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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. GCR Transport in the Brain: Assessment of Self-Shielding, Columnar Damage, and Nuclear Reactions on Cell Inactivation Rates

    Science.gov (United States)

    Shavers, M. R.; Atwell, W.; Cucinotta, F. A.; Badhwar, G. D. (Technical Monitor)

    1999-01-01

    Radiation shield design is driven by the need to limit radiation risks while optimizing risk reduction with launch mass/expense penalties. Both limitation and optimization objectives require the development of accurate and complete means for evaluating the effectiveness of various shield materials and body-self shielding. For galactic cosmic rays (GCR), biophysical response models indicate that track structure effects lead to substantially different assessments of shielding effectiveness relative to assessments based on LET-dependent quality factors. Methods for assessing risk to the central nervous system (CNS) from heavy ions are poorly understood at this time. High-energy and charge (HZE) ion can produce tissue events resulting in damage to clusters of cells in a columnar fashion, especially for stopping heavy ions. Grahn (1973) and Todd (1986) have discussed a microlesion concept or model of stochastic tissue events in analyzing damage from HZE's. Some tissues, including the CNS, maybe sensitive to microlesion's or stochastic tissue events in a manner not illuminated by either conventional dosimetry or fluence-based risk factors. HZE ions may also produce important lateral damage to adjacent cells. Fluences of high-energy proton and alpha particles in the GCR are many times higher than HZE ions. Behind spacecraft and body self-shielding the ratio of protons, alpha particles, and neutrons to HZE ions increases several-fold from free-space values. Models of GCR damage behind shielding have placed large concern on the role of target fragments produced from tissue atoms. The self-shielding of the brain reduces the number of heavy ions reaching the interior regions by a large amount and the remaining light particle environment (protons, neutrons, deuterons. and alpha particles) may be the greatest concern. Tracks of high-energy proton produce nuclear reactions in tissue, which can deposit doses of more than 1 Gv within 5 - 10 cell layers. Information on rates of

  3. [Thermal inactivation and stabilization of lysozyme substrate-- Micrococcus lysodeicticus cells].

    Science.gov (United States)

    Tarun, E I; Eremin, A N; Metelitsa, D I

    1986-01-01

    Heat inactivation of the acetonic powder of Micrococcus lysodeicticus cells suspended in phosphate buffer pH 6.2 was quantitatively characterized in the temperature range from 34 to 52 degrees. The total value of the rate constant for heat inactivation of the cells equals 2.88 X 10(8) exp(-18360/RT) sec-1. The activation parameters of the process at 34 degrees are the following: delta H* = 17.7 kcal/mole; delta S* = 21.8 E. U.; delta F* = 24.4 kcal/mole. The effect of ethylene glycol, mannitol, dextran, polyvinyl alcohol (PVA) and polyethylene glycols with different molecular weights on the lysis rate and cell stability was studied. Polyvinyl alcohol was found to be the most effective stabilizer. At concentrations of about 10(-5) it enhances the thermostability of the cells threefold.

  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. Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells.

    Science.gov (United States)

    Talukdar, Prabhat K; Udompijitkul, Pathima; Hossain, Ashfaque; Sarker, Mahfuzur R

    2017-01-01

    Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed. Copyright © 2016 American Society for Microbiology.

  6. 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

  7. 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.

  8. Synergism of UV Radiation and Heat for Cell Inactivation

    International Nuclear Information System (INIS)

    Kim, Jin-Kyu; Lee, Yun-Jong; Lee, Ju-Woon; Kim, Su-Hyoun; Petin, Vladislav G.

    2006-01-01

    Organisms including human beings are constantly exposed to UV radiation. The potential hazards of UV radiation have risen due to a depletion of the protective ozone layer in the stratosphere and the formation of ozone holes. Moreover, the effect of UV radiation may greatly increase due to synergistic interaction of UV radiation with other environmental factors. Fluence rate is known to constitute a very important parameter in photobiology. While it is generally accepted that lowering the UV radiation fluence rate results in a decrease of the cell killing or mutagenesis efficiency per unit dose, the matter is still unclear with regards to the synergistic interaction of UV radiation and another environmental agent. It is of great interest to investigate whether or not the synergistic interaction can take place at low intensities of such environmental factors. Heat is known to be an important modifier of UV radiation sensitivity. Exposure of skin to UV radiation is often encountered at hot ambient temperatures. Therefore, the elucidation of new fundamental aspects of the simultaneous action of UV radiation and heat is an actual task. Thus, the purpose of the present work was to establish whether the UV fluence rate alters the synergistic interaction between heat and UV radiation for cell inactivation

  9. 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.

  10. Inactivated Mesenchymal Stem Cells Maintain Immunomodulatory Capacity

    NARCIS (Netherlands)

    Luk, Franka; de Witte, Samantha F. H.; Korevaar, Sander S.; Roemeling, Marieke; Franquesa, Marcella; Strini, Tanja; van den Engel, Sandra; Gargesha, Madhusudhana; Roy, Debashish; Dor, Frank J. M. F.; Horwitz, Edwin M.; de Bruin, Ron W. F.; Betjes, Michiel G. H.; Baan, Carla C.; Hoogduijn, Martin J.

    2016-01-01

    Mesenchymal stem cells (MSC) are studied as a cell therapeutic agent for treatment of various immune diseases. However, therapy with living culture-expanded cells comes with safety concerns. Furthermore, development of effective MSC immunotherapy is hampered by lack of knowledge of the mechanisms of

  11. Inactivation of Escherichia coli Cells in Aqueous Solution by Atmospheric-Pressure N2, He, Air, and O2 Microplasmas.

    Science.gov (United States)

    Zhou, Renwu; Zhang, Xianhui; Bi, Zhenhua; Zong, Zichao; Niu, Jinhai; Song, Ying; Liu, Dongping; Yang, Size

    2015-08-01

    Atmospheric-pressure N2, He, air, and O2 microplasma arrays have been used to inactivate Escherichia coli cells suspended in aqueous solution. Measurements show that the efficiency of inactivation of E. coli cells is strongly dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2 and He microplasma arrays, air and O2 microplasma arrays may be utilized to more efficiently kill E. coli cells in aqueous solution. The efficiencies of inactivation of E. coli cells in water can be well described by using the chemical reaction rate model, where reactive oxygen species play a crucial role in the inactivation process. Analysis indicates that plasma-generated reactive species can react with E. coli cells in water by direct or indirect interactions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  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; P<0.001). Three adverse events occurred at a higher incidence in the two vaccine 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. 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

  14. 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.

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

    Science.gov (United States)

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

    2015-02-01

    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/I2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I2 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/I2 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.

  16. 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)

  17. 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.)

  18. Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-07-01

    Full Text Available Abstract Background A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source, and mRNA inactivation and protein degradation (the sink. Results Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc and of the enhanced green fluorescent protein (eGFP. Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. Conclusions The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in

  19. 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

  20. 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

  1. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation.

    Directory of Open Access Journals (Sweden)

    Li-Li Wen

    Full Text Available Perfluorinated chemicals (PFCs are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs. In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1 by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation.

  2. 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

  3. Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

    Science.gov (United States)

    1992-01-01

    Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process. PMID:1311019

  4. 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.

  5. Inactivation of myeloma cancer cells by helium and argon plasma jets: The effect comparison and the key reactive species

    Science.gov (United States)

    Chen, Zeyu; Cui, Qingjie; Chen, Chen; Xu, Dehui; Liu, Dingxin; Chen, H. L.; Kong, Michael G.

    2018-02-01

    In plasma cancer therapy, the inactivation of cancer cells under plasma treatment is closely related to the reactive oxygen and nitrogen species (RONS) induced by plasmas. Quantitative study on the plasma-induced RONS that related to cancer cells apoptosis is critical for advancing the research of plasma cancer therapy. In this paper, the effects of several reactive species on the inactivation of LP-1 myeloma cancer cells are comparatively studied with variable working gas composition, surrounding gas composition, and discharge power. The results show that helium plasma jet has a higher cell inactivation efficiency than argon plasma jet under the same discharge power. By comparing the concentration of aqueous phase reactive species and the cell inactivation efficiency under different working gases and discharge powers, it is demonstrated that the inactivation efficiency of LP-1 myeloma cancer cells is strongly correlated with the concentration of peroxynitrite (ONOOH/ONOO-).

  6. 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

  7. Inactivation of human T-cell lymphotropic virus, type III by heat, chemicals, and irradiation

    International Nuclear Information System (INIS)

    Quinnan, G.V. Jr.; Wells, M.A.; Wittek, A.E.; Phelan, M.A.; Mayner, R.E.; Feinstone, S.; Purcell, R.H.; Epstein, J.S.

    1986-01-01

    Infectivity of human T-cell lymphotropic virus, Type III (HTLV-III) was inactivated by heat more rapidly if in liquid medium than if lyophilized and more rapidly at 60 than 56 0 C. When HTLV-III was added to factor VIII suspension, then lyophilized and heated at 60 0 C for 2 hours or longer there was elimination of 1 X 10(6) in vitro infectious units (IVIU) of virus. Much of the viral inactivation appeared to result from lyophilization. The application of water-saturated chloroform to the lyophilized material containing virus also resulted in elimination of infectivity. HTLV-III was efficiently inactivated by formalin, beta-propiolactone, ethyl ether, detergent, and ultraviolet light plus psoralen. The results are reassuring regarding the potential safety of various biological products

  8. Caspase-8 inactivation in T cells increases necroptosis and suppresses autoimmunity in Bim−/− mice

    Science.gov (United States)

    Bohgaki, Toshiyuki; Mozo, Julien; Salmena, Leonardo; Matysiak-Zablocki, Elzbieta; Bohgaki, Miyuki; Sanchez, Otto; Strasser, Andreas

    2011-01-01

    Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of Bim−/− mice restrained their autoimmunity and extended their life span. We show that, similar to caspase-8−/− T cells, Bim−/− T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of Bim−/− T cells and restrains autoimmunity in Bim−/− mice. PMID:22006951

  9. 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

  10. 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

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

    Science.gov (United States)

    Sato, Takehiko; Yokoyama, Mayo; Johkura, Kohei

    2011-09-01

    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 H2O2 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 H2O2, we assessed the differences in the effects of plasma-treated medium and H2O2-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 H2O2 into the cells, response to H2O2 decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H2O2 is one of the main factors responsible for inactivation of HeLa cell viability.

  12. 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)

  13. 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.

  14. Sorption of grape proanthocyanidins and wine polyphenols by yeasts, inactivated yeasts, and yeast cell walls.

    Science.gov (United States)

    Mekoue Nguela, J; Sieczkowski, N; Roi, S; Vernhet, A

    2015-01-21

    Inactivated yeast fractions (IYFs) can be used in enology to improve the stability and mouthfeel of red wines. However, information concerning the mechanisms involved and the impact of the IYF characteristics is scarce. Adsorption isotherms were used to investigate interactions between grape proanthocyanidin fractions (PAs) or wine polyphenols (WP) and a commercial yeast strain (Y), the inactivated yeast (IY), the yeast submitted to autolyzis and inactivation (A-IY), and the cell walls obtained by mechanical disruption (CW). High affinity isotherms and high adsorption capacities were observed for grape PAs and whole cells (Y, IY, and A-IY). Affinity and adsorbed amount were lower with wine PAs, due to chemical changes occurring during winemaking. By contrast to whole cells, grape PAs and WP adsorption on CW remained very low. This raises the issue of the part played by cell walls in the interactions between yeast and proanthocyanidins and suggests the passage of the latter through the wall pores and their interaction with the plasma membrane.

  15. Solar radiation disinfection of drinking water at temperate latitudes: inactivation rates for an optimised reactor configuration.

    Science.gov (United States)

    Davies, C M; Roser, D J; Feitz, A J; Ashbolt, N J

    2009-02-01

    Solar radiation-driven inactivation of bacteria, virus and protozoan pathogen models was quantified in simulated drinking water at a temperate latitude (34 degrees S). The water was seeded with Enterococcus faecalis, Clostridium sporogenes spores, and P22 bacteriophage, each at ca 1x10(5) mL(-1), and exposed to natural sunlight in 30-L reaction vessels. Water temperature ranged from 17 to 39 degrees C during the experiments lasting up to 6h. Dark controls showed little inactivation and so it was concluded that the inactivation observed was primarily driven by non-thermal processes. The optimised reactor design achieved S90 values (cumulative exposure required for 90% reduction) for the test microorganisms in the range 0.63-1.82 MJ m(-2) of Global Solar Exposure (GSX) without the need for TiO2 as a catalyst. High turbidity (840-920 NTU) only reduced the S(90) value by 0.05). However, inactivation was significantly reduced for E. faecalis and P22 when the transmittance of UV wavelengths was attenuated by water with high colour (140 PtCo units) or a suboptimally transparent reactor lid (prob.SODIS type pasteurization were not produced, non-thermal inactivation alone appeared to offer a viable means for reliably disinfecting low colour source waters by greater than 4 orders of magnitude on sunny days at 34 degrees S latitude.

  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. 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

  18. 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

  19. UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

    OpenAIRE

    Johnson, Anne M.; Linden, Karl; Ciociola, Kristina M.; De Leon, Ricardo; Widmer, Giovanni; Rochelle, Paul A.

    2005-01-01

    The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be ...

  20. 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.

  1. 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

  2. Angiotensin II inactivation process in cultured mouse spinal cord cells

    Energy Technology Data Exchange (ETDEWEB)

    Allard, M.; Simonnet, G.; Dupouy, B.; Vincent, J.D.

    1987-05-01

    The pattern of hydrolysis of (/sup 3/H)angiotensin II ( (/sup 3/H)AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37 degrees C. In the absence of peptidase inhibitors, 70% of (/sup 3/H)AII was degraded, and the main labeled metabolite was (/sup 3/H)tyrosine (40% of total radioactivity). Minor quantities of (/sup 3/H)AII1-5 and (/sup 3/H)AII4-8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the AII-hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4.14.4) could play a critical role, as suggested by the formation of (/sup 3/H)Val3-Tyr4 and (/sup 3/H)-Tyr4-Ile5 in the presence of bestatin (2 X 10(-5) M). This hypothesis was confirmed by the potency of dipeptidyl amino-peptidase III inhibitors to inhibit both (/sup 3/H)AII hydrolysis and formation of these /sup 3/H-labeled dipeptides. An arylamidase-like activity could also be participating in (/sup 3/H)AII hydrolysis, because higher concentrations of bestatin (10(-4) M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited (/sup 3/H)tyrosine formation, increased protection of (/sup 3/H)AII and (/sup 3/H)AII1-7 formed, and provoked a slight accumulation of (/sup 3/H)AII2-8. These results suggest that the formation of (/sup 3/H)AII2-8 is due to the action of a bestatin-insensitive acidic aminopeptidase and that the Pro7-Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation.

    Science.gov (United States)

    Jones, Morgan; Osawa, Gail; Regal, Joshua A; Weinberg, Daniel N; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O; Keegan, Catherine E; Maillard, Ivan

    2014-01-01

    The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.

  4. 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

  5. Importance of cell damage causing growth delay for high pressure inactivation of Saccharomyces cerevisiae

    Science.gov (United States)

    Nanba, Masaru; Nomura, Kazuki; Nasuhara, Yusuke; Hayashi, Manabu; Kido, Miyuki; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru; Hirayama, Masao; Ueno, Shigeaki; Fujii, Tomoyuki

    2013-06-01

    A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular damage, which results in a growth delay.

  6. 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

  7. 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.)

  8. 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.

  9. 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

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

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    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.

  11. 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 ...

  12. 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

  13. 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

  14. The respiratory chain is the cell's Achilles' heel during UVA inactivation in Escherichia coli.

    Science.gov (United States)

    Bosshard, Franziska; Bucheli, Margarete; Meur, Yves; Egli, Thomas

    2010-07-01

    Solar disinfection (SODIS) is used as an effective and inexpensive tool to improve the microbiological quality of drinking water in developing countries where no other means are available. Solar UVA light is the agent that inactivates bacteria during the treatment. Damage to bacterial membranes plays a crucial role in the inactivation process. This study showed that even slightly irradiated cells (after less than 1 h of simulated sunlight) were strongly affected in their ability to maintain essential parts of their energy metabolism, in particular of the respiratory chain (activities of NADH oxidase, succinate oxidase and lactate oxidase were measured). The cells' potential to generate ATP was also strongly inhibited. Many essential enzymes of carbon metabolism (glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase) and defence against oxidative stress (catalases and glutathione-disulfide reductase) were reduced in their activity during SODIS. The work suggests that damage to membrane enzymes is a likely cause of membrane dysfunction (loss of membrane potential and increased membrane permeability) during UVA irradiation. In this study, the first targets on the way to cell death were found to be the respiratory chain and F(1)F(0) ATPase.

  15. Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

    Science.gov (United States)

    Fernández, Sonsire; Año, Gemma; Castaño, Jorge; Pino, Yadira; Uribarri, Evangelina; Riverón, Luis A; Cedré, Bárbara; Valmaseda, Tania; Falero, Gustavo; Pérez, José L; Infante, Juan F; García, Luis G; Solís, Rosa L; Sierra, Gustavo; Talavera, Arturo

    2013-01-01

    A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. 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.

  17. The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis.

    Science.gov (United States)

    Zbinden, Christina; Pilo, Paola; Frey, Joachim; Bruckmaier, Rupert M; Wellnitz, Olga

    2015-09-30

    Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Compound developmental eye disorders following inactivation of TGFβ signaling in neural-crest stem cells

    Directory of Open Access Journals (Sweden)

    Suter Ueli

    2005-12-01

    Full Text Available Abstract Background Development of the eye depends partly on the periocular mesenchyme derived from the neural crest (NC, but the fate of NC cells in mammalian eye development and the signals coordinating the formation of ocular structures are poorly understood. Results Here we reveal distinct NC contributions to both anterior and posterior mesenchymal eye structures and show that TGFβ signaling in these cells is crucial for normal eye development. In the anterior eye, TGFβ2 released from the lens is required for the expression of transcription factors Pitx2 and Foxc1 in the NC-derived cornea and in the chamber-angle structures of the eye that control intraocular pressure. TGFβ enhances Foxc1 and induces Pitx2 expression in cell cultures. As in patients carrying mutations in PITX2 and FOXC1, TGFβ signal inactivation in NC cells leads to ocular defects characteristic of the human disorder Axenfeld-Rieger's anomaly. In the posterior eye, NC cell-specific inactivation of TGFβ signaling results in a condition reminiscent of the human disorder persistent hyperplastic primary vitreous. As a secondary effect, retinal patterning is also disturbed in mutant mice. Conclusion In the developing eye the lens acts as a TGFβ signaling center that controls the development of eye structures derived from the NC. Defective TGFβ signal transduction interferes with NC-cell differentiation and survival anterior to the lens and with normal tissue morphogenesis and patterning posterior to the lens. The similarity to developmental eye disorders in humans suggests that defective TGFβ signal modulation in ocular NC derivatives contributes to the pathophysiology of these diseases.

