediate depths (0.4m) would be advisable in view of the high temperatures and thus evaporation recorded in sahelian regions.

  3. Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

    International Nuclear Information System (INIS)

    Wehrmaker, A.; Lehmann, V.; Droege, W.

    1986-01-01

    The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses

  4. Single-hit mechanism of tumour cell killing by radiation.

    Science.gov (United States)

    Chapman, J D

    2003-02-01

    To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells

  5. Inactivation of viruses in labile blood derivatives. II. Physical methods

    International Nuclear Information System (INIS)

    Horowitz, B.; Wiebe, M.E.; Lippin, A.; Vandersande, J.; Stryker, M.H.

    1985-01-01

    The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure of lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated

  6. Pulsed dielectric barrier discharge for Bacillus subtilis inactivation in water

    Science.gov (United States)

    Hernández-Arias, A. N.; Rodríguez-Méndez, B. G.; López-Callejas, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Peña-Eguiluz, R.; Barocio, S. R.; Muñoz-Castro, A. E.; de la Piedad Beneitez, A.

    2012-06-01

    The inactivation of Bacillus subtilis bacteria in water has been experimentally studied by means of a pulsed dielectric barrier discharge (PDBD) in a coaxial reactor endowed with an alumina dielectric. The plasma source is capable of operating at atmospheric pressure with gas, water or hybrid gas-liquid media at adjustable 25 kV pulses, 30 μs long and at a 500 Hz frequency. In order to evaluate the inactivation efficiency of the system, a set of experiments were designed on the basis of oxygen flow control. The initial data have showed a significant bacterial rate reduction of 103-107 CFU/mL. Additional results proved that applying an oxygen flow for a few seconds during the PDBD treatment inactivates the Bacillus subtilis population with 99.99% effectiveness. As a reference, without gas flow but with the same exposure times, this percentage is reduced to ~90%. The analysis of the relationship between inactivation rate and chemical species in the discharge has been carried out using optical emission spectroscopy as to identifying the main reactive species. Reactive oxygen species such as atomic oxygen and ozone tuned out to be the dominant germicidal species. Some proposed inactivation mechanisms of this technique are discussed.

  7. Pulsed dielectric barrier discharge for Bacillus subtilis inactivation in water

    International Nuclear Information System (INIS)

    Hernández-Arias, A N; López-Callejas, R; De la Piedad Beneitez, A; Rodríguez-Méndez, B G; Valencia-Alvarado, R; Mercado-Cabrera, A; Peña-Eguiluz, R; Barocio, S R; Muñoz-Castro, A E

    2012-01-01

    The inactivation of Bacillus subtilis bacteria in water has been experimentally studied by means of a pulsed dielectric barrier discharge (PDBD) in a coaxial reactor endowed with an alumina dielectric. The plasma source is capable of operating at atmospheric pressure with gas, water or hybrid gas-liquid media at adjustable 25 kV pulses, 30 μs long and at a 500 Hz frequency. In order to evaluate the inactivation efficiency of the system, a set of experiments were designed on the basis of oxygen flow control. The initial data have showed a significant bacterial rate reduction of 10 3 -10 7 CFU/mL. Additional results proved that applying an oxygen flow for a few seconds during the PDBD treatment inactivates the Bacillus subtilis population with 99.99% effectiveness. As a reference, without gas flow but with the same exposure times, this percentage is reduced to ∼90%. The analysis of the relationship between inactivation rate and chemical species in the discharge has been carried out using optical emission spectroscopy as to identifying the main reactive species. Reactive oxygen species such as atomic oxygen and ozone tuned out to be the dominant germicidal species. Some proposed inactivation mechanisms of this technique are discussed.

  8. Rate of renal cell carcinoma subtypes in different races

    Directory of Open Access Journals (Sweden)

    Alexander Sankin

    2011-02-01

    Full Text Available PURPOSE: We sought to identify racial differences among histological subtypes of renal cell carcinoma (RCC between black and non-black patients in an equal-access health care system. MATERIALS AND METHODS: We established a multi-institutional, prospective database of patients undergoing partial or radical nephrectomy between January 1, 2000 and Sept 31, 2009. For the purposes of this study, data captured included age at diagnosis, race, tumor size, presence of lymphovascular invasion, presence of capsular invasion, margin status, and tumor histology. RESULTS: 204 kidney tumors were identified (Table-1. Of these, 117 (57.4% were in black patients and 87 (42.6% were in non-black patients. Age at surgery ranged from 37 to 87 with a median of 62. Tumor size ranged from 1.0 to 22.0 cm with a median of 5.0 cm. Overall, tumors were composed of clear cell RCC in 97 cases (47.5%, papillary RCC in 65 cases (31.9%, chromophobe RCC in 13 cases (6.4%, collecting duct/medullary RCC in 2 cases (1.0%, RCC with multiple histological subtypes in 8 cases (3.9%, malignant tumors of other origin in 6 cases (2.9%, and benign histology in 13 cases (6.4%. Among black patients, papillary RCC was seen in 56 cases (47.9%, compared to 9 cases (10.3% among non-black patients (p < 0.001 (Table-2. Clear cell RCC was present in 38 (32.5% of black patients and in 59 (67.8% of non-blacks (p < 0.001. CONCLUSIONS: In our study, papillary RCC had a much higher occurrence among black patients compared to non-black patients. This is the first study to document such a great racial disparity among RCC subtypes.

  9. A single center, open label study of intradermal administration of an inactivated purified chick embryo cell culture rabies virus vaccine in adults.

    Science.gov (United States)

    Recuenco, Sergio; Warnock, Eli; Osinubi, Modupe O V; Rupprecht, Charles E

    2017-08-03

    In the USA, rabies vaccines (RVs) are licensed for intramuscular (IM) use only, although RVs are licensed for use by the intradermal (ID) route in many other countries. Recent limitations in supplies of RV in the USA reopened discussions on the more efficient use of available biologics, including utilization of more stringent risk assessments, and potential ID RV administration. A clinical trial was designed to compare the immunogenic and adverse effects of a purified chicken embryo cell (PCEC) RV administered ID or IM. Enrollment was designed in four arms, ID Pre-Exposure Prophylaxis (Pre-EP), IM Pre-EP, ID Booster, and IM Booster vaccination. Enrollment included 130 adult volunteers. The arms with IM administration received vaccine according to the current ACIP recommendations: Pre-EP, three 1mL (2.5 I.U.) RV doses, each on day 0, 7, and 21; or a routine Booster, one 1ml dose. The ID groups received the same schedule, but doses administered were in a volume of 0.1mL (0.25 I.U.). The rate of increase in rabies virus neutralizing antibody titers 14-21days after vaccination were similar in the ID and correspondent IM groups. The GMT values for ID vaccination were slightly lower than those for IM vaccination, for both naïve and booster groups, and these differences were statistically significant by t-test. Fourteen days after completing vaccination, all individuals developed RV neutralizing antibody titers over the minimum arbitrary value obtained with the rapid fluorescent focus inhibition test (RFFIT). Antibodies were over the set threshold until the end of the trial, 160days after completed vaccination. No serious adverse reactions were reported. Most frequent adverse reactions were erythema, induration and tenderness, localized at the site of injection. Multi use of 1mL rabies vaccine vials for ID doses of 0.1 was demonstrated to be both safe and inmunogenic. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Inactivation of the forkhead transcription factor FoxO3 is essential for PKB-mediated survival of hematopoietic progenitor cells by kit ligand

    DEFF Research Database (Denmark)

    Engström, Maria; Karlsson, Richard; Jönsson, Jan-Ingvar

    2003-01-01

    OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation of phosphatidylinos......OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation......, immunofluorescence, and subcellular fractionation, we analyzed the effects of KL on PKB and different forkhead family members in two factor-dependent cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow-derived Lin(-) progenitors. Forced overexpression of triple mutated form of FoxO3 by retroviral...

  11. In vitro studies of chlorin e6-assisted photodynamic inactivation of Helicobacter pylori

    Science.gov (United States)

    Simon, C.; Mohrbacher, C.; Hüttenberger, D.; Bauer-Marschall, Ina; Krickhahn, C.; Stachon, A.; Foth, H.-J.

    2014-03-01

    Helicobacter pylori (HP), a gram-negative microaerophilic bacterium located in gastric mucosa, plays an im- portant role in gastro carcinogenesis. Due to the increasing emergence of antibiotic resistance, photodynamic inactivation of bacteria presents a new approach to treat bacterial infections, like HP. In vitro experiments were performed to determine the irradiation conditions for a complete inactivation of HP with the photosensitizer Chlorin e6 (Ce6). The HP strain CCUG 38770 (Culture Collection, University of Gothenburg, Sweden) was routinely cultured under microaerophilic conditions, suspended in sodium chloride, incubated with Ce6 and irradiated briefly with red light of the appropriate wavelength of λ = 660 nm. Series of measurements of different Ce6-concentrations (0.1 μM - 100 μM) were carried out, whereby the incubation time was kept constant at 1 min. The absorbed energy dose has been selected in varying the irradiation time (1 s - 300 s) and the power density (4.5 mW/cm2 - 31 mW/cm2 ). Quantification of inactivation was performed by enumeration of the grown colonies. In addition, the accumulation of Ce6 in HP cells was studied more precisely by uorescence spectroscopy. With a Ce6 concentration of 100 μM and a power density of 9 mW cm2 , a 6-log10 reduction in the survival rate of HP was achieved within 30 seconds of irradiation. In conclusion the most relevant factor for the inactivation of HP is the exposure time of irradiation, followed by the concentration of Ce6 and the light intensity. Further studies with HP strains obtained from patient specimens are under current investigation.