  19. 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.

  20. 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.

  1. Radio-induced inactivation and transformation of rat epithelial cell by alpha particles

    International Nuclear Information System (INIS)

    Poncy, Jean-Luc; Tourdes, Francoise; Bailly, Isabelle

    2003-01-01

    Following alpha- and gamma-irradiations, radio-induced cell inactivation and cellular transformation were measured in primary cultures of tracheal epithelial cells from two rat strains, Sprague Dawley (SD) and Wistar Furth/Fisher F344 (WF/Fi) rats. The survival ratio for each of the two rat strain cells appeared to be statistically different after alpha irradiation. WF/Fi rat cells were 1.7 times more radiosensitive than SD rat cells, whereas no difference is observed following low LET irradiation. Comparison of survival of the cells yielded an RBE of 2.8 and 4.5 for SD and WF/Fi rat cells respectively. The relative transformation frequency (RTF) for WF/Fi primary cells was very close to the level of the spontaneous incidence and independent on the two irradiation types used. On the contrary, the RTF for the SD primary cells, exposed to α-particles of γ-rays, is significantly enhanced when the survival of cells at risk decreases under 40%. For a SD cell survival of about 10%, RTF after low-LET irradiation was approximately 3-fold higher than after exposure to high-LET radiation. In order to determine if the radiosensitivity and the radio-induced cellular transformation of the cells collected from the two rat strains, could be related to their respective antioxidant defenses, activities of the main antioxidant enzymes (superoxide dismutase, catalase and Glutathione peroxidase) have been measured in the primary cell in culture. In our cellular model, the basal level of antoxidant enzymes is not associated with the radiosensitivity induced by high LET radiation. (author)

  2. 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.

  3. 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 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.

  4. 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

  5. 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.

  6. Essential role of axonal VGSC inactivation in time-dependent deceleration and unreliability of spike propagation at cerebellar Purkinje cells

    Science.gov (United States)

    2014-01-01

    Background The output of the neuronal digital spikes is fulfilled by axonal propagation and synaptic transmission to influence postsynaptic cells. Similar to synaptic transmission, spike propagation on the axon is not secure, especially in cerebellar Purkinje cells whose spiking rate is high. The characteristics, mechanisms and physiological impacts of propagation deceleration and infidelity remain elusive. The spike propagation is presumably initiated by local currents that raise membrane potential to the threshold of activating voltage-gated sodium channels (VGSC). Results We have investigated the natures of spike propagation and the role of VGSCs in this process by recording spikes simultaneously on the somata and axonal terminals of Purkinje cells in cerebellar slices. The velocity and fidelity of spike propagation decreased during long-lasting spikes, to which the velocity change was more sensitive than fidelity change. These time-dependent deceleration and infidelity of spike propagation were improved by facilitating axonal VGSC reactivation, and worsen by intensifying VGSC inactivation. Conclusion Our studies indicate that the functional status of axonal VGSCs is essential to influencing the velocity and fidelity of spike propagation. PMID:24382121

  7. Plasminogen-dependent and -independent proteolytic activity of murine endothelioma cells with targeted inactivation of fibrinolytic genes.

    Science.gov (United States)

    Lijnen, H R; Wagner, E F; Collen, D

    1997-02-01

    Plasminogen-dependent and -independent proteolytic activity of marine endothelioma (End) cells that were derived from mice with targeted inactivation of the tissue-type plasminogen activator (t-PA-/-), urokinase-type plasminogen activator (u-PA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) genes was studied with the use of fibrin and extracellular matrix degradation assays. In a buffer milieu, the activation rate of plasminogen (final concentration 0.25 microM) with wild-type and t-PA-/- End cells (3 x 10(4) to 4 x 10(6) cells/ml) was comparable, but it was about 4-fold reduced with u-PA-/- End cells and 3-fold enhanced with PAI-1-/- End cells. Plasminogen activation was markedly reduced by addition of amiloride or of anti-murine u-PA antibodies but not by addition of anti-murine t-PA antibodies, and it was not stimulated by addition of fibrin. Lysis of 125I-fibrin labeled matrix in the presence of plasminogen was comparable with wild-type, t-PA-/- and PAI-1-/- End cells (50% lysis in 3 h with 0.7 to 1.5 x 10(6) cells/ml), but was significantly reduced with u-PA-/- End cells (50% lysis in 20 h with 0.87 x 10(6) cells/ml). Lysis of 3H-proline labeled extracellular matrix in the presence of plasminogen with wild-type, t-PA-/- and PAI-1-/- End cells (20% lysis in 48 h with 3 to 5 x 10(6) cells/ml) was comparable, but it was virtually abolished with u-PA-/- End cells. In the absence of plasminogen, lysis of both the fibrin and the extracellular matrix by all four cell types was drastically reduced and was virtually abolished by addition of phenylmethylsulfonylfluoride or 1,10 phenanthroline. These data indicate that the proteolytic activity of the transformed murine endothelioma cells, measured in plasminogen activation or matrix degradation assays, is essentially u-PA-related and largely plasminogen-dependent.

  8. Inhibition of telomere recombination by inactivation of KEOPS subunit Cgi121 promotes cell longevity.

    Directory of Open Access Journals (Sweden)

    Jing Peng

    2015-03-01

    Full Text Available DNA double strand break (DSB is one of the major damages that cause genome instability and cellular aging. The homologous recombination (HR-mediated repair of DSBs plays an essential role in assurance of genome stability and cell longevity. Telomeres resemble DSBs and are competent for HR. Here we show that in budding yeast Saccharomyces cerevisiae telomere recombination elicits genome instability and accelerates cellular aging. Inactivation of KEOPS subunit Cgi121 specifically inhibits telomere recombination, and significantly extends cell longevity in both telomerase-positive and pre-senescing telomerase-negative cells. Deletion of CGI121 in the short-lived yku80(tel mutant restores lifespan to cgi121Δ level, supporting the function of Cgi121 in telomeric single-stranded DNA generation and thus in promotion of telomere recombination. Strikingly, inhibition of telomere recombination is able to further slow down the aging process in long-lived fob1Δ cells, in which rDNA recombination is restrained. Our study indicates that HR activity at telomeres interferes with telomerase to pose a negative impact on cellular longevity.

  9. 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.

  10. Plumbagin from Plumbago Zeylanica L induces apoptosis in human non-small cell lung cancer cell lines through NF- κB inactivation.

    Science.gov (United States)

    Xu, Tong-Peng; Shen, Hua; Liu, Ling-Xiang; Shu, Yong-Qian

    2013-01-01

    To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-κB regulated apoptotic-related gene and activation of p65 and IκBκ. Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 μmol/L, 7.3 μmol/L, and 6.1 μmol/L for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-κB. In addition to inhibition of NF-κB/p65 nuclear translocation, the compound also suppressed the degradation of IκBκ. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-κB in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC. Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-κB-regulated mitochondrial-mediated pathway, involving activation of ROS.

  11. 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.

  12. Factors affecting the infectivity of tissues from pigs with classical swine fever: thermal inactivation rates and oral infectious dose.

    Science.gov (United States)

    Cowan, Lucie; Haines, Felicity J; Everett, Helen E; Crudgington, Bentley; Johns, Helen L; Clifford, Derek; Drew, Trevor W; Crooke, Helen R

    2015-03-23

    Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1 log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13 °C, 14 °C, 12 °C and 10 °C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  13. 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.

  14. 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

    2015-10-09

    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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. 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

  16. Reactivation of Escherichia coli cells, inactivated by ultraviolet rays, with cell extracts of propionic acid bacteria: Fractionation of the extract

    Energy Technology Data Exchange (ETDEWEB)

    Vorob`eva, L.I.; Khodzhaev, E.Yu.; Ponomareva, G.M.; Ambrosov, I.V. [M.V. Lomonosov Moscow State Univ. (Russian Federation)

    1995-01-01

    Separation of Propionibacterium shermanii extract into fractions and testing them for their reactivating effect on UV-inactivated Escherichia coli AB-1157 cells showed that the activity was associated with the fraction of soluble proteins. The activity was not demonstrated in the fractions of RNA, DNA, ribosomes, or cell walls. Fractional salting out and subsequent testing of the fractions showed two active protein fractions: fraction I (20-40% of ammonium sulfate saturating concentration) and fraction II (60-80%). These fractions were separated by HPLC into seven and eight subfractions, respectively. Reactivating activity was showed in subfraction 4 (fraction I) and subfractions 5 and 6 (fraction II). Electrophoresis showed five and four polypeptides in subfractions 4 and 5, respectively. Subfraction 6 (fraction II) contained one protein with a molecular mass of about 30 kDa. This protein, apparently, was responsible for the protective properties of fraction II. 9 refs., 2 figs., 4 tabs.

  17. Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization.

    Science.gov (United States)

    Ren, Siying; Luo, Yongfeng; Chen, Hui; Warburton, David; Lam, Hilaire C; Wang, Larry L; Chen, Ping; Henske, Elizabeth P; Shi, Wei

    2016-12-01

    The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus.

    Science.gov (United States)

    Yuan, L; Kang, S Y; Ward, L A; To, T L; Saif, L J

    1998-01-01

    Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075-3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. N'-formylkynurenine-photosensitized inactivation of bacteriophage

    International Nuclear Information System (INIS)

    Walrant, P.; Santus, R.; Redpath, J.L.; Pileni, M.P.

    1976-01-01

    Measurements have been made of the sensitizing properties of N'-formylkynurenine (FK) on bacteriophages, as part of a wider study of FK photosensitization of systems which have both protein and DNA components. Suspensions of bacteriophages T 6 and T 7 were near-U.V. (lambda > 320 nm) irradiated in solutions saturated with either O 2 or He in the presence of 5 x 10 -4 M FK. The survival curves obtained demonstrated that FK can act as a photosensitizer for biological inactivation. The involvement of singlet oxygen as one factor in this FK sensitized inactivation was clearly demonstrated by the increased rate of inactivation when the phage were suspended in O 2 -saturated D 2 O, in place of water, during irradiation. The complex mechanism of phage inactivation must involve direct interaction between excited FK and substrate, as well as singlet oxygen. FK is therefore a new natural photosensitizer of significance in cell photochemistry induced by sunlight. (U.K.)

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

    Science.gov (United States)

    Staudacher, Verena; Trujillo, Madia; Diederichs, Tim; Dick, Tobias P; Radi, Rafael; Morgan, Bruce; Deponte, Marcel

    2018-04-01

    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. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Deciphering von Hippel-Lindau (VHL/Vhl-associated pancreatic manifestations by inactivating Vhl in specific pancreatic cell populations.

    Directory of Open Access Journals (Sweden)

    H-C Jennifer Shen

    Full Text Available The von Hippel-Lindau (VHL syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in alpha-cells or an insulin promoter in beta-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL in terminally differentiated alpha-cells or beta-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model

  7. 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

  8. 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

  9. Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

    Science.gov (United States)

    Fan, Xiang; He, Lingli; Meng, Yao; Li, Gangrui; Li, Linli; Meng, Yanfa

    2015-05-01

    α‑Momorcharin (α‑MMC) and momordica anti‑human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome‑inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α‑MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α‑MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)‑sepharose fast flow, sephacryl S‑100 and macro‑Cap‑SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α‑MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α‑MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α‑MMC and MAP30 treatment in a dose‑ and time‑dependent manner; in addition, the results indicated that MAP30 had a more potent anti‑tumor activity compared with that of α‑MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α‑MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α‑MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α‑MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α‑MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer.

  10. Antibody-Secreting Cell Responses and Protective Immunity Assessed in Gnotobiotic Pigs Inoculated Orally or Intramuscularly with Inactivated Human Rotavirus†

    OpenAIRE

    Yuan, Lijuan; Kang, S.-Y.; Ward, Lucy A.; To, Thanh L.; Saif, Linda J.

    1998-01-01

    Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were dete...

  11. 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.

  12. 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.

  13. Peripheral blood complete remission after splenic irradiation in Mantle-Cell Lymphoma with 11q22-23 deletion and ATM inactivation

    Directory of Open Access Journals (Sweden)

    Galliano Marco

    2006-09-01

    Full Text Available Abstract Mantle Cell Lymphoma (MCL is a well-known histological and clinical subtype of B-cell non-Hodgkin's Lymphomas. It is usually characterized by an aggressive disease course, presenting with advanced stage disease at diagnosis and with low response rates to therapy. However few cases of indolent course MCL have been described. We herein report a case of MCL with splenomegaly and peripheral blood involvement as main clinical features. The patient underwent moderate dose splenic radiation therapy and achieved spleen downsizing and peripheral blood complete remission. Splenic irradiation has been extensively used in the past as palliative treatment in several lymphoproliferative disorders and a systemic effect and sometimes peripheral blood complete remissions have been observed. Mainly advocated mechanisms responsible for this phenomenon are considered direct radiation-induced apoptotic cell death, immune modulation via proportional changes of lymphocyte subsets due to known differences in intrinsic radiosensitivity and a radiation-induced cytokine release. The peculiar intrinsic radiosensitivity pattern of lymphoid cells could probably be explained by well-defined individual genetic and molecular features. In this context, among NHLs, MCL subtype has the highest rate of ATM (Ataxia Teleangiectasia Mutated inactivation. While the ATM gene is thought to play a key-role in detecting radiation-induced DNA damage (expecially Double Strand Breaks, recent in vitro data support the hypothesis that ATM loss may actually contribute to the radiosensitivity of MCL cells. ATM status was retrospectively investigated in our patient, with the tool of Fluorescence In Situ Hybridization, showing a complete inactivation of a single ATM allele secondary to the deletion of chromosomal region 11q22-23. The presence of this kind of cytogenetic aberration may be regarded in the future as a potential predictive marker of radiation response.

  14. 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

  15. The reduction of l-cystine to l-cysteine in the supernatant of A549 cell culture causes imipenem inactivation.

    Science.gov (United States)

    Takemura, Hiromu; Terakubo, Shigemi; Okamura, Ninyo; Nakashima, Hideki

    2018-02-26

    In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100 μg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3 h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  16. Protection of mice against gastric colonization of Helicobacter pylori by therapeutic immunization with systemic whole cell inactivated vaccines.

    Science.gov (United States)

    Suganya, K; Prem Kumar, A; Sekar, B; Sundaran, B

    2017-01-01

    The protective effect of therapeutic immunization with heat inactivated Helicobacter pylori cells administered with aluminum phosphate as an adjuvant was evaluated with "Swiss albino mice" infected with H. pylori Sydney strain 1 (SS1). The presence of bacteria in histological sections of the stomach was evaluated to confirm the colonization of H. pylori. The infection dose was determined to be 1 × 10 8  cells which resulted to be the optimal concentration to sustain infection for required time. Systemic immunization of H. pylori 26695 and SS1 Whole cell heat inactivated vaccine were induced on mice. The IgG titer levels of high dose adjuvant vaccine of both strains were proportionate on the 7th and 14th day. Subsequently on the 21st and 28th day SS1 high dose adjuvant revealed a higher titer value. The Probability values were pylori infection in mice. These results represent strong evidence for feasibility of therapeutic use of whole cell based vaccine formulations against H. pylori infection in animal model. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  17. Aberrant methylation inactivates somatostatin and somatostatin receptor type 1 in head and neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Kiyoshi Misawa

    Full Text Available The aim of this study was to define somatostatin (SST and somatostatin receptor type 1 (SSTR1 methylation profiles for head and neck squamous cell carcinoma (HNSCC tumors at diagnosis and follow up and to evaluate their prognostic significance and value as a biomarker.Gene expression was measured by quantitative RT-PCR. Promoter methylation status was determined by quantitative methylation-specific PCR (Q-MSP in HNSCC.Methylation was associated with transcription inhibition. SST methylation in 81% of HNSCC tumor specimens significantly correlated with tumor size (P = 0.043, stage (P = 0.008, galanin receptor type 2 (GALR2 methylation (P = 0.041, and tachykinin-1 (TAC1 (P = 0.040. SSTR1 hypermethylation in 64% of cases was correlated with tumor size (P = 0.037, stage (P = 0.037, SST methylation (P < 0.001, and expression of galanin (P = 0.03, GALR2 (P = 0.014, TAC1 (P = 0.023, and tachykinin receptor type 1 (TACR1 (P = 0.003. SST and SSTR1 promoter hypermethylation showed highly discriminating receiver operator characteristic curve profiles, which clearly distinguished HNSCC from adjacent normal mucosal tissues. Concurrent hypermethylation of galanin and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.0001. Among patients with oral cavity and oropharynx cancer, methylation of both SST and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.028. In multivariate logistic-regression analysis, concomitant methylation of galanin and SSTR1 was associated with an odds ratio for recurrence of 12.53 (95% CI, 2.62 to 59.8; P = 0.002.CpG hypermethylation is a likely mechanism of SST and SSTR1 gene inactivation, supporting the hypothesis that SST and SSTR1 play a role in the tumorigenesis of HNSCC and that this hypermethylation may serve as an important biomarker.

  18. 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.)

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

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Nobuto; Urade, Masahiro (Hahnemann Univ. School of Medicine, Philadelphia, PA (USA))

    1989-09-01

    Rubella virus is very sensitive to photodynamic action. When tested with 1.2 x 10{sup -5} M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m{sup 2}, inactivation kinetics showed a linear single hit curve with a k value of 1.48 min{sup -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).

  20. 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)

  1. Photodynamic inactivation of Klebsiella pneumoniae biofilms and planktonic cells by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

    Science.gov (United States)

    Liu, Chengcheng; Zhou, Yingli; Wang, Li; Han, Lei; Lei, Jin'e; Ishaq, Hafiz Muhammad; Nair, Sean P; Xu, Jiru

    2016-04-01

    The treatment of Klebsiella pneumoniae, particularly extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae, is currently a great challenge. Photodynamic antimicrobial chemotherapy is a promising approach for killing antibiotic-resistant bacteria. The aim of this study was to evaluate the capacity of 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL) in the presence of white light to cause photodynamic inactivation (PDI) of K. pneumoniae planktonic and biofilm cells. In the presence of white light, 5-ALA and MAL inactivated planktonic cells in a concentration-dependent manner. Biofilms were also sensitive to 5-ALA and MAL-mediated PDI. The mechanisms by which 5-ALA and MAL caused PDI of ESBL-producing K. pneumonia were also investigated. Exposure of K. pneumonia to light in the presence of either 5-ALA or MAL induced cleavage of genomic DNA and the rapid release of intracellular biopolymers. Intensely denatured cytoplasmic contents and aggregated ribosomes were also detected by transmission electron microscopy. Scanning electron microscopy showed that PDI of biofilms caused aggregated bacteria to detach and that the bacterial cell envelope was damaged. This study provides insights into 5-ALA and MAL-mediated PDI of ESBL-producing K. pneumoniae.

  2. Inactivation of Escherichia coli by superoxide radicals and their dismutation products

    NARCIS (Netherlands)

    Hemmen, J.J. van; Meuling, W.J.A.

    1977-01-01

    E. coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is superoxide. The rate of inactivation is decreased by superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen

  3. Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M-Phase Exit in Xenopus laevis Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Jacek Z. Kubiak

    2011-01-01

    Full Text Available Cyclin-Dependent Kinase 1 (CDK1 is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.

  4. Targeted light-inactivation of the Ki-67 protein using theranostic liposomes leads to death of proliferating cells

    Science.gov (United States)

    Rahmanzadeh, Ramtin; Rai, Prakash; Gerdes, Johannes; Hasan, Tayyaba

    2010-02-01

    Nanomedicine is beginning to impact the treatment of several diseases and current research efforts include development of integrated nano-constructs (theranostics) which serve as probes for imaging and therapy in addition to delivering macromolecules intracellularly. In cancer, there is a vital unmet need for effective alternative treatments with high specificity and low systemic toxicity. This can be achieved by targeting key molecular markers associated with cancer cells with reduced effective drug doses. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). Antibodies against pKi-67 are widely used as prognostic tools for tumor diagnosis. In this work, anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. After incubation of OVCAR-5 ovarian cancer cells with these liposomes, confocal microscopy confirmed the localization of the antibodies to the nucleoli of the cells. Irradiation with a 488 nm laser led to a significant loss of cell viability. The specificity of this approach for pKi-67 positive cells was demonstrated in confluent human lung fibroblasts (MRC-5) where only a small population of cells stain positive for pKi-67 and only minimal cell death was observed. Taken together, our findings suggest that pKi-67 targeted with nano-platform is an attractive therapeutic target in cancer therapy.

  5. Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

    Science.gov (United States)

    Pollex, Tim; Piolot, Tristan; Heard, Edith

    2013-01-01

    Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

  6. X chromosome inactivation in human pluripotent stem cells as a model for human development: back to the drawing board?

    Science.gov (United States)

    Geens, Mieke; Chuva De Sousa Lopes, Susana M

    2017-09-01

    Human pluripotent stem cells (hPSC), both embryonic and induced (hESC and hiPSC), are regarded as a valuable in vitro model for early human development. In order to fulfil this promise, it is important that these cells mimic as closely as possible the in vivo molecular events, both at the genetic and epigenetic level. One of the most important epigenetic events during early human development is X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female cells. XCI is important for proper development and aberrant XCI has been linked to several pathologies. Recently, novel data obtained using high throughput single-cell technology during human preimplantation development have suggested that the XCI mechanism is substantially different from XCI in mouse. It has also been suggested that hPSC show higher complexity in XCI than the mouse. Here we compare the available recent data to understand whether XCI during human preimplantation can be properly recapitulated using hPSC. We will summarize what is known on the timing and mechanisms of XCI during human preimplantation development. We will compare this to the XCI patterns that are observed during hPSC derivation, culture and differentiation, and comment on the cause of the aberrant XCI patterns observed in hPSC. Finally, we will discuss the implications of the aberrant XCI patterns on the applicability of hPSC as an in vitro model for human development and as cell source for regenerative medicine. Combinations of the following keywords were applied as search criteria in the PubMed database: X chromosome inactivation, preimplantation development, embryonic stem cells, induced pluripotent stem cells, primordial germ cells, differentiation. Recent single-cell RNASeq data have shed new light on the XCI process during human preimplantation development. These indicate a gradual inactivation on both XX chromosomes, starting from Day 4 of development and followed by a random choice

  7. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs.

    Science.gov (United States)

    Dhakal, Santosh; Hiremath, Jagadish; Bondra, Kathryn; Lakshmanappa, Yashavanth S; Shyu, Duan-Liang; Ouyang, Kang; Kang, Kyung-Il; Binjawadagi, Basavaraj; Goodman, Jonathan; Tabynov, Kairat; Krakowka, Steven; Narasimhan, Balaji; Lee, Chang Won; Renukaradhya, Gourapura J

    2017-02-10

    Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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.

  9. 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

  10. 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

  11. Kvbeta2 inhibits the Kvbeta1-mediated inactivation of K+ channels in transfected mammalian cells.

    Science.gov (United States)

    Xu, J; Li, M

    1997-05-02

    Cloned auxiliary beta-subunits (e.g. Kvbeta1) modulate the kinetic properties of the pore-forming alpha-subunits of a subset of Shaker-like potassium channels. Coexpression of the alpha-subunit and Kvbeta2, however, induces little change in channel properties. Since more than one beta-subunit has been found in individual K+ channel complexes and expression patterns of different beta-subunits overlap in vivo, it is important to test the possible physical and/or functional interaction(s) between different beta-subunits. In this report, we show that both Kvbeta2 and Kvbeta1 recognize the same region on the pore-forming alpha-subunits of the Kv1 Shaker-like potassium channels. In the absence of alpha-subunits the Kvbeta2 polypeptide interacts with additional beta-subunit(s) to form either a homomultimer with Kvbeta2 or a heteromultimer with Kvbeta1. When coexpressing alpha-subunits and Kvbeta1 in the presence of Kvbeta2, we find that Kvbeta2 is capable of inhibiting the Kvbeta1-mediated inactivation. Using deletion analysis, we have localized the minimal interaction region that is sufficient for Kvbeta2 to associate with both alpha-subunits and Kvbeta1. This mapped minimal interaction region is necessary and sufficient for inhibiting the Kvbeta1-mediated inactivation, consistent with the notion that the inhibitory activity of Kvbeta2 results from the coassembly of Kvbeta2 with compatible alpha-subunits and possibly with Kvbeta1. Together, these results provide biochemical evidence that Kvbeta2 may profoundly alter the inactivation activity of another beta-subunit by either differential subunit assembly or by competing for binding sites on alpha-subunits, which indicates that Kvbeta2 is capable of serving as an important determinant in regulating the kinetic properties of K+ currents.

  12. Role of p53 and CDKN2A Inactivation in Human Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Alessia Pacifico

    2007-01-01

    Several studies have shown that human SCCs harbour unique mutations in the p53 gene as well as inactivation of the CDKN2A gene. While mutations in the p53 gene are induced by UV radiation and represent tumor initiating events, the majority of alterations detected in the CDKN2A gene do not appear to be UV-dependent. In conclusion, in addition to p53 mutations, silencing of the CDKN2A gene might play a significant role in SCC development.

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

    Science.gov (United States)

    Ferreira, Roberta V.; Silva-Caldeira, Priscila P.; Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D.; Ardisson, José D.; Domingues, Rosana Z.

    2016-04-01

    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 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions

  14. Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines.

    Science.gov (United States)

    Sasaki, Sanae; Jaimes, Maria C; Holmes, Tyson H; Dekker, Cornelia L; Mahmood, Kutubuddin; Kemble, George W; Arvin, Ann M; Greenberg, Harry B

    2007-01-01

    Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.

  15. 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.

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

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    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.

  17. Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    Directory of Open Access Journals (Sweden)

    Peter C. Soema

    2018-03-01

    Full Text Available Influenza peptide antigens coding for conserved T cell epitopes have the capacity to induce cross-protective influenza-specific immunity. Short peptide antigens used as a vaccine, however, often show poor immunogenicity. In this study, we demonstrate that whole-inactivated influenza virus (WIV acts as an adjuvant for influenza peptide antigens, as shown by the induction of peptide-specific CD8+ T cells in HLA-A2.1 transgenic mice upon vaccination with the influenza-M1-derived GILGFVFTL peptide (GIL, formulated with WIV. By screening various concentrations of GIL and WIV, we found that both components contributed to the GIL-specific T cell response. Whereas co-localization of the peptide antigen and WIV adjuvant was found to be important, neither physical association between peptide and WIV nor fusogenic activity of WIV were relevant for the adjuvant effect of WIV. We furthermore show that WIV may adjuvate T cell responses to a variety of peptides, using pools of either conserved wild-type influenza peptides or chemically altered peptide ligands. This study shows the potential of WIV as an adjuvant for influenza peptides. The simple formulation process and the solid safety record of WIV make this an attractive adjuvant for T cell peptides, and may also be used for non-influenza antigens.

  18. 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

  19. 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...

  20. 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.

  1. Differential response of porcine immature monocyte-derived dendritic cells to virulent and inactivated transmissible gastroenteritis virus.

    Science.gov (United States)

    Zhao, Shanshan; Gao, Qi; Lin, Jian; Yan, Mengfei; Yu, Qinghua; Yang, Qian

    2014-12-01

    Exposure of piglets less than 2 weeks of age to virulent transmissible gastroenteritis virus (TGEV) gives rise to mortality as high as 100%, and adult pigs recovering from its infection often become TGEV carriers. These facts suggest an evasion of the immune system by virulent TGEV. In this study, we showed that a virulent TGEV SHXB strain could infect porcine immature monocyte-derived dendritic cells (Mo-DCs), and down-regulate cell surface markers (SLA-II-DR, CD1a and CD80/86). Moreover, SHXB-infected immature Mo-DCs showed low expression of IL-12 and IFN-γ, and also lost the ability to stimulate T cell proliferation. Finally, SHXB inhibited the activation of nuclear factor kappa B (NF-κB) in these cells. Instead, UV-inactivated SHXB (UV-SHXB) had the opposite effects in immature Mo-DCs. In conclusion, the virulent SHXB could severely impair immature Mo-DCs, which might be involved in the pathogenesis of virulent TGEV in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations.

    Science.gov (United States)

    Shen, Yin; Matsuno, Youko; Fouse, Shaun D; Rao, Nagesh; Root, Sierra; Xu, Renhe; Pellegrini, Matteo; Riggs, Arthur D; Fan, Guoping

    2008-03-25

    X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0-100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, approximately 12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.

  3. Photodynamic treatment with mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin for pathogen inactivation in cord blood stem cell products.

    Science.gov (United States)

    Trannoy, Laurence L; van Hensbergen, Yvette; Lagerberg, Johan W M; Brand, Anneke

    2008-12-01

    Hematopoietic stem cell transplants and culture of hematopoietic progenitor cells require pathogen-free conditions. The application of a method of pathogen inactivation in red blood cells using photodynamic treatment (PDT) was investigated for the decontamination of cord blood stem cell (CBSC) products. CBSC products, spiked with Gram-positive and Gram-negative bacteria, were treated with PDT using mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light. After PDT, in vitro and in vivo evaluation of the CBSC functions were performed. PDT of CBSC products resulted in the inactivation of the bacteria, with Staphylococcus aureus being the most resistant. Complete decontamination was achieved when CBSC products were contaminated with low titers of bacteria. PDT had no effect on white blood cell viability, the ex vivo expansion potential of the progenitor cells, and their capacity to differentiate to various hematopoietic cell lineages. However, PDT reduced the engraftment of human CBSCs in NOD/SCID mice, particularly affecting the B-cell lineage engraftment. Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible at contamination level up to 10 to 20 colony-forming units per mL and can be considered when ex vivo expansion culture is anticipated. However, this treatment is not recommended for transplantation purposes at this time. Further investigations may elucidate why engraftment is diminished.

  4. 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.

  5. 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

  6. 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.)

  7. Synergistic Effect of Atmospheric-pressure Plasma and TiO2 Photocatalysis on Inactivation of Escherichia coli Cells in Aqueous Media

    Science.gov (United States)

    Zhou, Renwu; Zhou, Rusen; Zhang, Xianhui; Li, Jiangwei; Wang, Xingquan; Chen, Qiang; Yang, Size; Chen, Zhong; Bazaka, Kateryna; (Ken) Ostrikov, Kostya

    2016-01-01

    Atmospheric-pressure plasma and TiO2 photocatalysis have been widely investigated separately for the management and reduction of microorganisms in aqueous solutions. In this paper, the two methods were combined in order to achieve a more profound understanding of their interactions in disinfection of water contaminated by Escherichia coli. Under water discharges carried out by microplasma jet arrays can result in a rapid inactivation of E. coli cells. The inactivation efficiency is largely dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2, He and air microplasma arrays, O2 microplasma had the highest activity against E. coli cells in aqueous solution, and showed >99.9% bacterial inactivation efficiency within 4 min. Addition of TiO2 photocatalytic film to the plasma discharge reactor significantly enhanced the inactivation efficiency of the O2 microplasma system, decreasing the time required to achieve 99.9% killing of E. coli cells to 1 min. This may be attributed to the enhancement of ROS generation due to high catalytic activity and stability of the TiO2 photocatalyst in the combined plasma-TiO2 systems. Present work demonstrated the synergistic effect of the two agents, which can be correlated in order to maximize treatment efficiency. PMID:28004829

  8. Synergistic Effect of Atmospheric-pressure Plasma and TiO2 Photocatalysis on Inactivation of Escherichia coli Cells in Aqueous Media

    Science.gov (United States)

    Zhou, Renwu; Zhou, Rusen; Zhang, Xianhui; Li, Jiangwei; Wang, Xingquan; Chen, Qiang; Yang, Size; Chen, Zhong; Bazaka, Kateryna; (Ken) Ostrikov, Kostya

    2016-12-01

    Atmospheric-pressure plasma and TiO2 photocatalysis have been widely investigated separately for the management and reduction of microorganisms in aqueous solutions. In this paper, the two methods were combined in order to achieve a more profound understanding of their interactions in disinfection of water contaminated by Escherichia coli. Under water discharges carried out by microplasma jet arrays can result in a rapid inactivation of E. coli cells. The inactivation efficiency is largely dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2, He and air microplasma arrays, O2 microplasma had the highest activity against E. coli cells in aqueous solution, and showed >99.9% bacterial inactivation efficiency within 4 min. Addition of TiO2 photocatalytic film to the plasma discharge reactor significantly enhanced the inactivation efficiency of the O2 microplasma system, decreasing the time required to achieve 99.9% killing of E. coli cells to 1 min. This may be attributed to the enhancement of ROS generation due to high catalytic activity and stability of the TiO2 photocatalyst in the combined plasma-TiO2 systems. Present work demonstrated the synergistic effect of the two agents, which can be correlated in order to maximize treatment efficiency.

  9. Retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of CD8+ T-cell migration to the porcine gut

    Science.gov (United States)

    Chen, Xiaojuan; Tu, Chongzhi; Qin, Tao; Zhu, Liqi; Yin, Yinyan; Yang, Qian

    2016-01-01

    The digestive tract is the entry site for transmissible gastroenteritis virus (TGEV). TGEV transmission can be prevented if local immunity is established with increased lymphocytes. The current parenteral mode of vaccination stimulates systemic immunity well, but it does not induce sufficient mucosal immunity. Retinoic acid (RA) plays an important role in the induction of cells that imprint gut-homing molecules. We examined whether RA assist parenteral vaccination of pigs could improve mucosal immunity. We demonstrated that elevated numbers of gut-homing CD8+ T cells (which express α4β7 and CCR9 molecules) were presented in porcine inguinal lymph nodes and were recruited to the small intestine by RA. Intestinal mucosal immunity (IgA titre) and systemic immunity (serum IgG titre) were enhanced by RA. Therefore, we hypothesized that RA could induce DCs to form an immature mucosal phenotype and could recruit them to the small intestinal submucosa. Porcine T-cells expressed β7 integrin and CCR9 receptors and migrated to CCL25 by a mechanism that was dependent of activation by RA-pretreated DCs, rather than direct activation by RA. Together, our results provide powerful evidence that RA can assist whole inactivated TGEV (WI-TGEV) via subcutaneous (s.c.) immunization to generate intestinal immunity, and offer new vaccination strategies against TGEV. PMID:27080036

  10. Retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of CD8(+) T-cell migration to the porcine gut.

    Science.gov (United States)

    Chen, Xiaojuan; Tu, Chongzhi; Qin, Tao; Zhu, Liqi; Yin, Yinyan; Yang, Qian

    2016-04-15

    The digestive tract is the entry site for transmissible gastroenteritis virus (TGEV). TGEV transmission can be prevented if local immunity is established with increased lymphocytes. The current parenteral mode of vaccination stimulates systemic immunity well, but it does not induce sufficient mucosal immunity. Retinoic acid (RA) plays an important role in the induction of cells that imprint gut-homing molecules. We examined whether RA assist parenteral vaccination of pigs could improve mucosal immunity. We demonstrated that elevated numbers of gut-homing CD8(+) T cells (which express α4β7 and CCR9 molecules) were presented in porcine inguinal lymph nodes and were recruited to the small intestine by RA. Intestinal mucosal immunity (IgA titre) and systemic immunity (serum IgG titre) were enhanced by RA. Therefore, we hypothesized that RA could induce DCs to form an immature mucosal phenotype and could recruit them to the small intestinal submucosa. Porcine T-cells expressed β7 integrin and CCR9 receptors and migrated to CCL25 by a mechanism that was dependent of activation by RA-pretreated DCs, rather than direct activation by RA. Together, our results provide powerful evidence that RA can assist whole inactivated TGEV (WI-TGEV) via subcutaneous (s.c.) immunization to generate intestinal immunity, and offer new vaccination strategies against TGEV.