  12. Light-driven photosensitizer uptake increases Candida albicans photodynamic inactivation.

    Science.gov (United States)

    Romano, Renan A; Pratavieira, Sebastião; Silva, Ana P da; Kurachi, Cristina; Guimarães, Francisco E G

    2017-11-01

    Photodynamic Inactivation (PDI) is based on the use of a photosensitizer (PS) and light that results mainly in the production of reactive oxygen species, aiming to produce microorganism cell death. PS incubation time and light dose are key protocol parameters that influence PDI response; the correct choice of them can increase the efficiency of inactivation. The results of this study show that a minor change in the PDI protocol, namely light-driven incubation leads to a higher photosensitizer and more uniform cell uptake inside the irradiated zone. Furthermore, as the uptake increases, the damage caused by PDI also increases. The proposed light-driven incubation prior to the inactivation illumination dose has advantages when compared to the traditional PDI treatments since it can be more selective and effective. Using a violet light as pre-illumination (light-driven incubation) source and a red-light system as PDI source, it was possible to demonstrate that when compared to the traditional protocol of dark incubation, the pre-illuminated cell culture showed an inactivation increase of 7 log units. These in vitro results performed in Candida albicans cells may result in the introduction of a new protocol for PDI. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Method of inactivation of viral and bacterial blood contaminants

    International Nuclear Information System (INIS)

    Hackett, R.; Goodrich, R.P.; Van Borssum Waalkes, M.; Wong, V.A.

    1992-01-01

    A method is provided for inactivating viral and/or bacterial contamination in blood cellular matter, such as erythrocytes and platelets, or protein fractions. The cells or protein fractions are mixed with chemical sensitizers and irradiated with, for example, gamma or X-ray radiation

  14. FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

    Science.gov (United States)

    Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

    2009-06-01

    We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

  15. A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Shima P Damodaran

    Full Text Available To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers and a significant subpopulation of slowly dividing cells (slow-growers. These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

  16. Physiology of inactivation of microbial cells by near-ultraviolet light: mode of action and application for the enrichment of mutants of Escherichia coli and saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Peters, J.

    1976-01-01

    The mode of action of near-ultraviolet (NUV) light was studied in Escherichia coli. NUV light (maximum emission at 365 nm) caused the photodestruction of ribonucleoside diphosphate (RDP) reductase activity in vivo. Evidence was presented for a model suggesting that the loss of RDP-reductase resulted in a metabolic state analogous to that produced during starvation for thymine. Some important properties of cells irradiated by NUV light, cell death, loss of the ability to support the replication of DNA phages and a delay in the onset of cell division in sublethally irradiated cells, were accounted for in terms of photoinactivation of RDP-reductase. Conditions were described under which NUV light was an effective counterselective agent for the enrichment of mutants of Escherichia coli and Saccharomyces cerevisiae

  17. Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry

    2007-01-01

    Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2......)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO...... activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell...

  18. [Kinetics of catalase inactivation induced by ultrasonic cavitation].

    Science.gov (United States)

    Potapovich, M V; Eremin, A N; Metelitsa, D I

    2003-01-01

    Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4-11) within the temperature range from 36 to 55 degrees C. Solutions of CAT were exposed to low-frequency (20.8 kHz) ultrasound (specific power, 48-62 W/cm2). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s-1) of total inactivation (kin), thermal inactivation (*kin), and ultrasonic inactivation (kin(us)). In all cases, the following inequality was valid: kin > *kin. The value of kin(us) increased with the ultrasound power (range, 48-62 W/cm2) and exhibited a strong dependence on pH of the medium. On increasing the initial concentration of CAT (0.4-4.0 nM), kin(us) decreased. The three rate constants were minimum within the range of pH 6.5-8; their values increased considerably at pH 9. At 36-55 degrees C, temperature dependence of kin(us) was characterized by an activation energy (Eact) of 19.7 kcal/mol, whereas the value of Eact for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine serum and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 micrograms/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (HO.), prevented sonication-induced CAT inactivation at 10% (kin and *kin increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.

  19. Microbial electrolytic disinfection process for highly efficient Escherichia coli inactivation

    DEFF Research Database (Denmark)

    Zhou, Shaofeng; Huang, Shaobin; Li, Xiaohu

    2018-01-01

    extensively studied for recalcitrant organics removal, its application potential towards water disinfection (e.g., inactivation of pathogens) is still unknown. This study investigated the inactivation of Escherichia coli in a microbial electrolysis cell based bio-electro-Fenton system (renamed as microbial......Water quality deterioration caused by a wide variety of recalcitrant organics and pathogenic microorganisms has become a serious concern worldwide. Bio-electro-Fenton systems have been considered as cost-effective and highly efficient water treatment platform technology. While it has been......]OH was identified as one potential mechanism for disinfection. This study successfully demonstrated the feasibility of bio-electro-Fenton process for pathogens inactivation, which offers insight for the future development of sustainable, efficient, and cost-effective biological water treatment technology....

  20. Inactivation of Listeria monocytogenes in milk by pulsed electric field.

    Science.gov (United States)

    Reina, L D; Jin, Z T; Zhang, Q H; Yousef, A E

    1998-09-01

    Pasteurized whole, 2%, and skim milk were inoculated with Listeria monocytogenes Scott A and treated with high-voltage pulsed electric field (PEF). The effects of milk composition (fat content) and PEF parameters (electric field strength, treatment time, and treatment temperature) on the inactivation of the bacterium were studied. No significant differences were observed in the inactivation of L. monocytogenes Scott A in three types of milk by PEF treatment. With treatment at 25 degrees C, 1- to 3-log reductions of L. monocytogenes were observed. PEF lethal effect was a function of field strength and treatment time. Higher field strength or longer treatment time resulted in a greater reduction of viable cells. A 4-log reduction of the bacterium was obtained by increasing the treatment temperature to 50 degrees C. Results indicate that the use of a high-voltage PEF is a promising technology for inactivation of foodborne pathogens.

  1. Gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles inhibit Panc-1 pancreatic cancer cell proliferation and migration by inactivating transcription factor ETS1.

    Science.gov (United States)

    Wang, Cailian; Zhang, Haijun; Chen, Yan; Shi, Fangfang; Chen, Baoan

    2012-01-01

    E26 transformation-specific sequence-1 (ETS1) transcription factor plays important roles in both carcinogenesis and the progression of a wide range of malignancies. Aberrant ETS1 expression correlates with aggressive tumor behavior and a poorer prognosis in patients with various malignancies. The aim of the current study was to evaluate the efficacy of a drug delivery system utilizing gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles (GA-MNP-Fe(3)O(4)) on the suppression of ETS1-mediated cell proliferation and migration in Panc-1 pancreatic cancer cells. The effects caused by GA-MNP-Fe(3)O(4) on the proliferation of Panc-1 pancreatic cancer cells were evaluated using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay while inhibition of tumor cell migration was investigated in a scratch assay. The expressions of ETS1, cyclin D1, urokinase-type plasminogen activator (u-PA), and VEGF (vascular endothelial growth factor) were examined by Western blot to elucidate the possible mechanisms involved. In Panc-1 pancreatic cancer cells, we observed that application of GA-MNP-Fe(3)O(4) was able to suppress cancer cell proliferation and prevent cells from migrating effectively. After treatment, Panc-1 pancreatic cancer cells showed significantly decreased expression of ETS1, as well as its downstream target genes for cyclin D1, u-PA, and VEGF. Our novel finding reaffirmed the significance of ETS1 in the treatment of pancreatic cancer, and application of GA-MNP-Fe(3)O(4) nanoparticles targeting ETS1 should be considered as a promising contribution for better pancreatic cancer care.

  2. Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection▿

    OpenAIRE

    Whiteside, Theresa L.; Piazza, Paolo; Reiter, Amanda; Stanson, Joanna; Connolly, Nancy C.; Rinaldo, Charles R.; Riddler, Sharon A.

    2008-01-01

    In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8+ and CD4+ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus is...