  11. Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation

    DEFF Research Database (Denmark)

    Ravnskjaer, Kim; Boergesen, Michael; Dalgaard, Louise T

    2006-01-01

    , the mechanism underlying this transcriptional repression by glucose remains unclear. Here we report that glucose-induced repression of PPARalpha gene expression in INS-1E cells is independent of beta-cell excitation and insulin secretion but requires activation of protein phosphatase 2A in a process involving...... but not AMPKalpha1 using RNAi suppressed PPARalpha expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARalpha expression in pancreatic beta-cells....... inactivation of the AMP-activated protein kinase (AMPK). Pharmacological activation of AMPK at high glucose concentrations interferes with glucose repression of PPARalpha and PPARalpha target genes in INS-1E cells as well as in rat islets. Specific knock-down of the catalytic AMPK-subunit AMPKalpha2...

  12. Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and Kupffer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Li-Jen Su

    Full Text Available BACKGROUND: Graptopetalum paraguayense (GP is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN- and carbon tetrachloride (CCl(4-induced liver injury rats. METHODS: Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs and Kupffer cells, respectively, were evaluated. RESULTS: Oral administration of MGP significantly alleviated DMN- or CCl(4-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. CONCLUSIONS: The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Emerging roles of Polycomb silencing in X-inactivation and stem cell maintenance

    NARCIS (Netherlands)

    Muyrers-Chen, [No Value; Hernandez-Munoz, [No Value; Lund, AH; Valk-Lingbeek, ME; Van der Stoop, P; Boutsma, E; Tolhuis, B; Bruggeman, SWM; Taghavi, P; Verhoeven, E; Hulsman, D; Noback, S; Tanger, E; Theunissen, H; van Lohuizen, M

    2004-01-01

    Maintenance of cell identity and cell fate depends on the tight regulation of gene expression patterns in correct time and space. Two families of proteins, the trithorax group (trxG) and the Polycomb group (PcG), use epigenetic mechanisms to faithfully ensure that designated genes are maintained on

  4. T cell inactivation by poxviral B22 family proteins increases viral virulence.

    Directory of Open Access Journals (Sweden)

    Dina Alzhanova

    2014-05-01

    Full Text Available Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197 caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

  5. Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

    Science.gov (United States)

    Sonoda, Masatoshi; Ide, Hiroaki; Nakayama, Shinya; Sasaki, Asako; Kitazaki, Shinei; Sato, Takahide; Nakagawa, Hiroki

    2003-04-01

    The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

  6. 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.

  7. 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).

  8. Inactivation of hypoxic cells by cisplatin and radiation at clinically relevant doses

    International Nuclear Information System (INIS)

    Korbelik, M.; Skov, K.A.

    1989-01-01

    We have examined the effects of exposure to cisplatin [cis-diamminedichloroplatinum(II)] on the response of exponentially growing V79 cells to low (0-4 Gy) and high (up to 30 Gy) doses of X rays under hypoxic and aerobic conditions. Survival in both dose regions was assessed by clonogenic assays; the low-dose studies were facilitated by a Cell Analyser. The results show that cisplatin, like its isomer trans-DDP, exhibits greater interaction with low than with high radiation doses in hypoxic cells. This increased interaction could be seen even with subtoxic exposures to cisplatin as low as 1 mumol dm-3. In contrast, with cells irradiated in air in the presence of either complex, the interaction seen with high doses of radiation is completely lost or greatly diminished in the low radiation dose region. Further experiments showed that enhanced interaction of hypoxic cells with low doses of radiation could be equally effective with cisplatin pretreatments in air or in hypoxia, even if the cells are exposed to cisplatin only after irradiation. In experiments with nonproliferating plateau-phase cultures, the same enhanced interaction was observed in the low-dose region. These results, for example enhancement ratios of 2.3 and 1.2 at low- and high-dose regions, respectively, for 5 mumol dm-3 cisplatin, are contrasted with those for nitroimidazoles which are better sensitizers in the high-dose region

  9. Edwardsiella tarda OmpA Encapsulated in Chitosan Nanoparticles Shows Superior Protection over Inactivated Whole Cell Vaccine in Orally Vaccinated Fringed-Lipped Peninsula Carp (Labeo fimbriatus

    Directory of Open Access Journals (Sweden)

    Saurabh Dubey

    2016-11-01

    Full Text Available The use of oral vaccination in finfish has lagged behind injectable vaccines for a long time as oral vaccines fall short of injection vaccines in conferring protective immunity. Biodegradable polymeric nanoparticles (NPs have shown potential to serve as antigen delivery systems for oral vaccines. In this study the recombinant outer membrane protein A (rOmpA of Edwardsiella tarda was encapsulated in chitosan NPs (NP-rOmpA and used for oral vaccination of Labeo fimbriatus. The rOmpA purity was 85%, nanodiameter <500 nm, encapsulation efficiency 60.6%, zeta potential +19.05 mV, and there was an in vitro release of 49% of encapsulated antigen within 48 h post incubation in phosphate-buffered saline. Empty NPs and a non-formulated, inactivated whole cell E. tarda (IWC-ET vaccine were used as controls. Post-vaccination antibody levels were significantly (p = 0.0458 higher in the NP-rOmpA vaccinated fish (Mean OD450 = 2.430 than in fish vaccinated with inactivated whole cell E. tarda (IWC-ET vaccine (Mean OD450 = 1.735, which corresponded with post-challenge survival proportions (PCSP of 73.3% and 48.28% for the NP-rOmpA and IWC-ET groups, respectively. Serum samples from NP-rOmpA-vaccinated fish had a higher inhibition rate for E. tarda growth on tryptic soy agar (TSA than the IWC-ET group. There was no significant difference (p = 0.989 in PCSPs between fish vaccinated with empty NPs and the unvaccinated control fish, while serum from both groups showed no detectable antibodies against E. tarda. Overall, these data show that the NP-rOmpA vaccine produced higher antibody levels and had superior protection over the IWC-ET vaccine, showing that encapsulating OmpA in chitosan NPs confer improved protection against E. tarda mortality in L. fimbriatus. There is a need to elucidate the possible adjuvant effects of chitosan NPs and the immunological mechanisms of protective immunity induced by OMPs administered orally to fish.

  10. Inactivation of PITX2 transcription factor induced apoptosis of gonadotroph tumoral cells.

    Science.gov (United States)

    Acunzo, Julie; Roche, Catherine; Defilles, Celine; Thirion, Sylvie; Quentien, Marie-Helene; Figarella-Branger, Dominique; Graillon, Thomas; Dufour, Henry; Brue, Thierry; Pellegrini, Isabelle; Enjalbert, Alain; Barlier, Anne

    2011-10-01

    Nonfunctioning pituitary adenomas (NFPA; gonadotroph derived), even not inducing hormonal hypersecretion, cause significant morbidity by compression neighboring structures. No effective and specific medical methods are available so far for treating these tumors. The pituitary homeobox 2 (PITX2) gene is a member of the bicoid-like homeobox transcription factor family, which is involved in the Wnt/Dvl/β-catenin pathway. PITX2 is overexpressed in NFPA. PITX2 mutations are known to be responsible for Axenfield Rieger syndrome, a genetic disorder in which pituitary abnormalities have been detected. The R91P mutant identified in Axenfeld Rieger syndrome is a dominant-negative factor, which is able to block the expression of several pituitary genes activated by PITX2. To better understand the role of Pitx2 on gonadotroph tumorigenesis and to explore new approach for inhibiting tumoral growth, the R91P mutant was transferred via a lentiviral vector in tumoral gonadotroph cells of two kinds: the αT3-1 cell line and human adenoma cells. R91P mutant and small interfering RNA directed against Pitx2 both decreased the viability of αT3-1 cells via an apoptotic mechanism involving the activation of executioner caspase. Similar effects of the R91P mutant were observed on human gonadotroph cells in primary culture. Therefore, Pitx2 overexpression may play an antiapoptotic role during NFPA tumorigenesis.

  11. 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.

  12. Photocatalytic Inactivation Effect of Gold-Doped TiO2 (Au/TiO2 Nanocomposites on Human Colon Carcinoma LoVo Cells

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    Zhiyu Jiang

    2007-11-01

    Full Text Available The photocatalytic inactivation effecting of gold-doped TiO2 (Au/TiO2 nanocomposites on human colon carcinoma LoVo cells was investigated for the first time. The Au/TiO2 samples containing different amounts of Au (1–4 wt% were prepared by deposition-precipitation (DP method. These synthesized Au/TiO2 nanocomposites were characterized by transmission electron microscopy (TEM and inductively coupled plasma atomic emission spectroscopy. It was found that the photocatalytic inactivation effect of TiO2 nanoparticles on LoVo cancer cells could be greatly improved by the surface modification of Au nanoparticles. Furthermore, the loading amount of Au on the surface of TiO2 nanoparticles affects the photocatalytic inactivation efficiency strongly, and it was found that the most efficient nanocomposites were TiO2 nanoparticles doped with 2 wt% Au. When 50 μg/mL 2 wt% Au/TiO2 nanocomposites were used, all of the LoVo cancer cells were killed under the irradiation of UV light (λmax = 365 nm, Intensity = 1.8 mW/cm2 within 100 minutes. But for 50 μg/mL TiO2 nanoparticles, only 40% cancer cells were killed under the same condition.

  13. 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.

  14. 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.

  15. 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.

  16. Coagulation Factor Xa inhibits cancer cell migration via LIMK1-mediated cofilin inactivation

    NARCIS (Netherlands)

    Borensztajn, Keren; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2010-01-01

    Previously, we showed that activated coagulation factor X (FXa) inhibits migration of breast, lung and colon cancer cells. We showed that the effect of FXa on migration was protease-activated receptor (PAR)-1-dependent, but the subsequent cellular signaling routes remained elusive. In the current

  17. Coagulation Factor Xa inhibits cancer cell migration via LIMK1-mediated cofilin inactivation

    NARCIS (Netherlands)

    Borensztajn, Keren; Peppelenbosch, Maikel P.; Spek, C. Arnold

    Previously, we showed that activated coagulation factor X (FXa) inhibits migration of breast, lung and colon cancer cells. We showed that the effect of FXa on migration was protease-activated receptor (PAR)-1-dependent, but the subsequent cellular signaling routes remained elusive. In the current

  18. 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

  19. Inactivation of SOCS3 in leptin receptor-expressing cells protects mice from diet-induced insulin resistance but does not prevent obesity a

    OpenAIRE

    Pedroso, João A.B.; Buonfiglio, Daniella C.; Cardinali, Lais I.; Furigo, Isadora C.; Ramos-Lobo, Angela M.; Tirapegui, Julio; Elias, Carol F.; Donato, Jose

    2014-01-01

    Therapies that improve leptin sensitivity have potential as an alternative treatment approach against obesity and related comorbidities. We investigated the effects of Socs3 gene ablation in different mouse models to understand the role of SOCS3 in the regulation of leptin sensitivity, diet-induced obesity (DIO) and glucose homeostasis. Neuronal deletion of SOCS3 partially prevented DIO and improved glucose homeostasis. Inactivation of SOCS3 only in LepR-expressing cells protected against lep...

  20. Efficacy of a single-dose regimen of inactivated whole-cell oral cholera vaccine: results from 2 years of follow-up of a randomised trial.

    Science.gov (United States)

    Qadri, Firdausi; Ali, Mohammad; Lynch, Julia; Chowdhury, Fahima; Khan, Ashraful Islam; Wierzba, Thomas F; Excler, Jean-Louis; Saha, Amit; Islam, Md Taufiqul; Begum, Yasmin A; Bhuiyan, Taufiqur R; Khanam, Farhana; Chowdhury, Mohiul I; Khan, Iqbal Ansary; Kabir, Alamgir; Riaz, Baizid Khoorshid; Akter, Afroza; Khan, Arifuzzaman; Asaduzzaman, Muhammad; Kim, Deok Ryun; Siddik, Ashraf U; Saha, Nirod C; Cravioto, Alejandro; Singh, Ajit P; Clemens, John D

    2018-03-14

    A single-dose regimen of inactivated whole-cell oral cholera vaccine (OCV) is attractive because it reduces logistical challenges for vaccination and could enable more people to be vaccinated. Previously, we reported the efficacy of a single dose of an OCV vaccine during the 6 months following dosing. Herein, we report the results of 2 years of follow-up. In this placebo-controlled, double-blind trial done in Dhaka, Bangladesh, individuals aged 1 year or older with no history of receipt of OCV were randomly assigned to receive a single dose of inactivated OCV or oral placebo. The primary endpoint was a confirmed episode of non-bloody diarrhoea for which the onset was at least 7 days after dosing and a faecal culture was positive for Vibrio cholerae O1 or O139. Passive surveillance for diarrhoea was done in 13 hospitals or major clinics located in or near the study area for 2 years after the last administered dose. We assessed the protective efficacy of the OCV against culture-confirmed cholera occurring 7-730 days after dosing with both crude and multivariable per-protocol analyses. This trial is registered at ClinicalTrials.gov, number NCT02027207. Between Jan 10, 2014, and Feb 4, 2014, 205 513 people were randomly assigned to receive either vaccine or placebo, of whom 204 700 (102 552 vaccine recipients and 102 148 placebo recipients) were included in the per-protocol analysis. 287 first episodes of cholera (109 among vaccine recipients and 178 among placebo recipients) were detected during the 2-year follow-up; 138 of these episodes (46 in vaccine recipients and 92 in placebo recipients) were associated with severe dehydration. The overall incidence rates of initial cholera episodes were 0·22 (95% CI 0·18 to 0·27) per 100 000 person-days in vaccine recipients versus 0·36 (0·31 to 0·42) per 100 000 person-days in placebo recipients (adjusted protective efficacy 39%, 95% CI 23 to 52). The overall incidence of severe cholera was 0·09 (0·07 to 0

  1. Tumor Induced Inactivation of Natural Killer Cell Cytotoxic Function; Implication in Growth, Expansion and Differentiation of Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Anahid Jewett, Han-Ching Tseng

    2011-01-01

    Full Text Available Accumulated evidence indicates that cytotoxic function of immune effectors is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and a potential mechanism for immunosuppression in cancer and to demonstrate the significance of such immunosuppression in cellular differentiation and progression of cancer. To that end, we have recently shown that NK cells mediate significant cytotoxicity against primary oral squamous carcinoma stem cells (OSCSCs as compared to their more differentiated oral squamous carcinoma cells (OSCCs. In addition, human embryonic stem cells (hESCs, Mesenchymal Stem Cells (hMSCs, dental pulp stem cells (hDPSCs and induced pluripotent stem cells (hiPSCs were all significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in primary monocytes in vivo significantly augmented NK cell function. Total population of monocytes and those depleted of CD16(+ subsets were able to substantially prevent NK cell mediated lysis of OSCSCs, MSCs and DPSCs. Taken together, our results suggest that stem cells are significant targets of the NK cell cytotoxicity. The concept of split anergy in NK cells and its contribution to tissue repair and regeneration and in tumor resistance and progression will be discussed in this review.

  2. 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.

  3. 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

  4. 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.

  5. Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

    Science.gov (United States)

    Dwivedi, Varun; Manickam, Cordelia; Dhakal, Santosh; Binjawadagi, Basavaraj; Ouyang, Kang; Hiremath, Jagadish; Khatri, Mahesh; Hague, Jacquelyn Gervay; Lee, Chang Won; Renukaradhya, Gourapura J

    2016-04-15

    Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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

  7. Removal of toxic metal ions from solution by inactivated cells of plants

    International Nuclear Information System (INIS)

    Raza, R.; Khattak, I.M.

    2006-01-01

    The aim of this paper to present the study of plant Larrea tridentate (bush) that grows abundantly in the desert environment of Hub, Balochistan toward the industrial area of Karachi (Pakistan). The binding of Ni(II), Cd(II), Pb(II), Zn(II), Cr(III), and Cr(VI) to Larrea tridentataroots. Stems, and leaves has been shown to be dependent upon pH, with best binding occurring between pH 5 and 6. This effect in pH suggests that the binding mechanism may be an ion exchange type process. Also, the binding mechanism for these metals is a stable, rapid process which implies that the binding is taking place on the cell wall surface of the creosote bush. Capacity and recovery experiments have demonstrated that Larrea tridentate possessed the ability to bind appreciable amounts of Ni(II), Pb(II), Zn(II), and Cr(III) as compared to other biosorbents. This ability to remove and recover heavy metals from solution indicates the tremendous potential that the creosote bush could have for cleansing the environment and industrial waste effluents from toxic metal ions. (author)

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

    Science.gov (United States)

    Liu, Yi; Zhang, Cuiping; Li, Zhenyu; Wang, Chi; Jia, Jianhang; Gao, Tianyan; Hildebrandt, Gerhard; Zhou, Daohong; Bondada, Subbarao; Ji, Peng; St Clair, Daret; Liu, Jinze; Zhan, Changguo; Geiger, Hartmut; Wang, Shuxia; Liang, Ying

    2017-04-11

    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. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. 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...... cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract...... for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature...

  10. 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

  11. Temperature-dependent rate models of vascular cambium cell mortality

    Science.gov (United States)

    Matthew B. Dickinson; Edward A. Johnson

    2004-01-01

    We use two rate-process models to describe cell mortality at elevated temperatures as a means of understanding vascular cambium cell death during surface fires. In the models, cell death is caused by irreversible damage to cellular molecules that occurs at rates that increase exponentially with temperature. The models differ in whether cells show cumulative effects of...

  12. Inactivation of a Gα(s)-PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis.

    Science.gov (United States)

    Iglesias-Bartolome, Ramiro; Torres, Daniela; Marone, Romina; Feng, Xiaodong; Martin, Daniel; Simaan, May; Chen, Min; Weinstein, Lee S; Taylor, Susan S; Molinolo, Alfredo A; Gutkind, J Silvio

    2015-06-01

    Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G protein, are associated with a large number of human diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signalling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal-cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs-PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signalling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumour suppressive function of Gαs-PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings could have broad implications in multiple pathophysiological conditions, including cancer.

  13. Inactivation of a Gαs-PKA tumor suppressor pathway in skin stem cells initiates basal-cell carcinogenesis

    Science.gov (United States)

    Iglesias-Bartolome, Ramiro; Torres, Daniela; Marone, Romina; Feng, Xiaodong; Martin, Daniel; Simaan, May; Chen, Min; Weinstein, Lee S.; Taylor, Susan S.; Molinolo, Alfredo A.; Gutkind, J. Silvio

    2015-01-01

    Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G-protein, are associated with a large number human of diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signaling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs-PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signaling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumor suppressive function of Gαs-PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings can have broad implications in multiple pathophysiological conditions, including cancer. PMID:25961504

  14. Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

    NARCIS (Netherlands)

    Kruijsen, Debby; Schijf, Marcel A.; Lukens, Michaël V.; van Uden, Nathalie O.; Kimpen, Jan L.; Coenjaerts, Frank E.; van Bleek, Grada M.

    2011-01-01

    Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza

  15. 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.

  16. 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)

  17. Quantitative description of radiation-induced cell inactivation. VII. Primary radiation lesions resulting in reproductive death of cells

    Energy Technology Data Exchange (ETDEWEB)

    Barsukov, V.S.

    1975-01-01

    The reproductive death of di- and polyploid cells is assumed to be caused mainly by asymmetrical exchanges of chromosomes. If a single DNA molecule is present in an interphase chromosome, the exchange is equivalent to cross-polymerization of two polynucleotide strands in which a double break in the DNA is inevitable. An hypothesis is advanced whereby injuries to chromosomes that result in changes are considered identical to double-strand breaks in DNA. To check the hypothesis, the yield or primary chromosomes injuries is equated with the number of double-strand breaks in DNA, which is determined from the DNA content of the cell. Experimental data are cited to support the hypothesis that primary chromosome lesions in eucaryotic cells in the G/sub 1/ phase are identical to double-strand breaks in DNA. (JPRS)

  18. 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

  19. [Impact of PRDM1 gene inactivation on C-MYC regulation in diffuse large B-cell lymphoma].

    Science.gov (United States)

    Zhang, X Y; Ma, Z P; Cui, W L; Pang, X L; Chen, R; Wang, L; Zhang, W; Li, X X

    2018-01-08

    in the two cell lines, C-MYC protein and gene expression were up-regulated in the transfection group, compared with the blank control group and negative control group by reverse transcription PCR and Western blot analyses. Moreover, PRDM1 expression was significantly associated with C-MYC(χ(2)=7.648, P =0.006) at mRNA level. Conclusion: The up-regulation of C-MYC gene expression induced by PRDM1 inactivation in DLBCL may play an important role for the development of DLBCL.PRDM1 protein and mRNA are associated with immunophenotyping and PRDM1 mRNA is a marker of poor prognosis.

  20. 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.

  1. 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

  2. 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.

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

    Science.gov (United States)

    Pilly, Praveen K; Grossberg, Stephen

    2013-01-01

    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 medial entorhinal cortex (MEC), have been interpreted to support oscillatory interference models of grid cell firing. The current article shows that an alternative self-organizing map (SOM) 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 and oscillatory dynamics.

  4. 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.

  5. CYTOTOXICITY AGAINST TUMOR CELL LINES OF A RIBOSOME-INACTIVATING PROTEIN (RIP-LIKE PROTEIN ISOLATED FROM LEAVES OF MIRABILIS JALAPA L.

    Directory of Open Access Journals (Sweden)

    ZULLIES IKAWATI

    2006-01-01

    Full Text Available The 30 kD protein fraction with properties like ribosome-inactivating protein (RIP was isolated from the leaves of Mirabilis jalapa L. and named MJ-30. This study investigated the cytotoxic effect of MJ-30 on normal and malignant cells. MJ-30 was isolated from the leave extract of M. jalapa L. using cation-exchange chromatography with CM Sepharose CL-6B column to obtain MJ-30. The fraction contain DNA cleaving ability were pooled and checked for cytotoxicity against breast cancer T47D cell line, cervical cancer SiHa cell line, and human mononuclear cells derived from peripheral blood of healthy volunteers. Results showed that the MJ-30 produced cytotoxic effect against T47D and SiHa cell line to different extent. The LC50 of the MJ-30 on T47D cell line and SiHa cell line were 0.36 μg/mL and 5.6 μg/mL, respectively. While in normal cells, represented by human mononuclear cells, MJ-30 was considerably less toxic, with LC50 of 21.04 μg/mL. The results suggest that MJ-30 produced more cytotoxic activity toward breast and cervical cancer cells (58-fold and 4-fold, respectively as compared to normal mononuclear cells.

  6. Human PIEZO1: removing inactivation.

    Science.gov (United States)

    Bae, Chilman; Gottlieb, Philip A; Sachs, Frederick

    2013-08-20

    PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Pseudomonas aeruginosa pyocyanin causes airway goblet cell hyperplasia and metaplasia and mucus hypersecretion by inactivating the transcriptional factor FoxA2.

    Science.gov (United States)

    Hao, Yonghua; Kuang, Zhizhou; Walling, Brent E; Bhatia, Shikha; Sivaguru, Mayandi; Chen, Yin; Gaskins, H Rex; Lau, Gee W

    2012-03-01

    The redox-active exotoxin pyocyanin (PCN) can be recovered in 100 µM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2. © 2011 Blackwell Publishing Ltd.

  8. Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells.

    Science.gov (United States)

    Kudryavtseva, Anna V; Fedorova, Maria S; Zhavoronkov, Alex; Moskalev, Alexey A; Zasedatelev, Alexander S; Dmitriev, Alexey A; Sadritdinova, Asiya F; Karpova, Irina Y; Nyushko, Kirill M; Kalinin, Dmitry V; Volchenko, Nadezhda N; Melnikova, Nataliya V; Klimina, Kseniya M; Sidorov, Dmitry V; Popov, Anatoly Y; Nasedkina, Tatiana V; Kaprin, Andrey D; Alekseev, Boris Y; Krasnov, George S; Snezhkina, Anastasiya V

    2016-12-22

    The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the "Warburg effect" and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear. The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis. We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

  9. Tyrosinase inactivation in organic solvents.

    Science.gov (United States)

    Warrington, J C; Saville, B A

    1999-11-05

    The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction. Copyright 1999 John Wiley & Sons, Inc.

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

    Science.gov (United States)

    Scaplen, Kristin M; Ramesh, Rohan N; Nadvar, Negin; Ahmed, Omar J; Burwell, Rebecca D

    2017-01-01

    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 navigation. FCM

  13. Inactivation of Rac1 reduces Trastuzumab resistance in PTEN deficient and insulin-like growth factor I receptor overexpressing human breast cancer SKBR3 cells.

    Science.gov (United States)

    Zhao, Yong; Wang, Zhishan; Jiang, Yiguo; Yang, Chengfeng

    2011-12-26

    Drug resistance remains to be a big challenge in applying anti-HER2 monoclonal antibody Trastuzumab for treating breast cancer with HER2 overexpression. Amplification of insulin-like growth factor I receptor (IGF-IR) and deletion of tumor suppressor phosphatase and tensin homolog (PTEN) are implicated in Trastuzumab resistance, however, the underlying mechanisms have not been clearly defined. Activation of Rac1, a member of Rho GTPase family, is capable of causing cytoskeleton reorganization, regulating gene expression and promoting cell proliferation. To investigate the mechanism of Trastuzumab resistance, PTEN knockdown and IGF-IR overexpressing stable cell lines were generated in HER2 overexpression human breast cancer SKBR3 cells. Rac1 was highly activated in PTEN deficient and IGF-IR overexpressing Trastuzumab-resistant cells in a HER2-independent manner. Inactivation of Rac1 by using a Rac1 inhibitor NSC23766 or siRNA knocking down the expression of Tiam1, a guanine nucleotide exchange factor for Rac, significantly reduced Trastuzumab resistance in SKBR3 cells. Inhibition of Rac1 had no effect on the levels of phosphor-HER2 and phosphor-Akt, but significantly decreased the levels of cyclin D1 in Trastuzumab-resistant cells. Inhibition of Akt with an Akt inhibitor also significantly reduced Trastuzumab resistance. However, simultaneous inhibition of both Rac1 and Akt resulted in a significantly more decrease of Trastuzumab resistance than inactivation of Rac1 or Akt alone. These results suggest that Rac1 activation is critically involved in Trastuzumab resistance caused by PTEN deletion or IGF-IR overexpression. Simultaneous inhibition of Rac1 and Akt may represent a promising strategy in reducing Trastuzumab resistance in HER2 overexpression breast cancer. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Odorants suppress T- and L-type Ca2+ currents in olfactory receptor cells by shifting their inactivation curves to a negative voltage.

    Science.gov (United States)

    Kawai, F

    1999-12-30

    Mechanisms underlying suppression of T- and L-type Ca2+ currents (I(Ca,T) and I(Ca,L)) by odorants were investigated in newt olfactory receptor cells (ORCs) using the whole-cell version of the patch-clamp technique. Under voltage clamp, odorants (amyl acetate, limonene and acetophenone) reversibly suppressed I(Ca,T) and I(Ca, L). These currents disappeared completely within 150 ms following amyl acetate puffs, and recovered in approximately 1 s after the washout. Hyperpolarization of the membrane greatly relieved the odorant block of I(Ca,T) and I(Ca,L). The activation curves of both currents were not changed significantly by odorants, while their inactivation curves were shifted to negative voltages. Half-inactivation voltages of I(Ca,T) were - 66 mV (control), - 102 mV (amyl acetate), - 101 mV (limonene) and - 105 mV (acetophenone) (all 0.3 mM); those of I(Ca,L) were -33 mV (control), - 61 mV (amyl acetate), - 59 mV (limonene), and - 63 mV (acetophenone) (all 0.3 mM). These phenomena are similar to the effects of local anesthetics on I(Ca) in various preparations and also similar to the effects of odorants on I(Na) in ORCs, suggesting that these types of suppression are caused by the same mechanism.

  15. Inactivated E. coli transformed with plasmids that produce dsRNA against infectious salmon anemia virus hemagglutinin show antiviral activity when added to infected ASK cells.

    Directory of Open Access Journals (Sweden)

    Katherine eGarcía

    2015-04-01

    Full Text Available Infectious salmon anemia virus (ISAV has caused great losses to the Chilean salmon industry, and the success of prevention and treatment strategies is uncertain. The use of RNA interference (RNAi is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm. We explored the capacity of E. coli transformed with plasmids that produce double-stranded RNA (dsRNA to induce antiviral activity when added to infected ASK cells. We transformed the non-pathogenic Escherichia coli HT115 (DE3 with plasmids that expressed highly conserved regions of the ISAV genes encoding the nucleoprotein (NP, fusion (F, hemagglutinin (HE and matrix (M proteins as dsRNA, which is the precursor of the RNAi mechanism. The inactivated transformed bacteria carrying dsRNA were tested for their capacity to silence the target ISAV genes, and the dsRNA that were able to inhibit gene expression were subsequently tested for their ability to attenuate the cytopathic effect (CPE and reduce the viral load. Of the four target genes tested, inactivated E. coli transformed with plasmids producing dsRNA targeting HE showed antiviral activity when added to infected ASK cells.

  16. [Characteristics of thermal inactivation of lysozyme in solution].

    Science.gov (United States)

    Tarun, E I; Eremin, A N; Metelitsa, D I

    1986-01-01

    In the buffer solution (pH 6,2) at 20-80 degrees, the lysozyme thermoinactivation was studied by monitoring of its activity decrease in the lysis of M. lysodeicticus cells. Protein inactivation was characterized by effective pseudofirst order rate constants which depend on enzyme concentration and are described by equation k = k0 . exp [-alpha 0 (1-gamma/T) [E]0], where k0 is inactivation rate constant at "infinite" enzyme dilution, [E0] is an initial lysozyme concentration, alpha 0 and gamma are the coefficients independent on [E0]. By extrapolation of the "k" dependencies on [E]0 the constants k0 were determined. In the range 40-70 degrees C, the rate constant k0 is equal 4,0 X 10(11) . exp (-24 200/RT) sec-1.

  17. 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

  18. 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).

  19. 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

  20. 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.

  1. 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)

  2. 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.

  3. 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 (pdidecyldimethylammonium 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.

  4. Sex-differential selection and the evolution of X inactivation strategies.

    Directory of Open Access Journals (Sweden)

    Tim Connallon

    2013-04-01

    Full Text Available X inactivation--the transcriptional silencing of one X chromosome copy per female somatic cell--is universal among therian mammals, yet the choice of which X to silence exhibits considerable variation among species. X inactivation strategies can range from strict paternally inherited X inactivation (PXI, which renders females haploid for all maternally inherited alleles, to unbiased random X inactivation (RXI, which equalizes expression of maternally and paternally inherited alleles in each female tissue. However, the underlying evolutionary processes that might account for this observed diversity of X inactivation strategies remain unclear. We present a theoretical population genetic analysis of X inactivation evolution and specifically consider how conditions of dominance, linkage, recombination, and sex-differential selection each influence evolutionary trajectories of X inactivation. The results indicate that a single, critical interaction between allelic dominance and sex-differential selection can select for a broad and continuous range of X inactivation strategies, including unequal rates of inactivation between maternally and paternally inherited X chromosomes. RXI is favored over complete PXI as long as alleles deleterious to female fitness are sufficiently recessive, and the criteria for RXI evolution is considerably more restrictive when fitness variation is sexually antagonistic (i.e., alleles deleterious to females are beneficial to males relative to variation that is deleterious to both sexes. Evolutionary transitions from PXI to RXI also generally increase mean relative female fitness at the expense of decreased male fitness. These results provide a theoretical framework for predicting and interpreting the evolution of chromosome-wide expression of X-linked genes and lead to several useful predictions that could motivate future studies of allele-specific gene expression variation.

  5. 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.

  6. 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.

  7. Riproximin: A type II ribosome inactivating protein with anti-neoplastic potential induces IL24/MDA-7 and GADD genes in colorectal cancer cell lines.

    Science.gov (United States)

    Pervaiz, Asim; Adwan, Hassan; Berger, Martin R

    2015-09-01

    Riproximin (Rpx) is a type II ribosome inactivating protein, which was extracted and purified from the seeds of Ximenia americana. Previous studies demonstrated cytotoxicity of Rpx against a variety of cell lines originating from solid and non-solid cancers. In this study, we investigated the mechanistic aspects of Rpx in selected human and rat colorectal cancer (CRC) cell lines. Cytotoxic levels of Rpx were determined by MTT assay, while cytostatic and apoptotic effects were investigated by flow cytometry and nuclear staining procedures. Effects of Rpx exposure on colony formation/migration of CRC cells and expressional modulations in anticancer/stress-related genes were also studied. Rpx showed significant and comparable levels of cytotoxicity in CRC cells as determined by inhibitory concentration (IC) values. Similar inhibitory effects were found for clonogenicity, while more pronounced inhibition of migration was observed in response to Rpx exposure. Profound arrest in S phases of the cell cycle was noted especially in primary CRC cells. Apoptotic effects were more prominent in rat CRC cells as indicated by Annexin V-FITC assay and Hoechst 33342 nuclear staining. Rpx exposure induced significantly increased levels of the IL24/MDA-7, a well characterized anticancer gene, in all CRC cells. In addition, following Rpx treatment, high expression levels of growth arrest and DNA damage (GADD family) genes were also observed. Increased expression of two additional GADD genes (34 and 153) only in rat CRC cells (CC531) conferred higher sensitivity towards Rpx and subsequent anti-proliferative/apoptotic effects as compared to human CRC cells (SW480 and SW620). The present investigation indicates the anticancer potential of Rpx in CRC and favor further evaluation of this natural compound as therapeutic agent.

  8. 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.

  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. Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis.

    Science.gov (United States)

    Huang, Ching-Shui; Lee, Yi-Ru; Chen, Ching-Shyang; Tu, Shih-Hsin; Wang, Ying-Jan; Lee, Chia-Hwa; Chen, Li-Ching; Chang, Hui-Wen; Chang, Chien-Hsi; Chih-Ming, Su; Wu, Chih-Hsiung; Ho, Yuan-Soon

    2010-08-01

    The aim of this study was to test whether long-term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol-treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20-40 passages with or without 10 mM ethanol (designated as E20-E40 and C20-C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long-term ethanol treatment was induced in the E40 cells through preferential acceleration of S-phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol-treated E40 cells. We further demonstrated that long-term ethanol-treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8 microM). Immunoblot analysis revealed that caspase-8-mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC-resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser-112) through its binding with 14-3-3 was detected more profoundly in the MMC-resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10 mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40- and C40-xenografted tumors. Significant reductions (>3-fold) in tumor growth were detected in MMC-treated C40-xenografted mice. In vivo and in vitro studies demonstrated that AKT- and extracellular signal-regulated kinase (ERK)-mediated survival factors inhibited the Bad-induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC. Copyright (c) 2010 Wiley-Liss, Inc.

  11. 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

  12. 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.