  3. Dose rate effect models for biological reaction to ionizing radiation in human cell lines

    International Nuclear Information System (INIS)

    Magae, Junji; Ogata, Hiromitsu

    2008-01-01

    Full text: Because of biological responses to ionizing radiation are dependent on irradiation time or dose rate as well as dose, simultaneous inclusion of dose and dose rate is required to evaluate the risk of long term irradiation at low dose rates. We previously published a novel statistical model for dose rate effect, modified exponential (MOE) model, which predicts irradiation time-dependent biological response to low dose rate ionizing radiation, by analyzing micronucleus formation and growth inhibition in a human osteosarcoma cell line, exposed to wide range of doses and dose rates of gamma-rays. MOE model demonstrates that logarithm of median effective dose exponentially increases in low dose rates, and thus suggests that the risk approaches to zero at infinitely low dose rate. In this paper, we extend the analysis in various kinds of human cell lines exposed to ionizing radiation for more than a year. We measured micronucleus formation and [ 3 H]thymidine uptake in human cell lines including an osteosarcoma, a DNA-dependent protein kinase-deficient glioma, a SV40-transformed fibroblast derived from an ataxia telangiectasia patient, a normal fibroblast, and leukemia cell lines. Cells were exposed to gamma-rays in irradiation room bearing 50,000 Ci of cobalt-60. After the irradiation, they were cultured for 24 h in the presence of cytochalasin B to block cytokinesis, and cytoplasm and nucleus were stained with DAPI and prospidium iodide. The number of binuclear cells bearing a micronucleus was counted under a fluorescence microscope. For proliferation inhibition, cells were cultured for 48 h after the irradiation and [ 3 H] thymidine was pulsed for 4 h before harvesting. We statistically analyzed the data for quantitative evaluation of radiation risk. While dose and dose rate relationship cultured within one month followed MOE model in cell lines holding wild-type DNA repair system, dose rate effect was greatly impaired in DNA repair-deficient cell lines

  4. A specific inactivator of mammalian C'4 isolated from nurse shark (Ginglymostoma cirratum) serum.

    Science.gov (United States)

    Jensen, J A

    1969-08-01

    A material which specifically inactivates mammalian C'4 was isolated from low ionic strength precipitates of nurse shark serum. The C'4 inactivator was not detected in whole serum. The conditions of its generation and its immunoelectrophoretic behavior seem to indicate that it is an enzymatically formed cleavage product of a precursor contained in whole shark serum. The inactivator was partially purified and characterized. It had an S-value of 3.3 (sucrose gradient) which was in agreement with its retardation on gel filtration, was stable between pH 5.0 and 10.0, had a half-life of 5 min at 56 degrees C, pH 7.5, was inactivated by trypsin and was nontoxic. Its powerful anticomplementary activity in vitro and in vivo was solely due to the rapid inactivation of C'4; no other complement components were affected. No cofactor requirement was observed for the equally rapid inactivation of highly purified human and guinea pig C'4. The kinetics of C'4 inactivation and TAME hydrolysis, the greater anodic mobility of inactivated human C'4, and the influence of temperature on the rate of inactivation suggest that the inactivator is an enzyme and C'4 its substrate. This conclusion was supported by the more recent detection of a split product of C'4. Intravenous administration of the C'4 inactivator could prevent lethal Forssman shock and suppress the Arthus reaction in guinea pigs; it prolonged significantly the rejection time of renal xenografts but had no detectable effect on passive cutaneous anaphylaxis. Anaphylatoxin could be generated in C'4 depleted guinea pig serum with the cobra venom factor, but not with immune precipitates. The possible relationship between C'1 esterase and the C'4 inactivator is discussed on the basis of similarities and dissimilarities.

  5. Inactivation of Coxiella burnetti by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Scott, G.H.; McCaul, T.F.; Williams, J.C.

    1989-01-01

    The gamma radiation inactivation kinetics for Coxiella burnetii at - 79 C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0-64 to 1.2 kGy depending on the phase of hte micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing C. burnetti was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

  6. Inactivation of Coxiella burnetii by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Scott, G.H.; McCaul, T.F. (Army Medical Research Inst. of Infectious Diseases, Fort Detrick, Frederick, MD (USA)); Williams, J.C. (National Inst. of Allergy and Infectious Diseases, Bethesda, MD (USA))

    1989-12-01

    The gamma radiation inactivation kinetics for Coxiella burnetii at - 79{sup 0}C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10{sup 11} C. burnetii ml{sup -1} was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes. (author).

  7. Inactivation of Coxiella burnetii by gamma irradiation

    International Nuclear Information System (INIS)

    Scott, G.H.; McCaul, T.F.; Williams, J.C.

    1989-01-01

    The gamma radiation inactivation kinetics for Coxiella burnetii at - 79 0 C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10 11 C. burnetii ml -1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes. (author)

  8. Saw Palmetto induces growth arrest and apoptosis of androgen-dependent prostate cancer LNCaP cells via inactivation of STAT 3 and androgen receptor signaling.

    Science.gov (United States)

    Yang, Yang; Ikezoe, Takayuki; Zheng, Zhixing; Taguchi, Hirokuni; Koeffler, H Phillip; Zhu, Wei-Guo

    2007-09-01

    PC-SPES is an eight-herb mixture that has an activity against prostate cancer. Recently, we purified Saw Palmetto (Serenoa repens) from PC-SPES and found that Saw Palmetto induced growth arrest of prostate cancer LNCaP, DU145, and PC3 cells with ED50s of approximately 2.0, 2.6, and 3.3 microl/ml, respectively, as measured by mitochondrial-dependent conversion of the the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Saw Palmetto induced apoptosis of LNCaP cells in a time- and dose-dependent manner as measured by TUNEL assays. Also, Saw Palmetto increased the expression of p21waf1 and p53 protein in LNCaP cells. In addition, we found that Saw Palmetto down-regulated DHT- or IL-6-induced expression of prostate specific antigen in conjunction with down-regulation of the level of androgen receptor in the nucleus as measured by Western blot analysis. Moreover, Saw Palmetto down-regulated the IL-6-induced level of the phosphorylated form of STAT 3 in LNCaP cells. Furthermore, Saw Palmetto inhibited the growth of LNCaP cells present as tumor xenografts in BALB/c nude mice without adverse effect. These results indicate that Saw Palmetto might be useful for the treatment of individuals with prostate cancer.

  9. Effects of Radiation on Germ Cells of Insects: Dominant Lethals, Gamete Inactivation and Gonial-Cell Killing; Effets des rayonnements sur les cellules germinales des insectes: letalite dominante, inactivation des gametes et destruction des cellules des gonades; Vozdejstvie radiatsii na polovye kletki nasekomykh: dominantnye letali, inaktivatsiya gamet i umershchvlenie polovykh kletok; Efectos de las radiaciones sobre las celulas germinales de los insectos: elementos letales dominantes, inactivacion de los gametos y exterminacion de las celulas gonadicas

    Energy Technology Data Exchange (ETDEWEB)

    Von Borstel, R. C. [Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN (United States)

    1963-09-15

    Radiations and chemical mutagens kill cells in numerous ways: by one of several kinds of induced dominant lethality, by a direct inactivation of function as with sperm, and by genetically undefinable types of death which may or may not be related to dominant lethality per se. Also, chemical mutagens appear to exert a curious enhancement of the fertilizing capacity of sperm. The different stages of oflgenesis and spermatogenesis respond with unequal sensitivity to radiation, and individual cells pass through stages conferring as much as a 50-fold difference in sensitivity. Where species of Diptera, Hymenoptera and Coleoptera can be compared, a striking similarity of response to radiation can be observed, both to stage sensitivity and degree of response with dose. The silkworm, Bombyx mori (Lepidoptera), seems to be similar in most respects to representatives of the other orders in response of germ cells to radiation, but differs sharply in types of dominant lethality induced. Species having atypical genetic mechanisms (e.g.,the lecanoid system of Planococcus citri (Hemipt. : Coccidae) are special cases, and their responses to radiation are considerably modified from those of other species. For insect population control by the irradiation-of-male method, dominant lethality is as advantageous in species where matings are multiple as in species where mating occurs once. Sperm inactivation and gonial killing can be regarded as instances of true sterility and are maximally effective only in species where mating occurs once. For most efficient control, doses should be chosen which would induce maximum dominant lethality, minimum sperm inactivation and complete killing of gonial cells. These parameters are simple to determine by gamete viability measurements, irradiated and unirradiated population competition experiments and histological examination of gonia. (author) [French] Les rayonnements et les agents chimiques de mutation detruisent les cellules de nombreuses facons

  10. Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

    Directory of Open Access Journals (Sweden)

    Carmen Clapp

    2011-07-01

    Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

  11. Maximum heart rate in brown trout (Salmo trutta fario) is not limited by firing rate of pacemaker cells.

    Science.gov (United States)

    Haverinen, Jaakko; Abramochkin, Denis V; Kamkin, Andre; Vornanen, Matti

    2017-02-01

    Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate (f H ), and at high temperatures Q̇ collapses due to heat-dependent depression of f H This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of f H in vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, f PM ), spontaneous beating rate of isolated sinoatrial preparations (f SA ), and in vivo f H of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, f PM steadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (T BP ) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum f PM at T BP was much higher [193 ± 21.0 beats per minute (bpm)] than the peak f SA (94.3 ± 6.0 bpm at 24.1°C) or peak f H (76.7 ± 2.4 at 15.7 ± 0.82°C) (P brown trout in vivo. Copyright © 2017 the American Physiological Society.

  12. Cellular metabolic rates from primary dermal fibroblast cells isolated from birds of different body masses.

    Science.gov (United States)

    Jimenez, Ana Gabriela; Williams, Joseph B

    2014-10-01

    The rate of metabolism is the speed at which organisms use energy, an integration of energy transformations within the body; it governs biological processes that influence rates of growth and reproduction. Progress at understanding functional linkages between whole organism metabolic rate and underlying mechanisms that influence its magnitude has been slow despite the central role this issue plays in evolutionary and physiological ecology. Previous studies that have attempted to relate how cellular processes translate into whole-organism physiology have done so over a range of body masses of subjects. However, the data still remains controversial when observing metabolic rates at the cellular level. To bridge the gap between these ideas, we examined cellular metabolic rate of primary dermal fibroblasts isolated from 49 species of birds representing a 32,000-fold range in body masses to test the hypothesis that metabolic rate of cultured cells scales with body size. We used a Seahorse XF-96 Extracellular flux analyzer to measure cellular respiration in fibroblasts. Additionally, we measured fibroblast size and mitochondrial content. We found no significant correlation between cellular metabolic rate, cell size, or mitochondrial content and body mass. Additionally, there was a significant relationship between cellular basal metabolic rate and proton leak in these cells. We conclude that metabolic rate of cells isolated in culture does not scale with body mass, but cellular metabolic rate is correlated to growth rate in birds. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Thin-film fixed-bed reactor (TFFBR for solar photocatalytic inactivation of aquaculture pathogen Aeromonas hydrophila

    Directory of Open Access Journals (Sweden)

    Khan Sadia J

    2012-01-01

    Full Text Available Abstract Background Outbreaks of infectious diseases by microbial pathogens can cause substantial losses of stock in aquaculture systems. There are several ways to eliminate these pathogens including the use of antibiotics, biocides and conventional disinfectants, but these leave undesirable chemical residues. Conversely, using sunlight for disinfection has the advantage of leaving no chemical residue and is particularly suited to countries with sunny climates. Titanium dioxide (TiO2 is a photocatalyst that increases the effectiveness of solar disinfection. In recent years, several different types of solar photocatalytic reactors coated with TiO2 have been developed for waste water and drinking water treatment. In this study a thin-film fixed-bed reactor (TFFBR, designed as a sloping flat plate reactor coated with P25 DEGUSSA TiO2, was used. Results The level of inactivation of the aquaculture pathogen Aeromonas hydrophila ATCC 35654 was determined after travelling across the TFFBR under various natural sunlight conditions (300-1200 W m-2, at 3 different flow rates (4.8, 8.4 and 16.8 L h-1. Bacterial numbers were determined by conventional plate counting using selective agar media, cultured (i under conventional aerobic conditions to detect healthy cells and (ii under conditions designed to neutralise reactive oxygen species (agar medium supplemented with the peroxide scavenger sodium pyruvate at 0.05% w/v, incubated under anaerobic conditions, to detect both healthy and sub-lethally injured (oxygen-sensitive cells. The results clearly demonstrate that high sunlight intensities (≥ 600 W m-2 and low flow rates (4.8 L h-1 provided optimum conditions for inactivation of A. hydrophila ATCC 3564, with greater overall inactivation and fewer sub-lethally injured cells than at low sunlight intensities or high flow rates. Low sunlight intensities resulted in reduced overall inactivation and greater sub-lethal injury at all flow rates. Conclusions This

  14. Dose-rate effects and chronological changes of chromosome aberration rates in spleen cells from mice that are chronically exposed to gamma-ray at low dose rates

    International Nuclear Information System (INIS)

    Tanaka, Kimio; Kohda, Atsushi; Ichinohe, Kazuaki; Matsumoto, Tsuneya; Oghiso, Yoichi

    2006-01-01

    Dose-rate effects have not been examined in the low dose-rate regions of less than 60-600 mGy/h. Mice were chronically exposed to gamma-ray at 20 mGy/day (approximately 1 mGy/h) up to 700 days and at 1 mGy/day (approximately 0.05 mGy/h) for 500 days under SPF conditions. Chronological changes of chromosome aberration rates in spleen cells were observed along with accumulated doses at both low dose-rates. Unstable aberrations increased in a biphasic manner within 0-2 Gy and 4-14 Gy in 20 mGy/day irradiation. They slightly increased up to 0.5 Gy in 1 mGy/day irradiation. Chromosome aberration rates at 20 mGy/day and 1 mGy/day were compared at the same total doses of 0.5 Gy and 0.25 Gy. They were 2.0 vs. 0.53, and 1.0 vs. 0.47 respectively. Thus, dose-rate effects were observed in these low dose-rate regions. (author)

  15. Rate of oxygen consumption of hamster melanoma cells as a factor influencing their radioresistance

    International Nuclear Information System (INIS)

    Pajak, S.; Subczynski, W.; Panz, T.; Lukiewicz, S.

    1980-01-01

    It has been reported in recent years that the level of radiosensitivity of neoplasmic cells in vivo and of sphaeroids in vitro can be modified by controlling their rate of oxygen consumption. Thus, an attempt was made to compare this rate in the case of the melanotic and amelanotic lines of Bomirski hamster melanoma in vitro, as it is known that these two lines distinctly differ in their reactivity to ionizing radiations. The measurements carried out by the use of a new ESR method revealed that pigmented and pigmentless cells consume oxygen at significantly different rates. This means that oxygen utilization may contribute to the overall level of radioresistance of melanoma cells. (author)

  16. The biological effect of 125I seed continuous low dose rate irradiation in CL187 cells

    Directory of Open Access Journals (Sweden)

    Zhuang Hong-Qing

    2009-01-01

    Full Text Available Abstract Background To investigate the effectiveness and mechanism of 125I seed continuous low-dose-rate irradiation on colonic cell line CL187 in vitro. Methods The CL187 cell line was exposed to radiation of 60Coγ ray at high dose rate of 2 Gy/min and 125I seed at low dose rate of 2.77 cGy/h. Radiation responses to different doses and dose rates were evaluated by colony-forming assay. Under 125I seed low dose rate irradiation, a total of 12 culture dishes were randomly divided into 4 groups: Control group, and 2, 5, and 10 Gy irradiation groups. At 48 h after irradiation, apoptosis was detected by Annexin and Propidium iodide (PI staining. Cell cycle arrests were detected by PI staining. In order to investigate the influence of low dose rate irradiation on the MAPK signal transduction, the expression changes of epidermal growth factor receptor (EGFR and Raf under continuous low dose rate irradiation (CLDR and/or EGFR monoclonal antibodies were determined by indirect immunofluorescence. Results The relative biological effect (RBE for 125I seeds compared with 60Co γ ray was 1.41. Apoptosis rates of CL187 cancer cells were 13.74% ± 1.63%, 32.58% ± 3.61%, and 46.27% ± 3.82% after 2 Gy, 5 Gy, and 10 Gy irradiation, respectively; however, the control group apoptosis rate was 1.67% ± 0.19%. G2/M cell cycle arrests of CL187 cancer cells were 42.59% ± 3.21%, 59.84% ± 4.96%, and 34.61% ± 2.79% after 2 Gy, 5 Gy, and 10 Gy irradiation, respectively; however, the control group apoptosis rate was 26.44% ± 2.53%. P 2/M cell cycle arrest. After low dose rate irradiation, EGFR and Raf expression increased, but when EGFR was blocked by a monoclonal antibody, EGFR and Raf expression did not change. Conclusion 125I seeds resulted in more effective inhibition than 60Co γ ray high dose rate irradiation in CL187 cells. Apoptosis following G2/M cell cycle arrest was the main mechanism of cell-killing effects under low dose rate irradiation. CLDR could

  17. Radiation inactivation of T7 phage

    International Nuclear Information System (INIS)

    Becker, D.; Redpath, J.L.; Grossweiner, L.I.

    1978-01-01

    The radiation inactivation of T7 phage by 25-MeV electron pulses has been measured in various media containing a wide concentration range of radical scavenging solutes and in the presence of protective and sensitizing agents. The dependence of sensitivity on pulse dose, from 1 mrad to 3.6 krad, is attributed to radical depletion via bimolecular processes. The survival data are analyzed by extending target theory to include diffusive reactions of primary and secondary radicals generated in the medium. It is concluded that OH radicals are the principal primary inactivating species and that secondary radicals from Br - , CNS - , uracil, glucose, ribose, sucrose, tyrosine, and histidine are lethal to some extent. In nutrient broth or 100 mM histidine, psoralen derivatives, Actinomycin D, and Mitomycin C are anoxic sensitizers. It is proposed that the psoralens promote the formation of non-strand break lesions as the sensitization mechanism. The target theory based on diffusional kinetics is applicable to other systems including single cells

  18. Performance of PEM fuel cells stack as affected by number of cell and gas flow-rate

    Science.gov (United States)

    Syampurwadi, A.; Onggo, H.; Indriyati; Yudianti, R.

    2017-03-01

    The proton exchange membrane fuel cell (PEMFC) is a promising technology as an alternative green energy due to its high power density, low operating temperatures, low local emissions, quiet operation and fast start up-shutdown. In order to apply fuel cell as portable power supply, the performance investigation of small number of cells is needed. In this study, PEMFC stacks consisting of 1, 3, 5 and 7-cells with an active area of 25 cm2 per cell have been designed and developed. Their was evaluated in variation of gas flow rate. The membrane electrode assembly (MEA) was prepared by hot-pressing commercial gas diffusion electrodes (Pt loading 0.5 mg/cm2) on pre-treated Nafion 117 membrane. The stacks were constructed using bipolar plates in serpentine pattern and Z-type gas flow configuration. The experimental results were presented as polarization and power output curves which show the effects of varying number of cells and H2/O2 flow-rates on the PEMFC performance. The experimental results showed that not only number of cells and gas flow-rates affected the fuel cells performance, but also the operating temperature as a result of electrochemistry reaction inside the cell.

  19. Using micro-patterned sensors and cell self-assembly for measuring the oxygen consumption rate of single cells

    International Nuclear Information System (INIS)

    Etzkorn, James R; Parviz, Babak A; Wu, Wen-Chung; Tian, Zhiyuan; Kim, Prince; Jang, Sei-Hum; Jen, Alex K-Y; Meldrum, Deirdre R

    2010-01-01

    We present a method for self-assembling arrays of live single cells on a glass chip using a photopatternable polymer to form micro-traps. We have studied the single-cell self-assembly method and optimized the process to obtain a 52% yield of single-trapped cells. We also report a method to measure the oxygen consumption rate of a single cell using micro-patterned sensors. These molecular oxygen sensors were fabricated around each micro-trap allowing optical interrogation of oxygen concentration in the immediate environment of the trapped cell. Micromachined micro-wells were then used to seal the trap, sensor and cell in order to determine the oxygen consumption rate of single cells. These techniques reported here add to the collection of tools for performing 'singe-cell' biology. An oxygen consumption rate of 1.05 ± 0.28 fmol min −1 was found for a data set consisting of 25 single A549 cells.