  13. From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects.

    Science.gov (United States)

    Ben-Ari, Meital; Schick, Revital; Barad, Lili; Novak, Atara; Ben-Ari, Erez; Lorber, Avraham; Itskovitz-Eldor, Joseph; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2014-10-01

    We previously reported that induced pluripotent stem cell-derived cardiomyocytes manifest beat rate variability (BRV) resembling heart rate variability (HRV) in the human sinoatrial node. We now hypothesized the BRV-HRV continuum originates in pacemaker cells. To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized 3 levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells, and (3) the in situ sinoatrial node. We measured BRV/HRV properties in single pacemaker cells, induced pluripotent stem cell-derived contracting embryoid bodies (EBs), and electrocardiograms from the same individual. Pronounced BRV/HRV was present at all 3 levels. The coefficient of variance of interbeat intervals and Poincaré plot indices SD1 and SD2 for single cells were 20 times greater than those for EBs (P heart (the latter two were similar; P > .05). We also compared BRV magnitude among single cells, small EBs (~5-10 cells), and larger EBs (>10 cells): BRV indices progressively increased with the decrease in the cell number (P heart rhythm. The decreased BRV magnitude in transitioning from the single cell to the EB suggests that the HRV of in situ hearts originates from the summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca(2+) handling determine HRV in humans and cardiomyocyte networks. Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  14. Cranberry proanthocyanidins inhibit esophageal adenocarcinoma in vitro and in vivo through pleiotropic cell death induction and PI3K/AKT/mTOR inactivation

    Science.gov (United States)

    Kresty, Laura A.; Weh, Katherine M.; Zeyzus-Johns, Bree; Perez, Laura N.; Howell, Amy B.

    2015-01-01

    Cranberries are rich in bioactive constituents known to improve urinary tract health and more recent evidence supports cranberries possess cancer inhibitory properties. However, mechanisms of cancer inhibition by cranberries remain to be elucidated, particularly in vivo. Properties of a purified cranberry-derived proanthocyanidin extract (C-PAC) were investigated utilizing acid-sensitive and acid-resistant human esophageal adenocarcinoma (EAC) cell lines and esophageal tumor xenografts in athymic NU/NU mice. C-PAC induced caspase-independent cell death mainly via autophagy and low levels of apoptosis in acid-sensitive JHAD1 and OE33 cells, but resulted in cellular necrosis in acid-resistant OE19 cells. Similarly, C-PAC induced necrosis in JHAD1 cells pushed to acid-resistance via repeated exposures to an acidified bile cocktail. C-PAC associated cell death involved PI3K/AKT/mTOR inactivation, pro-apoptotic protein induction (BAX, BAK1, deamidated BCL-xL, Cytochrome C, PARP), modulation of MAPKs (P-P38/P-JNK) and G2-M cell cycle arrest in vitro. Importantly, oral delivery of C-PAC significantly inhibited OE19 tumor xenograft growth via modulation of AKT/mTOR/MAPK signaling and induction of the autophagic form of LC3B supporting in vivo efficacy against EAC for the first time. C-PAC is a potent inducer of EAC cell death and is efficacious in vivo at non-toxic behaviorally achievable concentrations, holding promise for preventive or therapeutic interventions in cohorts at increased risk for EAC, a rapidly rising and extremely deadly malignancy. PMID:26378019

  15. Mitochondrial reactive oxygen species perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of PP2A in lowdose irradiated human fibroblasts.

    Science.gov (United States)

    Shimura, Tsutomu; Sasatani, Megumi; Kamiya, Kenji; Kawai, Hidehiko; Inaba, Yohei; Kunugita, Naoki

    2016-01-19

    Here we investigated the cellular response of normal human fibroblasts to repeated exposure to low-dose radiation. In contrast to acute single radiation, low-dose fractionated radiation (FR) with 0.01 Gy/fraction or 0.05 Gy/fraction for 31 days increased in mitochondrial mass, decreased cellular levels of the antioxidant glutathione and caused persistent accumulation of mitochondrial reactive oxygen species (ROS). Excess ROS promoted oxidative inactivation of protein phosphatase PP2A which in turn led to disruption of normal negative feed-back control of AKT/cyclin D1 signaling in cells treated with long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 causes growth retardation, cellular senescence and genome instability in low-dose irradiated cells. Thus, loss of redox control and subsequently elevated levels of ROS perturb signal transduction as a result of oxidative stress. Our study highlights a specific role of mitochondrial ROS in perturbation of AKT/cyclin D1 cell cycle signaling after low-dose long-term FR. The antioxidants N-acetyl-L-cysteine, TEMPO and mitochondrial-targeted antioxidant Mito-TEMPO provided protection against the harmful cell cycle perturbations induced by low-dose long-term FR.

  16. 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.

  17. Glomerular filtration rate in steady state children with sickle cell ...

    African Journals Online (AJOL)

    Background: Sickle cell anaemia (SCA) affects mostly people of African origin. It causes kidney problems termed Sickle cell nephropathy (SCN). Increased glomerular filtration rate (GFR) has been documented as one of the functional abnormalities seen in young SCA patients. Objective: To estimate GFR in Nigerian ...

  18. Short Communication: Inhibition of DC-SIGN-Mediated HIV-1 Infection by Complementary Actions of Dendritic Cell Receptor Antagonists and Env-Targeting Virus Inactivators.

    Science.gov (United States)

    Pustylnikov, Sergey; Dave, Rajnish S; Khan, Zafar K; Porkolab, Vanessa; Rashad, Adel A; Hutchinson, Matthew; Fieschi, Frank; Chaiken, Irwin; Jain, Pooja

    2016-01-01

    The DC-SIGN receptor on human dendritic cells interacts with HIV gp120 to promote both infection of antigen-presenting cells and transinfection of T cells. We hypothesized that in DC-SIGN-expressing cells, both DC-SIGN ligands such as dextrans and gp120 antagonists such as peptide triazoles would inhibit HIV infection with potential complementary antagonist effects. To test this hypothesis, we evaluated the effects of dextran (D66), isomaltooligosaccharides (D06), and several peptide triazoles (HNG156, K13, and UM15) on HIV infection of B-THP-1/DC-SIGN cells. In surface plasmon resonance competition assays, D66 (IC50 = 35.4 μM) and D06 (IC50 = 3.4 mM) prevented binding of soluble DC-SIGN to immobilized mannosylated bovine serum albumin (BSA). An efficacious dose-dependent inhibition of DC-SIGN-mediated HIV infection in both pretreatment and posttreatment settings was observed, as indicated by inhibitory potentials (EC50) [D66 (8 μM), D06 (48 mM), HNG156 (40 μM), UM15 (100 nM), and K13 (25 nM)]. Importantly, both dextrans and peptide triazoles significantly decreased HIV gag RNA levels [D66 (7-fold), D06 (13-fold), HNG156 (7-fold), K-13 (3-fold), and UM15 (6-fold)]. Interestingly, D06 at the highest effective concentration showed a 14-fold decrease of infection, while its combination with 50 μM HNG156 showed a 26-fold decrease. Hence, these compounds can combine to inactivate the viruses and suppress DC-SIGN-mediated virus-cell interaction that as shown earlier leads to dendritic cell HIV infection and transinfection dependent on the DC-SIGN receptor.

  19. 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

  20. The effectiveness of radiant catalytic ionization in inactivation of Listeria monocytogenes planktonic and biofilm cells from food and food contact surfaces as a method of food preservation.

    Science.gov (United States)

    Skowron, Krzysztof; Grudlewska, Katarzyna; Krawczyk, Agnieszka; Gospodarek-Komkowska, Eugenia

    2018-02-02

    The aim of the study was to evaluate the microbicidal effectiveness of Radiant Catalytic Ionization (RCI) against L. monocytogenes strains in the form of planktonic cells and biofilm on food products and food contact surfaces as a method of food preservation. The study material comprised six strains of L. monocytogenes, isolated from food. Samples of different types of food available by retail (raw carrot, frozen salmon filets, soft cheese) and the fragments of surfaces (stainless steel AISI 304, rubber, milled rock tiles, polypropylene) were used in the experiment. The obtained results showed the effectiveness of RCI in the inactivation of both forms of the tested L. monocytogenes strains on all the surfaces. The effectiveness of RCI for biofilm forms was lower as compared with planktonic forms. The PRR value ranged from 18,19% to 99,97% for planktonic form and from 3,92% to 70,10% for biofilm. The RCI phenomenon induces the inactivation of L. monocytogenes on surfaces of food and materials used in the processing industry to a varying degree, depending on the manner of surface contamination, the properties of the contaminated materials as well as on the origin of the strain and the properties of surrounding dispersive environment in which the microorganisms were suspended. Searching of new actions aimed at reduction of the microbial contamination of food and food contact surfaces are extremely important. RCI method has been already described as an effective technique of microbial and abiotic pollution removal from air. However, our studies provide new, additional data related to evaluation the RCI efficacy against microbes on different surfaces, both in planktonic and biofilm form. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. In vitro photodynamic inactivation effects of cationic benzylidene cyclopentanone photosensitizers on clinical fluconazole-resistant Candida albicans planktonic cells and biofilms

    Science.gov (United States)

    Zhou, Shaona; Fang, Yanyan; Ye, Zulin; Wang, Ying; Zhao, Yuxia; Gu, Ying

    2016-10-01

    Background: An increasing prevalence of Candida infections has emerged with the wide use of immune-suppressants and antibiotics. Photodynamic inactivation (PDI) as a new approach to treat localized Candida infections is an emerging and promising field nowadays. This study evaluated the efficacy of photodynamic therapy using two new Cationic benzylidene cyclopentanone photosensitizers(P1 and P2) against strains of clinical fluconazole-resistant Candida albicans. Methods: Suspensions and biofilms of Candida species were incubated with P1 and P2 concentrations (0.25 50 μM) for 30 min followed by 532nm laser irradiation. For planktonic suspensions, viability of cells was assayed by CFU counting. For biofilms, the metabolic activity was evaluated by XTT. Results: In PDI of a planktonic culture of clinical fluconazole-resistant Candida albicans, P2 showed the higher efficacy. After incubation with 25 μM of P2 for 30 min and irradiation with 532nm laser (36 J cm-2), the viability of C. albicans planktonic cells decreased by 3.84 log10. For biofilm cells, a higher light dose of 75 mW cm-2 was necessary to achieve 97.71% metabolic activity reduction. Conclusions: The results of this investigation demonstrated that benzylidene cyclopentanone photosensitizer, P2, is an efficient photosensitizer to kill C. albicans. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.

  2. Arhgef15 promotes retinal angiogenesis by mediating VEGF-induced Cdc42 activation and potentiating RhoJ inactivation in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Sentaro Kusuhara

    Full Text Available BACKGROUND: Drugs inhibiting vascular endothelial growth factor (VEGF signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs. Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF and evaluate its role in retinal angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas. CONCLUSIONS/SIGNIFICANCE: Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.

  3. 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.

  4. 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.

  5. 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.

  6. Inactivation of Vhl in Osteochondral Progenitor Cells Causes High Bone Mass Phenotype and Protects Against Age-Related Bone Loss in Adult Mice

    Science.gov (United States)

    Weng, Tujun; Xie, Yangli; Huang, Junlan; Luo, Fengtao; Yi, Lingxian; He, Qifen; Chen, Di; Chen, Lin

    2014-01-01

    Previous studies have shown that disruption of von Hippel–Lindau gene (Vhl) coincides with activation of hypoxia-inducible factor α (HIFα) signaling in bone cells and plays an important role in bone development, homeostasis, and regeneration. It is known that activation of HIF1α signaling in mature osteoblasts is central to the coupling between angiogenesis and bone formation. However, the precise mechanisms responsible for the coupling between skeletal angiogenesis and osteogenesis during bone remodeling are only partially elucidated. To evaluate the role of Vhl in bone homeostasis and the coupling between vascular physiology and bone, we generated mice lacking Vhl in osteochondral progenitor cells (referred to as Vhl cKO mice) at postnatal and adult stages in a tamoxifen-inducible manner and changes in skeletal morphology were assessed by micro–computed tomography (µCT), histology, and bone histomorphometry. We found that mice with inactivation of Vhl in osteochondral progenitor cells at the postnatal stage largely phenocopied that of mice lacking Vhl in mature osteoblasts, developing striking and progressive accumulation of cancellous bone with increased microvascular density and bone formation. These were accompanied with a significant increase in osteoblast proliferation, upregulation of differentiation marker Runx2 and osteocalcin, and elevated expression of vascular endothelial growth factor (VEGF) and phosphorylation of Smad1/5/8. In addition, we found that Vhl deletion in osteochondral progenitor cells in adult bone protects mice from aging-induced bone loss. Our data suggest that the VHL-mediated signaling in osteochondral progenitor cells plays a critical role in bone remodeling at postnatal/adult stages through coupling osteogenesis and angiogenesis. © 2014 American Society for Bone and Mineral Research. PMID:23999831

  7. 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.

  8. A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

    Directory of Open Access Journals (Sweden)

    Lejeune Laurence

    2006-11-01

    Full Text Available Abstract Background T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2 promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. Results First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg. Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA. This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. Conclusion These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be

  9. 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

  10. Platinum Nanoparticles with Adsorptive Layer of Chlorella vulgaris Polysaccharides Inactivate Tumor Cells of Ascitic Ehrlich Carcinoma, Ovarian Cancer and Leukemia

    Science.gov (United States)

    Estrela-Llopis, V. R.; Chevichalova, A. V.; Tregubova, N. A.; Shishko, E. D.; Litvin, P. M.

    In comparison (with) nanogold, not much work on the application of nanoplatinum in cancer therapy is known. The purpose of this work is to investigate the aggregation of platinum nanoparticles with different cancer cells and its effects on cells' vital activity.

  11. 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

  12. 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)

  13. The biological effect of large single doses: a possible role for non-targeted effects in cell inactivation.

    Directory of Open Access Journals (Sweden)

    Marlon R Veldwijk

    Full Text Available BACKGROUND AND PURPOSE: Novel radiotherapy techniques increasingly use very large dose fractions. It has been argued that the biological effect of large dose fractions may differ from that of conventional fraction sizes. The purpose was to study the biological effect of large single doses. MATERIAL AND METHODS: Clonogenic cell survival of MCF7 and MDA-MB-231 cells was determined after direct X-ray irradiation, irradiation of feeder cells, or transfer of conditioned medium (CM. Cell-cycle distributions and the apoptotic sub-G1 fraction were measured by flow cytometry. Cytokines in CM were quantified by a cytokine antibody array. γH2AX foci were detected by immunofluorescence microscopy. RESULTS: The surviving fraction of MCF7 cells irradiated in vitro with 12 Gy showed an 8.5-fold decrease (95% c.i.: 4.4-16.3; P<0.0001 when the density of irradiated cells was increased from 10 to 50×10(3 cells per flask. Part of this effect was due to a dose-dependent transferrable factor as shown in CM experiments in the dose range 5-15 Gy. While no effect on apoptosis and cell cycle distribution was observed, and no differentially expressed cytokine could be identified, the transferable factor induced prolonged expression of γH2AX DNA repair foci at 1-12 h. CONCLUSIONS: A dose-dependent non-targeted effect on clonogenic cell survival was found in the dose range 5-15 Gy. The dependence of SF on cell numbers at high doses would represent a "cohort effect" in vivo. These results support the hypothesis that non-targeted effects may contribute to the efficacy of very large dose fractions in radiotherapy.

  14. 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

  15. 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

  16. 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.

  17. Discovery of dehydroabietic acid sulfonamide based derivatives as selective matrix metalloproteinases inactivators that inhibit cell migration and proliferation.

    Science.gov (United States)

    Huang, Ri-Zhen; Liang, Gui-Bin; Huang, Xiao-Chao; Zhang, Bin; Zhou, Mei-Mei; Liao, Zhi-Xin; Wang, Heng-Shan

    2017-09-29

    A series of dehydroabietic acid (DHAA) dipeptide derivatives containing the sulfonamide moiety were designed, synthesized and evaluated for inhibition of MMPs as well as the effects of in vitro cell migration. These compounds exhibited relatively good inhibition activity against MMPs with IC 50 values in low micromolar range. A docking study of the most active compound 8k revealed key interactions between 8k and MMP-3 in which the sulfonamide moiety and the dipeptide group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU. In particular, compound 8k appeared to be the most potent compound against the HepG2 cell line, at least partly, by inhibition of the activity of MMP-3 and apoptosis induction. The treatment of HepG2 cells with compound 8k resulted in inhibition of in vitro cell migration through wound healing assay and G1 phase of cell cycle arrested. In addition, 8k-induced apoptosis was significantly facilitated in HepG2 cells. Thus, we conclude that DHAA dipeptide derivatives containing the sulfonamide moiety may be the potential MMPs inhibitors with the ability to suppress cells migration. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. 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.

  19. 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.

  20. Berberine and a Berberis lycium extract inactivate Cdc25A and induce {alpha}-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Musa [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Giessrigl, Benedikt; Vonach, Caroline; Madlener, Sibylle [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Prinz, Sonja [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Herbaceck, Irene; Hoelzl, Christine [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Bauer, Sabine; Viola, Katharina [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Mikulits, Wolfgang [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Quereshi, Rizwana Aleem [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Knasmueller, Siegfried; Grusch, Michael [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Kopp, Brigitte [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Krupitza, Georg, E-mail: georg.krupitza@meduniwien.ac.at [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2010-01-05

    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{sub 2}O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 {mu}g BuOH extract (that was gained from 1 mg dried root) contained 2.0 {mu}g berberine and 0.3 {mu}g/ml palmatine. 1.2 {mu}g/ml berberine inhibited cell proliferation significantly, while 0.5 {mu}g/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 {alpha}-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.

  1. 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...

  2. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells.

    Science.gov (United States)

    Maillard, Pierre V; Van der Veen, Annemarthe G; Deddouche-Grass, Safia; Rogers, Neil C; Merits, Andres; Reis E Sousa, Caetano

    2016-12-01

    RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Data analysis of the inactivation of foodborne microorganisms under high hydrostatic pressure to establish global kinetic parameters and influencing factors

    NARCIS (Netherlands)

    Santillana Farakos, S.M.; Zwietering, M.H.

    2011-01-01

    The inactivation rate of foodborne microorganisms under high hydrostatic pressure (HHP) is influenced by factors such as substrate, species, strain, temperature, pH, and stage of growth of the cell. In this study, 445 DP-values from previously published data were analyzed, including those from

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

    International Nuclear Information System (INIS)

    Cho, O Yeon; Hwang, Hye Sook; Lee, Bok Soon; Oh, Young Taek; Kim, Chul Ho; Chun, Mi Son

    2015-01-01

    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

  9. 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.

  10. Triazole-dithiocarbamate based selective lysine specific demethylase 1 (LSD1) inactivators inhibit gastric cancer cell growth, invasion, and migration.