  20. The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology.

    Science.gov (United States)

    Zhao, Yu; Li, Yang; Mao, Shuangshuang; Sun, Wei; Yao, Rui

    2015-11-02

    Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

  1. Thermal inactivation of eight Salmonella serotypes on dry corn flour.

    OpenAIRE

    VanCauwenberge, J E; Bothast, R J; Kwolek, W F

    1981-01-01

    Dry heat was used to inactivate Salmonella newington, Salmonella typhimurium, Salmonella anatum, Salmonella kentucky, Salmonella cubana, Salmonella seftenberg, Salmonella thompson, and Salmonella tennessee in corn flour at 10 and 15% moisture. The flour was spray inoculated at 10(5) Salmonella cells per g and then stored at 49 degrees C (120 degrees F); viable Salmonella cells were counted on Trypticase (BBL Microbiology Systems) soy agar plates every 30 min for the first 4 h and then at 4-h ...

  2. Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

    Science.gov (United States)

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

    2016-09-01

    Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields.

    Science.gov (United States)

    Wouters, P C; Bos, A P; Ueckert, J

    2001-07-01

    Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.

  4. Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

    Science.gov (United States)

    Wouters, Patrick C.; Bos, Ad P.; Ueckert, Joerg

    2001-01-01

    Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products. PMID:11425727

  5. Sinularin Induces Apoptosis through Mitochondria Dysfunction and Inactivation of the pI3K/Akt/mTOR Pathway in Gastric Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Wu

    2016-07-01

    Full Text Available Sinularin is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinularin on two human gastric cancer cell lines, AGS and NCI-N87. Our results demonstrated that sinularin suppressed the proliferation of gastric cancer cells in a dose-dependent manner and induced apoptosis. In addition, the loss of mitochondrial membrane potential, the release of cytochrome C, the activation of Bax, Bad and caspase-3/9, and the suppression of p-Bad, Bcl-xL and Bcl-2 were observed in the cells treated with sinularin. This finding suggests that sinularin-induced apoptosis is associated with mitochondria-mediated apoptosis and occurs through caspase-dependent pathways. Furthermore, sinularin inhibited the phosphoinositol 3-kinase/Akt/mechanistic target of the rapamycin signaling pathway. Taken together, our results show that sinularin-induced apoptosis is mediated by activation of the caspase cascade and mitochondrial dysfunction. Our findings suggest that sinularin merits further evaluation as a chemotherapeutic agent for human gastric cancer.

  6. Low dose rate radiation favors apoptosis as a mechanism of cell death

    International Nuclear Information System (INIS)

    Murtha, Albert D.; Rupnow, Brent; Knox, Susan J.

    1997-01-01

    Purpose/Objective: Radioimmunotherapy (RIT) has demonstrated promising results in the treatment of chemotherapy refractory non-Hodgkin's lymphoma. The radiation associated with this therapy is emitted in a continuous fashion at low dose rates (LDR). Results from studies comparing the relative efficacy of LDR radiation and high dose rate (HDR) radiation on malignant cell killing have been variable. This variability may be due in part to the relative contribution of different mechanisms of cell killing (apoptosis or necrosis) at different dose rates. Materials and Methods: In order to test this hypothesis, the relative efficacy of LDR (16.7 cGy/hr) and HDR radiation (422 cGy/min) were compared using a human B cell lymphoma cell line (PW) and a PW clone (c26) stably transfected to overexpress the anti-apoptotic gene Bcl-2. The endpoints evaluated included the relative amount of cell killing, the fraction of cell killing attributable to apoptosis versus necrosis, and the impact of Bcl-2 overexpression on both overall cell killing and the fraction of killing attributable to apoptosis. Results: HDR and LDR radiation resulted in similar overall cell killing in the PW wild type cell line. In contrast, killing of clone c26 cells was dose rate dependent. One third less killing was seen following LDR irradiation of c26 cells compared with equivalent doses of HDR radiation. Analysis of the relative mechanisms of killing following LDR irradiation revealed a relative increase in the proportion of killing attributable to apoptosis. Conclusion: These findings support the hypothesis that in PW cells, LDR radiation appears to be highly dependent on apoptosis as a mechanism of cell death. These findings may have implications for the selection of patients for RIT, and for the treatment of tumors that overexpress Bcl-2. They may also help form the basis for future rational design of effective combined modality therapies utilizing RIT

  7. The effect of age at exposure on the inactivating mechanisms and relative contributions of key tumor suppressor genes in radiation-induced mouse T-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Sunaoshi, Masaaki [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Department of Biological Sciences, College of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512 (Japan); Amasaki, Yoshiko; Hirano-Sakairi, Shinobu; Blyth, Benjamin J. [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Morioka, Takamitsu [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Kaminishi, Mutsumi [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Shang, Yi [Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Nishimura, Mayumi; Shimada, Yoshiya [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Tachibana, Akira [Department of Biological Sciences, College of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512 (Japan); and others

    2015-09-15

    Highlights: • T-cell lymphoma incidence, latency and weight did not change with age at exposure. • Lymphomas had frequent loss of heterozygosity on chromosomes 4, 11 and 19. • These lesions targeted the Cdkn2a, Ikaros and Pten tumor suppressor genes. • Age at exposure may influence which tumor suppressor genes are lost in each tumor. • The mechanisms of tumor suppressor gene loss were different at each locus. - Abstract: Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2 Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult-irradiation group, with Ikaros frequently (50%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc-finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult-irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2

  8. The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms.

    Science.gov (United States)

    Behar, Joachim; Ganesan, Ambhighainath; Zhang, Jin; Yaniv, Yael

    2016-01-01

    Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect

  9. Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Cristiana Ossaille Beltrame

    Full Text Available Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR. Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

  10. Inactivation disinfection property of Moringa Oleifera seed extract: optimization and kinetic studies

    Science.gov (United States)

    Idris, M. A.; Jami, M. S.; Hammed, A. M.

    2017-05-01

    This paper presents the statistical optimization study of disinfection inactivation parameters of defatted Moringa oleifera seed extract on Pseudomonas aeruginosa bacterial cells. Three level factorial design was used to estimate the optimum range and the kinetics of the inactivation process was also carried. The inactivation process involved comparing different disinfection models of Chicks-Watson, Collins-Selleck and Homs models. The results from analysis of variance (ANOVA) of the statistical optimization process revealed that only contact time was significant. The optimum disinfection range of the seed extract was 125 mg/L, 30 minutes and 120rpm agitation. At the optimum dose, the inactivation kinetics followed the Collin-Selleck model with coefficient of determination (R2) of 0.6320. This study is the first of its kind in determining the inactivation kinetics of pseudomonas aeruginosa using the defatted seed extract.

  11. Cell Growth Rate Dictates the Onset of Glass to Fluidlike Transition and Long Time Superdiffusion in an Evolving Cell Colony

    Science.gov (United States)

    Malmi-Kakkada, Abdul N.; Li, Xin; Samanta, Himadri S.; Sinha, Sumit; Thirumalai, D.

    2018-04-01

    Collective migration dominates many phenomena, from cell movement in living systems to abiotic self-propelling particles. Focusing on the early stages of tumor evolution, we enunciate the principles involved in cell dynamics and highlight their implications in understanding similar behavior in seemingly unrelated soft glassy materials and possibly chemokine-induced migration of CD 8+T cells. We performed simulations of tumor invasion using a minimal three-dimensional model, accounting for cell elasticity and adhesive cell-cell interactions, as well as cell birth and death, to establish that cell-growth-rate-dependent tumor expansion results in the emergence of distinct topological niches. Cells at the periphery move with higher velocity perpendicular to the tumor boundary, while the motion of interior cells is slower and isotropic. The mean-square displacement Δ (t ) of cells exhibits glassy behavior at times comparable to the cell cycle time, while exhibiting superdiffusive behavior, Δ (t )≈tα (α >1 ), at longer times. We derive the value of α ≈1.33 using a field theoretic approach based on stochastic quantization. In the process, we establish the universality of superdiffusion in a class of seemingly unrelated nonequilibrium systems. Superdiffusion at long times arises only if there is an imbalance between cell birth and death rates. Our findings for the collective migration, which also suggest that tumor evolution occurs in a polarized manner, are in quantitative agreement with in vitro experiments. Although set in the context of tumor invasion, the findings should also hold in describing the collective motion in growing cells and in active systems, where creation and annihilation of particles play a role.

  12. Development of a moderate rate lithium/thionyl-chloride D cell

    Science.gov (United States)

    Cieslak, Wendy R.; Street, Henry K.

    1990-05-01

    We have designed a lithium/thionyl chloride D cell for efficient performance at the moderate rate of approximately 500 mA (6.25 omega load). The SNL-MR-D cell has 345 sq cm of active electrode area, 1.0 M LiAlCl4 electrolyte that may have SO2 additive, and a cathode blended of Shawinigan Acetylene Black, Cabot Black Pearls 2000, and Teflon binder. The average performance of cells built in-house and discharged at 25 C and 6.25 omega has been 14.9 Ah (50 Wh). We have aged the cells at 30 C and 50 C, and measured complex impedance and microcalorimetry during the aging period. The cells have been discharged after the aging period at 25 C and 0 C. This preliminary study has allowed us to establish an initial cell design and estimate the rate of capacity loss on storage or long-term usage.