    Science.gov (United States)

    Zheng, Yi-Chao; Duan, Ying-Chao; Ma, Jin-Lian; Xu, Rui-Min; Zi, Xiaolin; Lv, Wen-Lei; Wang, Meng-Meng; Ye, Xian-Wei; Zhu, Shun; Mobley, David; Zhu, Yan-Yan; Wang, Jun-Wei; Li, Jin-Feng; Wang, Zhi-Ru; Zhao, Wen; Liu, Hong-Min

    2013-11-14

    Lysine specific demethylase 1 (LSD1), the first identified histone demethylase, plays an important role in epigenetic regulation of gene activation and repression. The up-regulated LSD1's expression has been reported in several malignant tumors. In the current study, we designed and synthesized five series of 1,2,3-triazole-dithiocarbamate hybrids and screened their inhibitory activity toward LSD1. We found that some of these compounds, especially compound 26, exhibited the most specific and robust inhibition of LSD1. Interestingly, compound 26 also showed potent and selective cytotoxicity against LSD1 overexpressing gastric cancer cell lines MGC-803 and HGC-27, as well as marked inhibition of cell migration and invasion, compared to 2-PCPA. Furthermore, compound 26 effectively reduced the tumor growth bared by human gastric cancer cells in vivo with no signs of adverse side effects. These findings suggested that compound 26 deserves further investigation as a lead compound in the treatment of LSD1 overexpressing gastric cancer.

  11. 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.

  12. 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).

  13. 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

  14. Polyphyllin G exhibits antimicrobial activity and exerts anticancer effects on human oral cancer OECM-1 cells by triggering G2/M cell cycle arrest by inactivating cdc25C-cdc2.

    Science.gov (United States)

    Cai, Xiaoqing; Guo, Lele; Pei, Fei; Chang, Xiaoyun; Zhang, Rui

    2018-04-15

    Plant natural products have long been considered to be important sources of bioactive molecules. A large number of antimicrobial and anticancer agents have been isolated form plants. In the present study we evaluated the antimicrobial and anticancer activity of a plant derived secondery metabolite, Polyphyllin G. The results of antibacterial assays showed that Polyphyllin G prevented the growth of both Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MICs) ranging from 13.1 to 78 μg/ml. Antifungal activity measured as inhibition of mycelium growth ranged between 38.32 and 56.50%. Further Polyphyllin G was also evaluated against a panel of cancer cell lines. The IC 50 of Polyphyllin G ranged from 10 to 65 μM. However the IC 50 of Polyphyllin G was found to be comparatively high (120 μM) against the normal FR2 cancer cell line. The lowest IC 50 of 10 μM was found against the oral cancer cell line OECM-1. Therefore further studies were carried out on this cell line only. Our results indicated that Polyphyllin G induced cell arrest in oral cancer OECM-1 cells by inactivation of cdc25C-cdc22 via ATM-Chk 1/2 stimulation. Therefore, we propose that Polyphyllin G might prove a lead molecule in the management of oral cancers and at the same time may prevent the growth of opportunistic microbes. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass.

    Science.gov (United States)

    Roccisana, Jennifer; Reddy, Vasumathi; Vasavada, Rupangi C; Gonzalez-Pertusa, Jose A; Magnuson, Mark A; Garcia-Ocaña, Adolfo

    2005-07-01

    Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.

  16. Evaluation of cell proliferation rate in non-dysplastic leukoplakias

    OpenAIRE

    Hildebrand, Laura de Campos; Carrard, Vinicius C.; Lauxen, Isabel da Silva; Quadros, Onofre Francisco de; Chaves, Anna Cecília Moraes; Sant'ana Filho, Manoel

    2010-01-01

    Objective: Analyze whether the most frequent cases of non-dysplastic leukoplakias, hyperkeratosis (H), acanthosis (A), and hyperkeratosis with acanthosis (HA) have similar cell proliferation rates and to compare them with epithelial dysplastic (ED) leukoplakias and normal oral epithelium (NOE).Study design: The sample comprised 10 cases of normal oral epithelium, 10 cases of hyperkeratosis, 10 cases of acanthosis, 10 cases of hyperkeratosis with acanthosis and 10 cases of epithelial dyspl...

  17. 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

  18. 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.

  19. Suicide inactivation of horseradish peroxidase by excess hydrogen ...

    African Journals Online (AJOL)

    In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher ...

  20. Effectiveness of ultrasound, UV-C, and photocatalysis on inactivation kinetics of Aeromonas hydrophila.

    Science.gov (United States)

    Kaur, Jasjeet; Karthikeyan, Raghupathy; Pillai, Suresh D

    2015-01-01

    In this study, bactericidal effects of 24 kHz ultrasound, ultraviolet (UV-C) irradiation, and titanium dioxide (TiO2) photocatalyst were studied on inactivation of Aeromonas hydrophila, an emerging pathogen listed on the US Environmental Protection Agency's (US EPA) candidate contaminant list. Metabolic activity (using the AlamarBlue dye) assays were performed to assess the residual activity of the microbial cells after the disinfection treatments along with culture-based methods. A faster inactivation rate of 1.52 log min(-1) and inactivation of 7.62 log10 was observed within 5 min of ultrasound exposure. Ultrasound treated cells repaired by 1.4 log10 in contrast to 5.3 log10 repair for UV-C treated cells. Ultrasound treatment significantly lowered the reactivation of Aeromonas hydrophila in comparison to UV-C- and UV-C-induced photocatalysis. Ultrasound appeared to be an effective means of inactivating Aeromonas hydrophila and could be used as a potential disinfection method for water and wastewater reuse.

  1. Safety and immunogenicity of an inactivated cell culture-derived H7N9 influenza vaccine in healthy adults: A phase I/II, prospective, randomized, open-label trial.

    Science.gov (United States)

    Wu, Un-In; Hsieh, Szu-Min; Lee, Wen-Sen; Wang, Ning-Chi; Kung, Hsiang-Chi; Ou, Tsong-Yih; Chen, Fu-Lun; Lin, Te-Yu; Chen, Yee-Chun; Chang, Shan-Chwen

    2017-07-24

    We conducted a phase I/II clinical trial to evaluate the safety and immunogenicity of a Madin-Darby canine kidney (MDCK) cell-grown inactivated H7N9 influenza vaccine for pandemic preparedness purposes. Between April 7, 2015 and May 27, 2016, healthy adults aged 20-60years were enrolled sequentially in phase I (n=40) and phase II (n=160) from three hospitals in Taiwan and randomized to receive 2 doses of whole-virus H7N9 vaccine (15 or 30μg hemagglutinin antigen (HA) with or without an aluminum hydroxide adjuvant) at 21-day intervals. Safety up to 180days and changes in hemagglutinin inhibition (HI) titers at 21days after each vaccination were determined. Of the 200 randomized subjects, 193 (96.5%) received 2 doses of the study vaccine and were included in the intention-to-treat analysis for safety, and 190 (95%) were included in the per-protocol analysis for immunogenicity. Most adverse events were mild and transient; no death or vaccine-related serious adverse events were reported. Overall, higher immune responses were observed in the groups administered with 30μgHA formulation than in the other two groups administered with 15μgHA formulation. The highest immune response was observed in subjects who received 2 doses of the adjuvanted vaccine containing 30μgHA with HI titer, seroprotection rate, seroconversion rate, and seroconversion factor of 36.2, 64.6%, 64.6% and 5.7, respectively. Our study demonstrated that the H7N9 influenza vaccine containing 30µgHA with aluminum hydroxide adjuvant was immunogenic and safe in adults aged 20-60years. CLINICALTRIALS.GOV identifier: NCT02436928. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Thermal Inactivation of Viruses

    Science.gov (United States)

    1977-10-01

    production. Proc. Soc. Exptl. Biol. Med. 116:174-177. Mayer, V. 1965. Study of the virulence of tick-borne encephalitis virus. IV. Thermosensitivity...inactivation of rabies and other rhabrtoviruses: stabilization of the chelating agent Ethylenediaminetetraacetic acid at physiological temperatures. Infec

  13. Evaluation of cell proliferation rate in non-dysplastic leukoplakias.

    Science.gov (United States)

    Hildebrand, Laura-de Campos; Carrard, Vinicius-Coelho; Lauxen, Isabel-da Silva; de Quadros, Onofre-Francisco; Chaves, Anna-Cecília-Moraes; Sant' Ana-Filho, Manoel

    2010-03-01

    Analyze whether the most frequent cases of non-dysplastic leukoplakias, hyperkeratosis (H), acanthosis (A), and hyperkeratosis with acanthosis (HA) have similar cell proliferation rates and to compare them with epithelial dysplastic (ED) leukoplakias and normal oral epithelium (NOE). The sample comprised 10 cases of normal oral epithelium, 10 cases of hyperkeratosis, 10 cases of acanthosis, 10 cases of hyperkeratosis with acanthosis and 10 cases of epithelial dysplasia. The mean number of AgNORs per nucleus (mAgNOR) and the mean percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were recorded. The results of mAgNOR showed differences between disorders in the evaluation of the basal layer, of the parabasal layer, and in the overall evaluation. mAgNOR and pAgNOR=2 increased progressively from normal oral epithelium to hyperkeratosis with acanthosis, hyperkeratosis, acanthosis and epithelial dysplasia (pdysplastic leukoplakias and this group presented a higher proliferative behavior when compared to normal oral epithelium. It may be suggested that non-dysplastic leukoplakias had different characteristics regarding cell proliferation rates and sometimes showed a proliferative behavior similar to that found in epithelial dysplasia. More studies should be conduced to increase knowledge about the biological profile of non-dysplastic leukoplakias, especially as it pertains to acanthosis.

  14. 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.

  15. Pneumococcal vaccination rates in children with sickle cell disease.

    Science.gov (United States)

    Nero, Alecia C; Akuete, Kwei; Leasure Reeves, Sarah; Dombkowski, Kevin J

    2014-01-01

    Sickle cell disease (SCD) confers an increased risk of invasive pneumococcal disease, especially among young children. Pneumococcal vaccination decreases this risk, but the completion rate of age-appropriate vaccinations is not well defined in SCD. The goal of this study was to assess whether pneumococcal vaccines are administered to high-risk children with SCD according to recommended vaccine schedules. A case-control design was used to conduct this study. Administrative data were obtained on Michigan Medicaid or Children's Special Health Care Services programs enrollees. In addition, Michigan Newborn Screening and Michigan Care Improvement Registry records were used to confirm diagnosis and vaccine administration. This study compared pneumococcal vaccination rates in a cohort of 179 children with SCD with 537 age-matched non-SCD controls (1:3) enrolled in the Michigan Medicaid Program between 2001 and 2008. Study subjects were born in the state of Michigan between 2001 and 2005. The main outcome measure was the proportion of children defined as up to date for pneumococcal vaccines at defined milestone ages. Children with SCD had significantly higher vaccination rates than controls, yet these values were much lower than state and national immunization survey rates. Barriers to completing age-appropriate recommended pneumococcal immunizations should be identified and addressed to further reduce invasive pneumococcal disease in this high-risk patient population.

  16. 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.

  17. 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.

  18. 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...

  19. 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 ...

  20. [Polyphenolic antioxidants efficiently protect urease from inactivation by ultrasonic cavitation].

    Science.gov (United States)

    Metelitsa, D I; Tarun, E I; Losev, Iu P

    2002-01-01

    Inactivation of urease (25 nM) in aqueous solutions (pH 5.0-6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 Wt/cm2, 36-56 degrees C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 Wt/cm2, 36 or 56 degrees C) has been characterized quantitatively, using first-order rate constants: kin, aggregate inactivation; kin*, thermal inactivation; and kin* (US), ultrasonic inactivation. Within the range from 1 nM to 10 microM, propyl gallate (PG) decreases approximately threefold the rate of LFUS-induced inactivation of urease (56 degrees C), whereas resorcinol poly-2-disulfide prevents this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36 degrees C) or 10 nM (56 degrees C). At 0.2-10 microM, human serum albumin (HSA) increases the resistance of urease (at 56 degrees C) treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS-HSA), formed by conjugation of 1.0 nM PGDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56 degrees C).

  1. 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...... electrolytic-Fenton cell) with the aim to broad the application of microbial electrochemistry. Results showed that a 4-log reduction of Escherichia coli (107 to hundreds CFU/mL) was achieved with an external applied voltage of 0.2 V, 0.3 mM Fe2+ and cathodic pH of 3.0. However, non-notable inactivation...

  2. 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.

  3. 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.

  4. 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

  5. Safety and immunogenicity of an inactivated whole cell tuberculosis vaccine booster in adults primed with BCG: A randomized, controlled trial of DAR-901.

    Directory of Open Access Journals (Sweden)

    C Fordham von Reyn

    Full Text Available Development of a tuberculosis vaccine to boost BCG is a major international health priority. SRL172, an inactivated whole cell booster derived from a non-tuberculous mycobacterium, is the only new vaccine against tuberculosis to have demonstrated efficacy in a Phase 3 trial. In the present study we sought to determine if a three-dose series of DAR-901 manufactured from the SRL172 master cell bank by a new, scalable method was safe and immunogenic.We performed a single site, randomized, double-blind, controlled, Phase 1 dose escalation trial of DAR-901 at Dartmouth-Hitchcock Medical Center in the United States. Healthy adult subjects age 18-65 with prior BCG immunization and a negative interferon-gamma release assay (IGRA were enrolled in cohorts of 16 subjects and randomized to three injections of DAR-901 (n = 10 per cohort, or saline placebo (n = 3 per cohort, or two injections of saline followed by an injection of BCG (n = 3 per cohort; 1-8 x 106 CFU. Three successive cohorts were enrolled representing DAR-901 at 0.1, 0.3, and 1 mg per dose. Randomization was performed centrally and treatments were masked from staff and volunteers. Subsequent open label cohorts of HIV-negative/IGRA-positive subjects (n = 5 and HIV-positive subjects (n = 6 received three doses of 1 mg DAR-901. All subjects received three immunizations at 0, 2 and 4 months administered as 0.1 mL injections over the deltoid muscle alternating between right and left arms. The primary outcomes were safety and immunogenicity. Subjects were followed for 6 months after dose 3 for safety and had phlebotomy performed for safety studies and immune assays before and after each injection. Immune assays using peripheral blood mononuclear cells included cell-mediated IFN-γ responses to DAR-901 lysate and to Mycobacterium tuberculosis (MTB lysate; serum antibody to M. tuberculosis lipoarabinomannan was assayed by ELISA.DAR-901 had an acceptable safety profile and was well-tolerated at all

  6. Low Survival Rates of Oral and Oropharyngeal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Anna Carolina Omena Vasconcellos Le Campion

    2017-01-01

    Full Text Available Aim. To assess the epidemiological and clinical factors that influence the prognosis of oral and oropharyngeal squamous cell carcinoma (SCC. Methods. One hundred and twenty-one cases of oral and oropharyngeal SCC were selected. The survival curves for each variable were estimated using the Kaplan-Meier method. The Cox regression model was applied to assess the effect of the variables on survival. Results. Cancers at an advanced stage were observed in 103 patients (85.1%. Cancers on the tongue were more frequent (23.1%. The survival analysis was 59.9% in one year, 40.7% in two years, and 27.8% in 5 years. There was a significant low survival rate linked to alcohol intake (p=0.038, advanced cancer staging (p=0.003, and procedures without surgery (p<0.001. When these variables were included in the Cox regression model only surgery procedures (p=0.005 demonstrated a significant effect on survival. Conclusion. The findings suggest that patients who underwent surgery had a greater survival rate compared with those that did not. The low survival rates and the high percentage of patients diagnosed at advanced stages demonstrate that oral and oropharyngeal cancer patients should receive more attention.

  7. 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.

  8. 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

  9. Molecular Viability Testing of UV-Inactivated Bacteria.

    Science.gov (United States)

    Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

    2017-05-15

    PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli , Aeromonas hydrophila , and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment. IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection. Copyright © 2017 American Society for Microbiology.

  10. 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.

  11. 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.

  12. 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.

  13. 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...

  14. Inactivation of Aujeszky's disease virus in slurry at various temperatures

    DEFF Research Database (Denmark)

    Bøtner, Anette

    1991-01-01

    Survival of Aujeszky's disease virus in pig slurry was investigated during anaerobic storage at 5, 20, 35, 40, 45, 50 and 55°C using 100-ml laboratory models simulating the conditions in slurry tanks during winter and summer seasons and during anaerobic digestion in batch reactors. The inactivation...... rate was found to increase with increasing temperature. Virus was inactivated at 5 and 20°C in 15 weeks and 2 weeks, respectively. At 35°C (mesophilic conditions) the virus was inactivated in 5 hours and at 55°C (thermophilic conditions) no virus could be detected after 10 minutes....

  15. Pulsed-dose-rate and low-dose-rate brachytherapy : Comparison of sparing effects in cells of a radiosensitive and a radioresistant cell line

    NARCIS (Netherlands)

    Pomp, J; Woudstra, EC; Kampinga, HH

    Pulsed-dose-rate regimens are an attractive alternative to continuous low-dose-rate brachytherapy. However, apart from data obtained from modeling, only a few irt vitro results are available for comparing the biological effectiveness of both modalities. Cells of two human cell lines with survival

  16. 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.

  17. The Effect of Shear Rate on Dissolution of Gas and Cell Density in Continuous Foaming Process

    Directory of Open Access Journals (Sweden)

    M.H.N. Famili

    2009-12-01

    Full Text Available The effect of shear rate on dissolution of carbon dioxide in viscoelastic wheat flour matrix and cell density in a glass barrel twin screw extruder is investigated. It is found that by increasing the shear rate there will be a decrease in the required thermodynamic conditions and hence, it improves blowing agent dissolution and increases the cell density. Shear rate breaks up big bubbles and helps to better distribute the blowing agent in the matrix and hence it increases the cell density. Cell density decreases by dropping foam cooling rate. For a given cell density, a higher screw speed is needed, ifthe cooling rate is decreased.