  13. Reduction of the Earth's magnetic field inhibits growth rates of model cancer cell lines.

    Science.gov (United States)

    Martino, Carlos F; Portelli, Lucas; McCabe, Kevin; Hernandez, Mark; Barnes, Frank

    2010-12-01

    Small alterations in static magnetic fields have been shown to affect certain chemical reaction rates ex vivo. In this manuscript, we present data demonstrating that similar small changes in static magnetic fields between individual cell culture incubators results in significantly altered cell cycle rates for multiple cancer-derived cell lines. This change as assessed by cell number is not a result of apoptosis, necrosis, or cell cycle alterations. While the underlying mechanism is unclear, the implications for all cell culture experiments are clear; static magnetic field conditions within incubators must be considered and/or controlled just as one does for temperature, humidity, and carbon dioxide concentration. Copyright © 2010 Wiley-Liss, Inc.

  14. Can deaths in police cells be prevented? Experience from Norway and death rates in other countries.

    Science.gov (United States)

    Aasebø, Willy; Orskaug, Gunnar; Erikssen, Jan

    2016-01-01

    To describe the changes in death rates and causes of deaths in Norwegian police cells during the last 2 decades. To review reports on death rates in police cells that have been published in medical journals and elsewhere, and discuss the difficulties of comparing death rates between countries. Data on deaths in Norwegian police cells were collected retrospectively in 2002 and 2012 for two time periods: 1993-2001 (period 1) and 2003-2012 (period 2). Several databases were searched to find reports on deaths in police cells from as many countries as possible. The death rates in Norwegian police cells reduced significantly from 0.83 deaths per year per million inhabitants (DYM) in period 1 to 0.22 DYM in period 2 (p police cells reduced by about 75% over a period of approximately 10 years. This is probably mainly due to individuals with severe alcohol intoxication no longer being placed in police cells. However, there remain large methodology difficulties in comparing deaths rates between countries. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  15. Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

    Science.gov (United States)

    Yamazaki, Hiroki; Lai, Yu-Chang; Tateno, Morihiro; Setoguchi, Asuka; Goto-Koshino, Yuko; Endo, Yasuyuki; Nakaichi, Munekazu; Tsujimoto, Hajime; Miura, Naoki

    2017-01-01

    We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

  16. Inactivation of tumor suppressor genes and cancer therapy: An evolutionary game theory approach.

    Science.gov (United States)

    Khadem, Heydar; Kebriaei, Hamed; Veisi, Zahra

    2017-06-01

    Inactivation of alleles in tumor suppressor genes (TSG) is one of the important issues resulting in evolution of cancerous cells. In this paper, the evolution of healthy, one and two missed allele cells is modeled using the concept of evolutionary game theory and replicator dynamics. The proposed model also takes into account the interaction rates of the cells as designing parameters of the system. Different combinations of the equilibrium points of the parameterized nonlinear system is studied and categorized into some cases. In each case, the interaction rates' values are suggested in a way that the equilibrium points of the replicator dynamics are located on an appropriate region of the state space. Based on the suggested interaction rates, it is proved that the system doesn't have any undesirable interior equilibrium point as well. Therefore, the system will converge to the desirable region, where there is a scanty level of cancerous cells. In addition, the proposed conditions for interaction rates guarantee that, when a trajectory of the system reaches the boundaries, then it will stay there forever which is a desirable property since the equilibrium points have been already located on the boundaries, appropriately. The simulation results show the effectiveness of the suggestions in the elimination of the cancerous cells in different scenarios. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Dose-rate dependent stochastic effects in radiation cell-survival models

    International Nuclear Information System (INIS)

    Sachs, R.K.; Hlatky, L.R.

    1990-01-01

    When cells are subjected to ionizing radiation the specific energy rate (microscopic analog of dose-rate) varies from cell to cell. Within one cell, this rate fluctuates during the course of time; a crossing of a sensitive cellular site by a high energy charged particle produces many ionizations almost simultaneously, but during the interval between events no ionizations occur. In any cell-survival model one can incorporate the effect of such fluctuations without changing the basic biological assumptions. Using stochastic differential equations and Monte Carlo methods to take into account stochastic effects we calculated the dose-survival rfelationships in a number of current cell survival models. Some of the models assume quadratic misrepair; others assume saturable repair enzyme systems. It was found that a significant effect of random fluctuations is to decrease the theoretically predicted amount of dose-rate sparing. In the limit of low dose-rates neglecting the stochastic nature of specific energy rates often leads to qualitatively misleading results by overestimating the surviving fraction drastically. In the opposite limit of acute irradiation, analyzing the fluctuations in rates merely amounts to analyzing fluctuations in total specific energy via the usual microdosimetric specific energy distribution function, and neglecting fluctuations usually underestimates the surviving fraction. The Monte Carlo methods interpolate systematically between the low dose-rate and high dose-rate limits. As in other approaches, the slope of the survival curve at low dose-rates is virtually independent of dose and equals the initial slope of the survival curve for acute radiation. (orig.)

  18. DNA precursor compartmentation in mammalian cells: distribution and rates of equilibration between nucleus and cytoplasm

    International Nuclear Information System (INIS)

    Leeds, J.M.

    1986-01-01

    A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Experiments comparing the labeling kinetics of 3 H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of 3 H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells

  19. X-chromosome inactivation in development and cancer.

    Science.gov (United States)

    Chaligné, Ronan; Heard, Edith

    2014-08-01

    X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development. Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combination of chromatin associated proteins, DNA methylation and nuclear organization. However, sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells and disappearance of the Barr body is frequently observed in cancer cells. In this review we summarise current knowledge on the epigenetic changes that accompany X inactivation and discuss the extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast cancer. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Thermal inactivation of enzymes and pathogens in biosamples for MS analysis.

    Science.gov (United States)

    Ahnoff, Martin; Cazares, Lisa H; Sköld, Karl

    2015-01-01

    Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (≈1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

  1. Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans.

    Science.gov (United States)

    Mercx, Sébastien; Smargiasso, Nicolas; Chaumont, François; De Pauw, Edwin; Boutry, Marc; Navarre, Catherine

    2017-01-01

    Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N -glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker ( bar ), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells.

  2. Overview of Radiosensitivity of Human Tumor Cells to Low-Dose-Rate Irradiation

    International Nuclear Information System (INIS)

    Williams, Jerry R.; Zhang Yonggang; Zhou Haoming; Gridley, Daila S.; Koch, Cameron J.; Slater, James M.; Little, John B.

    2008-01-01

    Purpose: We compared clonogenic survival in 27 human tumor cell lines that vary in genotype after low-dose-rate (LDR) or high-dose rate (HDR) irradiation. We measured susceptibility to LDR-induced redistribution in the cell cycle in eight of these cell lines. Methods and Materials: We measured clonogenic survival after up to 96 hours of LDR (0.25 Gy/h) irradiation. We compared these with clonogenic survival after HDR irradiation (50 Gy/h). Using flow cytometry, we measured LDR-induced redistribution as a function of time during LDR irradiation in eight of these cell lines. Results: Coefficients that describe clonogenic survival after both LDR and HDR irradiation segregate into four radiosensitivity groups that associate with cell genotype: mutant (mut)ATM, wild-type TP53, mutTP53, and an unidentified gene in radioresistant glioma cells. The LDR and HDR radiosensitivity correlates at lower doses (∼2 Gy HDR, ∼6 Gy LDR), but not at higher doses (HDR > 4 Gy; LDR > 6 Gy). The rate of LDR-induced loss of clonogenic survival changes at approximately 24 hours; wild-type TP53 cells become more resistant and mutTP53 cells become more sensitive. Redistribution induced by LDR irradiation also changes at approximately 24 hours. Conclusions: Radiosensitivity of human tumor cells to both LDR and HDR irradiation is genotype dependent. Analysis of coefficients that describe cellular radiosensitivity segregates 27 cell lines into four statistically distinct groups, each associating with specific genotypes. Changes in cellular radiosensitivity and redistribution in the cell cycle are strongly time dependent. Our data establish a genotype-dependent time-dependent model that predicts clonogenic survival, explains the inverse dose-rate effect, and suggests possible clinical applications

  3. Effect and Analysis of Sustainable Cell Rate using MPEG video Traffic in ATM Networks

    Directory of Open Access Journals (Sweden)

    Sakshi Kaushal

    2006-04-01

    Full Text Available The broadband networks inhibit the capability to carry multiple types of traffic – voice, video and data, but these services need to be controlled according to the traffic contract negotiated at the time of the connection to maintain desired Quality of service. Such control techniques use traffic descriptors to evaluate its performance and effectiveness. In case of Variable Bit Rate (VBR services, Peak Cell Rate (PCR and its Cell Delay Variation Tolerance (CDVTPCR are mandatory descriptors. In addition to these, ATM Forum proposed Sustainable Cell Rate (SCR and its Cell delay variation tolerance (CDVTSCR. In this paper, we evaluated the impact of specific SCR and CDVTSCR values on the Usage Parameter Control (UPC performance in case of measured MPEG traffic for improving the efficiency

  4. Gamma irradiation inactivates honey bee fungal, microsporidian, and viral pathogens and parasites.

    Science.gov (United States)

    Simone-Finstrom, Michael; Aronstein, Kate; Goblirsch, Michael; Rinkevich, Frank; de Guzman, Lilia

    2018-03-01

    Managed honey bee (Apis mellifera) populations are currently facing unsustainable losses due to a variety of factors. Colonies are challenged with brood pathogens, such as the fungal agent of chalkbrood disease, the microsporidian gut parasite Nosema spp., and several viruses. These pathogens may be transmitted horizontally from worker to worker, vertically from queen to egg and via vectors like the parasitic mite, Varroa destructor. Despite the fact that these pathogens are widespread and often harbored in wax comb that is reused from year to year and transferred across beekeeping operations, few, if any, universal treatments exist for their control. In order to mitigate some of these biological threats to honey bees and to allow for more sustainable reuse of equipment, investigations into techniques for the sterilization of hive equipment and comb are of particular significance. Here, we investigated the potential of gamma irradiation for inactivation of the fungal pathogen Ascosphaera apis, the microsporidian Nosema ceranae and three honey bee viruses (Deformed wing virus [DWV], Black queen cell virus [BQCV], and Chronic bee paralysis virus [CBPV]), focusing on the infectivity of these pathogens post-irradiation. Results indicate that gamma irradiation can effectively inactivate A. apis, N. ceranae, and DWV. Partial inactivation was noted for BQCV and CBPV, but this did not reduce effects on mortality at the tested, relatively high doses. These findings highlight the importance of studying infection rate and symptom development post-treatment and not simply rate or quantity detected. These findings suggest that gamma irradiation may function as a broad treatment to help mitigate colony losses and the spread of pathogens through the exchange of comb across colonies, but raises the question why some viruses appear to be unaffected. These results provide the basis for subsequent studies on benefits of irradiation of used comb for colony health and productivity

  5. ATM phosphorylation in HepG2 cells following continuous low dose-rate irradiation

    International Nuclear Information System (INIS)

    Mei Quelin; Du Duanming; Chen Zaizhong; Liu Pengcheng; Yang Jianyong; Li Yanhao

    2008-01-01

    Objective: To investigate the change of ATM phosphorylation in HepG2 cells following a continuous low dose-rate irradiation. Methods: Cells were persistently exposed to low dose-rate (8.28 cGy/h) irradiation. Indirect immunofluorescence and Western blot were used to detect the expression of ATM phosphorylated proteins. Colony forming assay was used to observe the effect of a low dose-rate irradiation on HepG2 cell survival. Results: After 30 min of low dose-rate irradiation, the phosphorylation of ATM occurred. After 6 h persistent irradiation, the expression of ATM phosphorylated protein reached the peak value, then gradually decreased. After ATM phosphorylation was inhibited with Wortmannin, the surviving fraction of HepG2 cells was lower than that of the irradiation alone group at each time point (P<0.05). Conclusions: Continuous low dose-rate irradiation attenuated ATM phosphorylation, suggesting that continuous low dose-rate irradiation has a potential effect for increasing the radiosensitivity of HepG2 cells. (authors)

  6. Inactivation kinetics and efficiencies of UV-LEDs against Pseudomonas aeruginosa, Legionella pneumophila, and surrogate microorganisms.

    Science.gov (United States)

    Rattanakul, Surapong; Oguma, Kumiko

    2018-03-01

    To demonstrate the effectiveness of UV light-emitting diodes (UV-LEDs) to disinfect water, UV-LEDs at peak emission wavelengths of 265, 280, and 300 nm were adopted to inactivate pathogenic species, including Pseudomonas aeruginosa and Legionella pneumophila, and surrogate species, including Escherichia coli, Bacillus subtilis spores, and bacteriophage Qβ in water, compared to conventional low-pressure UV lamp emitting at 254 nm. The inactivation profiles of each species showed either a linear or sigmoidal survival curve, which both fit well with the Geeraerd's model. Based on the inactivation rate constant, the 265-nm UV-LED showed most effective fluence, except for with E. coli which showed similar inactivation rates at 265 and 254 nm. Electrical energy consumption required for 3-log 10 inactivation (E E,3 ) was lowest for the 280-nm UV-LED for all microbial species tested. Taken together, the findings of this study determined the inactivation profiles and kinetics of both pathogenic bacteria and surrogate species under UV-LED exposure at different wavelengths. We also demonstrated that not only inactivation rate constants, but also energy efficiency should be considered when selecting an emission wavelength for UV-LEDs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Effects of growth rate, cell size, motion, and elemental stoichiometry on nutrient transport kinetics.

    Science.gov (United States)

    Flynn, Kevin J; Skibinski, David O F; Lindemann, Christian

    2018-04-01

    Nutrient acquisition is a critical determinant for the competitive advantage for auto- and osmohetero- trophs alike. Nutrient limited growth is commonly described on a whole cell basis through reference to a maximum growth rate (Gmax) and a half-saturation constant (KG). This empirical application of a Michaelis-Menten like description ignores the multiple underlying feedbacks between physiology contributing to growth, cell size, elemental stoichiometry and cell motion. Here we explore these relationships with reference to the kinetics of the nutrient transporter protein, the transporter rate density at the cell surface (TRD; potential transport rate per unit plasma-membrane area), and diffusion gradients. While the half saturation value for the limiting nutrient increases rapidly with cell size, significant mitigation is afforded by cell motion (swimming or sedimentation), and by decreasing the cellular carbon density. There is thus potential for high vacuolation and high sedimentation rates in diatoms to significantly decrease KG and increase species competitive advantage. Our results also suggest that Gmax for larger non-diatom protists may be constrained by rates of nutrient transport. For a given carbon density, cell size and TRD, the value of Gmax/KG remains constant. This implies that species or strains with a lower Gmax might coincidentally have a competitive advantage under nutrient limited conditions as they also express lower values of KG. The ability of cells to modulate the TRD according to their nutritional status, and hence change the instantaneous maximum transport rate, has a very marked effect upon transport and growth kinetics. Analyses and dynamic models that do not consider such modulation will inevitably fail to properly reflect competitive advantage in nutrient acquisition. This has important implications for the accurate representation and predictive capabilities of model applications, in particular in a changing environment.

  8. Effectiveness and economic analysis of the whole cell/recombinant B subunit (WC/rbs inactivated oral cholera vaccine in the prevention of traveller's diarrhoea

    Directory of Open Access Journals (Sweden)

    Diez-Diaz Rosa

    2009-05-01

    Full Text Available Abstract Background Nowadays there is a debate about the indication of the oral whole-cell/recombinant B-subunit cholera vaccine (WC/rBS in traveller's diarrhoea. However, a cost-benefit analysis based on real data has not been published. Methods A cost-effectiveness and cost-benefit study of the oral cholera vaccine (WC/rBS, Dukoral® for the prevention of traveller's diarrhoea (TD was performed in subjects travelling to cholera risk areas. The effectiveness of WC/rBS vaccine in the prevention of TD was analyzed in 362 travellers attending two International Vaccination Centres in Spain between May and September 2005. Results The overall vaccine efficacy against TD was 42,6%. Direct healthcare-related costs as well as indirect costs (lost vacation days subsequent to the disease were considered. Preventive vaccination against TD resulted in a mean saving of 79.26 € per traveller. Conclusion According to the cost-benefit analysis performed, the recommendation for WC/rBS vaccination in subjects travelling to zones at risk of TD is beneficial for the traveller, regardless of trip duration and visited continent.

  9. Facility for gamma irradiations of cultured cells at low dose rates: design, physical characteristics and functioning

    International Nuclear Information System (INIS)

    Esposito, Giuseppe; Anello, Pasquale; Pecchia, Ilaria; Tabocchini, Maria Antonella; Campa, Alessandro

    2016-01-01

    We describe a low dose/dose rate gamma irradiation facility (called LIBIS) for in vitro biological systems, for the exposure, inside a CO_2 cell culture incubator, of cells at a dose rate ranging from few μGy/h to some tens of mGy/h. Three different "1"3"7Cs sources are used, depending on the desired dose rate. The sample is irradiated with a gamma ray beam with a dose rate uniformity of at least 92% and a percentage of primary 662 keV photons greater than 80%. LIBIS complies with high safety standards. - Highlights: • A gamma irradiation facility for chronic exposures of cells was set up at the Istituto Superiore di Sanità. • The dose rate uniformity and the percentage of primary 662 keV photons on the sample are greater than 92% and 80%, respectively. • The GEANT4 code was used to design the facility. • Good agreement between simulation and experimental dose rate measurements has been obtained. • The facility will allow to safely investigate different issues about low dose rate effects on cultured cells.

  10. Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

    Science.gov (United States)

    Freiburghaus, C; Janicke, B; Lindmark-Månsson, H; Oredsson, S M; Paulsson, M A

    2009-06-01

    Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

  11. Predicting radiation effects on the development of leukemic stem cells based on studies of leukemias induced by high- and low-dose-rate radiation

    International Nuclear Information System (INIS)

    Hirouchi, Tokuhisa

    2012-01-01

    One of the most important causes of radiation-induced cancers, particularly leukemia, is gene mutations resulting from single and double strand breaks in the DNA. Tanaka et al. (2003) reported life shortening in specific pathogen free male and female B6C3F1 mice continuously exposed to γ rays at a low dose rate of 20 mGy/22 h/d for 400 days from 8 weeks of age. Early death due to cancer, mostly malignant lymphomas, was observed in both sexes. A significant increase in the incidence of myeloid leukemia, resulting in early death, was also reported in males. It is expected however, that at 20 mGy/22 h/d, which is equivalent to a dose of 15 μGy/min, DNA strand breaks induced in these cells are repaired soon after they occur. Murine leukemias induced by high-dose-rate radiation were also found in males, and 80% of the mice with leukemia had hemizygous deletions in chromosome 2 around the PU.1 gene and they appeared to be derived from DNA strand breaks. Majority of these leukemia showing hemizygous deletions in chromosome 2 revealed point mutations in the remaining alleles resulting in PU.1 inactivation, which was reported to be related to leukemogenesis. These point mutations are assumed to be independent of DNA strand breaks that occur immediately after irradiation, as they appear at later time after irradiation. This review discusses the effect of radiation-induced DNA strand breaks and also mutagenesis induced independently of DNA strand breaks in hematopoietic cells contributing to the development of the first leukemic stem cell. (author)

  12. Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

    Science.gov (United States)

    Mega, T; Nishijima, T; Ikenaka, T

    1990-04-01

    beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

  13. Efficient Bacteria Inactivation by Ultrasound in Municipal Wastewater

    Directory of Open Access Journals (Sweden)

    Leonel Ernesto Amabilis-Sosa

    2018-04-01

    Full Text Available The reuse of treated wastewaters could contribute to reducing water stress. In this research, ultrasound application on bacterial inactivation in municipal wastewater (MWW was evaluated. Total and fecal coliforms were used as standard fecal indicators; volatile suspended solids (VSS were analyzed too. Samples were taken from the effluent of secondary clarifiers. In addition, inactivation tests were carried out on pure cultures of E. coli (EC and B. subtilis (BS. Sonication was performed at 20 kHz, 35% amplitude and 600 W/L for 15, 30 and 45 min. After 15 min of sonication, bacterial density was reduced by 1.85 Log10 MPN/100 mL for EC and 3.16 Log10 CFU/mL for BS. After 30 min, no CFU/mL of BS were observed in MWW and, after 45 min, the reduction of total and fecal coliforms was practically 6.45 Log10 MPN/100mL. Inactivation mechanism was made by cavitation, which causes irreversible damage to the cell wall. Although high bacterial densities were employed, percentages of inactivation >99% were reached at 45 min. This research contributes to the implementation of ultrasound as a disinfection technique with high potential due to its high efficiency without producing byproducts. In fact, the water meets the guidelines for reuse in direct human contact services.

  14. Inactivation of bacteria in sewage sludge by gamma radiation

    International Nuclear Information System (INIS)

    Pandya, G.A.; Kapila, Smita; Kelkar, V.B.; Negi, Shobha; Modi, V.V.

    1987-01-01

    The survival of certain bacterial cultures suspended in sewage sludge and exposed to gamma-radiation was studied. The inactivation patterns of most of the organisms were significantly different when irradiation was performed using sewage samples collected in the summer and monsoon seasons. The summer sample collected from the anaerobic digester afforded significant protection to both Gram negative and Gram positive organisms. This was evident by the increase in dose required to bring about a 6 log cycle reduction in viable count of the bacterial cultures, when suspended in sewage samples instead of phosphate buffer. The observations made using monsoon digester samples were quite different. This sewage sludge greatly enhanced inactivation by gamma-radiation in most cases. The effects of certain chemicals on the inactivation patterns of two organisms - Salmonella typhi and Shigella flexneri - were examined. Arsenate, mercury and lead salts sensitised S. typhi, while barium acetate and sodium sulphide protected this culture against gamma-radiation. In the case of Sh. flexneri, barium acetate and iodacetamide proved to be radioprotectors. The effects of some chemicals on the inactivation pattern of Sh. flexneri cells irradiated in sludge are also discussed. (author)

  15. Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate

    NARCIS (Netherlands)

    Kamminga, Tjerko; Slagman, Simen Jan; Bijlsma, Jetta J.E.; Martins dos Santos, Vitor A.P.; Suarez-Diez, Maria; Schaap, Peter J.

    2017-01-01

    Mycoplasma hyopneumoniae is cultured on large-scale to produce antigen for inactivated whole-cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for

  16. Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals.

    Science.gov (United States)

    Lucena, Rafael; Alcaide-Gavilán, Maria; Schubert, Katherine; He, Maybo; Domnauer, Matthew G; Marquer, Catherine; Klose, Christian; Surma, Michal A; Kellogg, Douglas R

    2018-01-22

    The size of all cells, from bacteria to vertebrates, is proportional to the growth rate set by nutrient availability, but the underlying mechanisms are unknown. Here, we show that nutrients modulate cell size and growth rate via the TORC2 signaling network in budding yeast. An important function of the TORC2 network is to modulate synthesis of ceramide lipids, which play roles in signaling. TORC2-dependent control of ceramide signaling strongly influences both cell size and growth rate. Thus, cells that cannot make ceramides fail to modulate their growth rate or size in response to changes in nutrients. PP2A associated with the Rts1 regulatory subunit (PP2A Rts1 ) is embedded in a feedback loop that controls TORC2 signaling and helps set the level of TORC2 signaling to match nutrient availability. Together, the data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals arising from the TORC2 network. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Development of methods to measure virus inactivation in fresh waters.

    Science.gov (United States)

    Ward, R L; Winston, P E

    1985-11-01

    This study concerns the identification and correction of deficiencies in methods used to measure inactivation rates of enteric viruses seeded into environmental waters. It was found that viable microorganisms in an environmental water sample increased greatly after addition of small amounts of nutrients normally present in the unpurified seed virus preparation. This burst of microbial growth was not observed after seeding the water with purified virus. The use of radioactively labeled poliovirus revealed that high percentages of virus particles, sometimes greater than 99%, were lost through adherence to containers, especially in less turbid waters. This effect was partially overcome by the use of polypropylene containers and by the absence of movement during incubation. Adherence to containers clearly demonstrated the need for labeled viruses to monitor losses in this type of study. Loss of viral infectivity in samples found to occur during freezing was avoided by addition of broth. Finally, microbial contamination of the cell cultures during infectivity assays was overcome by the use of gentamicin and increased concentrations of penicillin, streptomycin, and amphotericin B.

  18. UK-18,892: resistance to modification by aminoglycoside-inactivating enzymes.

    Science.gov (United States)

    Andrews, R J; Brammer, K W; Cheeseman, H E; Jevons, S

    1978-12-01

    UK-18,892, a new semisynthetic aminoglycoside, was active against bacteria possessing aminoglycoside-inactivating enzymes, with the exception of some known to possess AAC(6') or AAD(4') enzymes. This activity has been rationalized by using cell-free extracts of bacteria containing known inactivating enzymes, where it was shown that UK-18,892 was not a substrate for the APH(3'), AAD(2''), AAC(3), and AAC(2') enzymes. It was also demonstrated that UK-18,892 protected mice against lethal infections caused by organisms possessing aminoglycoside-inactivating enzymes.

  19. Mixed effects modeling of proliferation rates in cell-based models: consequence for pharmacogenomics and cancer.

    Directory of Open Access Journals (Sweden)

    Hae Kyung Im

    2012-02-01

    Full Text Available The International HapMap project has made publicly available extensive genotypic data on a number of lymphoblastoid cell lines (LCLs. Building on this resource, many research groups have generated a large amount of phenotypic data on these cell lines to facilitate genetic studies of disease risk or drug response. However, one problem that may reduce the usefulness of these resources is the biological noise inherent to cellular phenotypes. We developed a novel method, termed Mixed Effects Model Averaging (MEM, which pools data from multiple sources and generates an intrinsic cellular growth rate phenotype. This intrinsic growth rate was estimated for each of over 500 HapMap cell lines. We then examined the association of this intrinsic growth rate with gene expression levels and found that almost 30% (2,967 out of 10,748 of the genes tested were significant with FDR less than 10%. We probed further to demonstrate evidence of a genetic effect on intrinsic growth rate by determining a significant enrichment in growth-associated genes among genes targeted by top growth-associated SNPs (as eQTLs. The estimated intrinsic growth rate as well as the strength of the association with genetic variants and gene expression traits are made publicly available through a cell-based pharmacogenomics database, PACdb. This resource should enable researchers to explore the mediating effects of proliferation rate on other phenotypes.

  20. Ontogeny of metabolic rate and red blood cell size in eyelid geckos: species follow different paths.

    Directory of Open Access Journals (Sweden)

    Zuzana Starostová

    Full Text Available While metabolism is a fundamental feature of all organisms, the causes of its scaling with body mass are not yet fully explained. Nevertheless, observations of negative correlations between red blood cell (RBC size and the rate of metabolism suggest that size variation of these cells responsible for oxygen supply may play a crucial role in determining metabolic rate scaling in vertebrates. Based on a prediction derived from the Cell Metabolism Hypothesis, metabolic rate should increase linearly with body mass in species with RBC size invariance, and slower than linearly when RBC size increases with body mass. We found support for that prediction in five species of eyelid geckos (family Eublepharidae with different patterns of RBC size variation during ontogenetic growth. During ontogeny, metabolic rate increases nearly linearly with body mass in those species of eyelid geckos where there is no correlation between RBC size and body mass, whereas non-linearity of metabolic rate scaling is evident in those species with ontogenetic increase of RBC size. Our findings provide evidence that ontogenetic variability in RBC size, possibly correlating with sizes of other cell types, could have important physiological consequences and can contribute to qualitatively different shape of the intraspecific relationship between metabolic rate and body mass.

  1. Ontogeny of metabolic rate and red blood cell size in eyelid geckos: species follow different paths.

    Science.gov (United States)

    Starostová, Zuzana; Konarzewski, Marek; Kozłowski, Jan; Kratochvíl, Lukáš

    2013-01-01

    While metabolism is a fundamental feature of all organisms, the causes of its scaling with body mass are not yet fully explained. Nevertheless, observations of negative correlations between red blood cell (RBC) size and the rate of metabolis