  18. Significance of Inactivated Genes in Leukemia: Pathogenesis and Prognosis

    Science.gov (United States)

    Heidari, Nazanin; Abroun, Saeid; Bertacchini, Jessika; Vosoughi, Tina; Rahim, Fakher; Saki, Najmaldin

    2017-01-01

    Epigenetic and genetic alterations are two mechanisms participating in leukemia, which can inactivate genes involved in leukemia pathogenesis or progression. The purpose of this review was to introduce various inactivated genes and evaluate their possible role in leukemia pathogenesis and prognosis. By searching the mesh words “Gene, Silencing AND Leukemia” in PubMed website, relevant English articles dealt with human subjects as of 2000 were included in this study. Gene inactivation in leukemia is largely mediated by promoter’s hypermethylation of gene involving in cellular functions such as cell cycle, apoptosis, and gene transcription. Inactivated genes, such as ASPP1, TP53, IKZF1 and P15, may correlate with poor prognosis in acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), respectively. Gene inactivation may play a considerable role in leukemia pathogenesis and prognosis, which can be considered as complementary diagnostic tests to differentiate different leukemia types, determine leukemia prognosis, and also detect response to therapy. In general, this review showed some genes inactivated only in leukemia (with differences between B-ALL, T-ALL, CLL, AML and CML). These differences could be of interest as an additional tool to better categorize leukemia types. Furthermore; based on inactivated genes, a diverse classification of Leukemias could represent a powerful method to address a targeted therapy of the patients, in order to minimize side effects of conventional therapies and to enhance new drug strategies. PMID:28580304

  19. 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

  20. Inactivation of orange pectinesterase by combined high-pressure and -temperature treatments: a kinetic study.

    Science.gov (United States)

    Van den Broeck, I; Ludikhuyze, L R; Van Loey, A M; Hendrickx, M E

    2000-05-01

    Pressure and/or temperature inactivation of orange pectinesterase (PE) was investigated. Thermal inactivation showed a biphasic behavior, indicating the presence of labile and stable fractions of the enzyme. In a first part, the inactivation of the labile fraction was studied in detail. The combined pressure-temperature inactivation of the labile fraction was studied in the pressure range 0.1-900 MPa combined with temperatures from 15 to 65 degrees C. Inactivation in the pressure-temperature domain specified could be accurately described by a first-order fractional conversion model, estimating the inactivation rate constant of the labile fraction and the remaining activity of the stable fraction. Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the Eyring and Arrhenius relations, respectively. By replacing in the latter equation the pressure-dependent parameters (E(a), k(ref)(T)()) by mathematical expressions, a global model was formulated. This mathematical model could accurately predict the inactivation rate constant of the labile fraction of orange PE as a function of pressure and temperature. In a second part, the stable fraction was studied in more detail. The stable fraction inactivated at temperatures exceeding 75 degrees C. Acidification (pH 3.7) enhanced thermal inactivation of the stable fraction, whereas addition of Ca(2+) ions (1 M) suppressed inactivation. At elevated pressure (up to 900 MPa), an antagonistic effect of pressure and temperature on the inactivation of the stable fraction was observed. The antagonistic effect was more pronounced in the presence of a 1 M CaCl(2) solution as compared to the inactivation in water, whereas it was less pronounced for the inactivation in acid medium.

  1. 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

  2. [Ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions].

    Science.gov (United States)

    Karaseva, E I; Tarun, E I; Metelitsa, D I

    2009-01-01

    The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37-59 degrees C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constants k(in), k(in)*, and k(in)(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO* -DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.

  3. 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

    1996-02-01

    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.

  4. Changes of respiration and of specific growth rate during cell cycle of yeast cells of different genealogical age.

    Science.gov (United States)

    Vraná, D

    1988-01-01

    When investigating changes of respiratory activity during the cell cycle of mother and daughter Candida cells significant oscillations of specific rate of oxygen consumption were detected; specific growth rate also varied. The oscillations were less pronounced when the inoculum was obtained from the chemostat at the high dilution rates of 0.25 and 0.35/h.

  5. Rating

    OpenAIRE

    Karas, Vladimír

    2006-01-01

    Charakteristika ratingu. Dělení a druhy ratingu (rating emise × rating emitenta; dlouhodobý rating × krátkodobý rating; mezinárodní rating × lokální rating). Obecné požadavky kladené na rating. Proces tvorby ratingu. Vyžádaný rating. Nevyžádaný rating. Ratingový proces na bázi volně přístupných informací. Uplatňované ratingové systémy. Ratingová kriteria. Využití a interpretace ratingové známky. Funkce ratingu. Rating v souvislosti s BASEL II. Rating v souvislosti s hospodářskými krizemi....

  6. In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process

    NARCIS (Netherlands)

    Klei, Ida J. van der; Veenhuis, Marten; Nicolay, Klaas; Harder, Willem

    1988-01-01

    The fate of alcohol oxidase (AO) in chemostat-grown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors.

    Science.gov (United States)

    Carmo, M; Dias, J D; Panet, A; Coroadinha, A S; Carrondo, M J T; Alves, P M; Cruz, P E

    2009-10-01

    Lentiviral vectors are an important tool for gene transfer research and gene therapy purposes. However, the low stability of these vectors affects their production, storage, and efficacy in preclinical and clinical settings. In the present work the mechanism underlying the thermosensitivity of lentiviral vectors was evaluated. For lentiviral vectors pseudotyped with amphotropic and RDpro envelopes, the capacity to perform reverse transcription was lost rapidly at 37 degrees C, in high correlation with the loss of infectivity. The vector with RDpro envelope presented a higher level of stability than that with amphotropic envelope for both the reverse transcription process and viral infectivity. Reverse transcriptase enzyme inactivation and viral template RNA degradation were not implicated in the loss of the viral capacity to perform reverse transcription. Furthermore, early entry steps in the infection process do not determine the rate of viral inactivation, as the amount of viral RNA and p24 protein entering the cells decreased slowly for both vectors. Taken together, it can be concluded that the reverse transcription process is thermolabile and thus determines the rate of lentiviral inactivation. Strategies to stabilize the reverse transcription process should be pursued to improve the applicability of lentiviral vectors in gene therapy.

  12. 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.

  13. Inactivation of pectin methylesterase by immobilized trypsins from cunner fish and bovine pancreas.

    Science.gov (United States)

    Li, Dan; Matos, Madyu; Simpson, Benjamin K

    2013-01-01

    Immobilized cunner fish trypsin was used to inactivate pectin methylesterase (PME). The effects of different reaction conditions (e.g., incubation time, PME concentration, and temperature) on PME inactivation and kinetics of inactivation were investigated. Temperature, incubation time, and PME concentration significantly affected the extent of PME inactivation. Generally, higher temperature, longer incubation time, and low PME concentration caused more PME inactivation. The immobilized fish trypsin had higher capacity to inactivate PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was about 20% higher than that of its bovine counterpart. However, PME inactivated by both trypsins regained partial activity during storage at 4°C, with immobilized fish trypsin-treated PME regaining more of its original activity than the immobilized bovine trypsin-treated PME. Heat-denatured PME was hydrolyzed more extensively by immobilized fish trypsin than by its bovine counterpart. The rate constants increased, whereas the D-values decreased with temperature for both immobilized fish and bovine trypsins. The inactivation rate constants of immobilized fish trypsin at all the temperatures investigated (i.e., 15-35°C) were higher than those of immobilized bovine trypsin. Furthermore, the activation energy (Ea ) of PME inactivation by immobilized fish trypsin was lower than that of immobilized bovine trypsin. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  14. The Effect of Shear Rate on Dissolution of Gas and Cell Density in Continuous Foaming Process

    OpenAIRE

    M.H.N. Famili; M. Ako

    2009-01-01

    The effect of shear rate on dissolution of carbon dioxide in viscoelastic wheat flour matrix and cell density in a glass barrel twin screw extruder is investigated. It is found that by increasing the shear rate there will be a decrease in the required thermodynamic conditions and hence, it improves blowing agent dissolution and increases the cell density. Shear rate breaks up big bubbles and helps to better distribute the blowing agent in the matrix and hence it increases the cell density. Ce...

  15. Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

    Science.gov (United States)

    Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

    2015-01-01

    Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

  16. Responses of cultured mammalian cells to prolonged low dose rate gamma irradiation

    International Nuclear Information System (INIS)

    Kal, H.B.; Rongen, E. van; Welleweerd, J.

    1992-01-01

    Experiments were performed to verify the finding of others that prolonged irradiation caused sudden death of cells. Continuous irradiation (dose rate 0.09 Gy/h) was applied to the R-1, RUC-2, V79 and T-1g cells for up to 140 days. End points were: Population growth rate, plating efficiency and radiosensitivity. The population doubling times were increased and plating efficiencies decreased; cell survival after single doses at acute dose rate were lower than that of control cells. Part of the cell parameters returned to control values within three weeks after prolonged irradiation had been terminated. Sudden death after prolonged exposure was not observed. (orig.) [de

  17. 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.

  18. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    Science.gov (United States)

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  19. 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

  20. Formulation and Cytotoxicity of Ribosome-Inactivating Protein Mirabilis Jalapa L. Nanoparticles Using Alginate-Low Viscosity Chitosan Conjugated with Anti-Epcam Antibodies in the T47D Breast Cancer Cell Line.

    Science.gov (United States)

    Wicaksono, Psycha Anindya; Name, Sismindari; Martien, Ronny; Ismail, Hilda

    2016-01-01

    Ribosome-inactivating protein (RIP) from Mirabilis jalapa L. leaves has cytotoxic effects on breast cancer cell lines but is less toxic towards normal cells. However, it can easily be degraded after administration so it needs to be formulated into nanoparticles to increase its resistance to enzymatic degradation. The objectives of this study were to develop a protein extract of M. jalapa L. leaves (RIP-MJ) incorporated into nanoparticles conjugated with Anti-EpCAM antibodies, and to determine its cytotoxicity and selectivity in the T47D breast cancer cell line. RIP-MJ was extracted from red-flowered M. jalapa L. leaves. Nanoparticles were formulated based on polyelectrolyte complexation using low viscosity chitosan and alginate, then chemically conjugated with anti-EpCAM antibody using EDAC based on carbodiimide reaction. RIP-MJ nanoparticles were characterised for the particle size, polydispersity index, zeta potential, particle morphology, and entrapment efficiency. The cytotoxicity of RIP-MJ nanoparticles against T47D and Vero cells was then determined with MTT assay. The optimal formula of RIP-MJ nanoparticles was obtained at the concentration of RIP-MJ, low viscosity chitosan and alginate respectively 0.05%, 1%, and 0.4% (m/v). RIP-MJ nanoparticles are hexagonal with high entrapment efficiency of 98.6%, average size of 130.7 nm, polydispersity index of 0.380 and zeta potential +26.33 mV. The IC50 values of both anti-EpCAM-conjugated and non-conjugated RIP-MJ nanoparticles for T47D cells (13.3 and 14.9 μg/mL) were lower than for Vero cells (27.8 and 33.6 μg/mL). The IC50 values of conjugated and non- conjugated RIP-MJ for both cells were much lower than IC50 values of non-formulated RIP-MJ (>500 μg/mL).

  1. 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.

  2. 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.

  3. Gamma ray inactivation of some animal viruses.

    Science.gov (United States)

    Thomas, F C; Davies, A G; Dulac, G C; Willis, N G; Papp-Vid, G; Girard, A

    1981-10-01

    Twenty samples of animal viruses comprising 14 different viruses in 12 families were subjected to varying doses of gamma irradiation from a 60Co source in a Gamma Cell 220 (Atomic Energy of Canada Limited) to determine lethal dose levels. The dose responses appeared linear throughout inactivation. The D10 values, that is the dose necessary to reduce infectivity by one log10, ranged from less than 0.20 Megarads to approximately 0.55 Megarads. There was not a complete inverse correlation between the target size (virion core) and the D10 value.

  4. Gamma ray inactivation of some animal viruses.

    OpenAIRE

    Thomas, F C; Davies, A G; Dulac, G C; Willis, N G; Papp-Vid, G; Girard, A

    1981-01-01

    Twenty samples of animal viruses comprising 14 different viruses in 12 families were subjected to varying doses of gamma irradiation from a 60Co source in a Gamma Cell 220 (Atomic Energy of Canada Limited) to determine lethal dose levels. The dose responses appeared linear throughout inactivation. The D10 values, that is the dose necessary to reduce infectivity by one log10, ranged from less than 0.20 Megarads to approximately 0.55 Megarads. There was not a complete inverse correlation betwee...

  5. Sunlight inactivation of somatic coliphage in the presence of natural organic matter.

    Science.gov (United States)

    Sun, Chen-Xi; Kitajima, Masaaki; Gin, Karina Yew-Hoong

    2016-01-15

    Long wavelengths of sunlight spectrum (UVA and visible light), as well as natural organic matter (NOM) are important environmental factors affecting survival of viruses in aquatic environment through direct and indirect inactivation. In order to understand the virus inactivation kinetics under such conditions, this study investigated the effects of Suwannee River natural organic matter (NOM) on the inactivation of a somatic coliphage, phiX174, by UVA and visible light. Experiments were carried out to examine the virucidal effects of UVA/visible light, assess the influence of SRNOM at different concentrations, and identify the effective ROS in virus inactivation. The results from this study showed that the presence of NOM could either enhance virus inactivation or reduce virus inactivation depending on the concentration, where the inactivation rate followed a parabolic relationship against NOM concentration. The results indicated that moderate levels of NOM (11 ppm) had the strongest antiviral activity, while very low or very high NOM concentrations prolonged virus survival. The results also showed that OH▪ was the primary ROS in causing phiX174 (ssDNA virus) inactivation, unlike previous findings where (1)O2 was the primary ROS causing MS2 (ssRNA virus) inactivation. The phiX174 inactivation by OH∙ could be described as k=3.7 ✕ 10(13)[OH∙]+1.404 (R(2)=0.8527). Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. One-year immunogenicity kinetics and safety of a purified chick embryo cell rabies vaccine and an inactivated Vero cell-derived Japanese encephalitis vaccine administered concomitantly according to a new, 1-week, accelerated primary series.

    Science.gov (United States)

    Cramer, Jakob P; Jelinek, Tomas; Paulke-Korinek, Maria; Reisinger, Emil C; Dieckmann, Sebastian; Alberer, Martin; Bühler, Silja; Bosse, Dietrich; Meyer, Seetha; Fragapane, Elena; Costantini, Marco; Pellegrini, Michele; Lattanzi, Maria; Dovali, Claudia

    2016-03-01

    Conventional rabies pre-exposure prophylaxis (PrEP) and Japanese encephalitis (JE) primary series vaccination regimens each require up to 4 weeks to complete and, thus, may not be feasible for individuals who need these immunizations on short notice. This Phase 3b, randomized, controlled, observer-blind study evaluated the immunogenicity and safety of concomitant administration of a purified chick embryo cell culture rabies vaccine and an inactivated, adsorbed JE vaccine according to an accelerated (1 week) regimen when compared with the conventional regimens (4 weeks). This report describes the kinetics of immune responses up to 1 year after vaccination. A total of 661 healthy adults (18 to ≤65 years) were randomized into the following accelerated or conventional vaccine regimens: Rabies + JE-Conventional, Rabies + JE-Accelerated, Rabies-Conventional and JE-Conventional. Immunogenicity was assessed by virus neutralization tests. Safety and tolerability were also evaluated. Irrespective of rabies vaccination regimen, ≥97% of subjects had adequate levels of rabies virus neutralizing antibody (RVNA) concentrations (≥0.5 IU/ml) up to Day 57, with percentages of subjects with RVNA concentrations ≥0.5 IU/ml at Day 366 ranging between 68% in the Rabies + JE-Accelerated group and 80% of subjects in the Rabies-Conventional group. The Rabies + JE-Accelerated group revealed high JE neutralizing antibody titers at all-time points. At Day 366, the percentage of subjects with antibody titers indicative of seroprotection (PRNT50 titers ≥1:10) remained high across JE vaccine groups (86-94%). The accelerated PrEP rabies and JE vaccination regimens, once licensed, could represent a valid alternative in the short-term to currently recommended conventional regimens. The concomitant administration of these two vaccines does not compromise immune responses to any of the vaccine antigens particularly when aiming for short-term protection. Further evidence

  9. In vivo measurement of DNA synthesis rates of colon epithelial cells in carcinogenesis

    International Nuclear Information System (INIS)

    Kim, Sylvia Jeewon; Turner, Scott; Killion, Salena; Hellerstein, Marc K.

    2005-01-01

    We describe here a highly sensitive technique for measuring DNA synthesis rates of colon epithelial cells in vivo. Male SD rats were given 2 H 2 O (heavy water). Colon epithelial cells were isolated, DNA was extracted, hydrolyzed to deoxyribonucleosides, and the deuterium enrichment of the deoxyribose moiety was determined by gas chromatographic/mass spectrometry. Turnover time of colon crypts and the time for migration of cells from basal to top fraction of the crypts were measured. These data were consistent with cell cycle analysis and bromodeoxyuridine labeling. By giving different concentrations of a promoter, dose-dependent increases in DNA synthesis rates were detected, demonstrating the sensitivity of the method. Administration of a carcinogen increased DNA synthesis rates cell proliferation in all fractions of the crypt. In conclusion, DNA synthesis rates of colon epithelial cells can be measured directly in vivo using stable-isotope labeling. Potential applications in humans include use as a biomarker for cancer chemoprevention studies

  10. Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries.

    Science.gov (United States)

    Schultze, K K; Linton, R H; Cousin, M A; Luchansky, J B; Tamplin, M L

    2007-06-01

    Preinoculation growth conditions and fat levels were evaluated for effects on the heat resistance of Listeria monocytogenes strain MFS 102 in formulated frankfurter slurries and on frankfurter surfaces. Comparison of linear inactivation rates (D-values) for cells heated in frankfurter slurry showed that growth conditions were significant (Pdeath rate of L. monocytogenes. Cells heated in 8.5% fat slurry had a significantly higher (P0.05) affecting thermal inactivation rates on frankfurter surfaces. Heat inactivation rates were consistently higher on frankfurter surfaces compared to similar treatments done in frankfurter slurry. On frankfurter surfaces, a 2.3- to 5.1-log(10) reduction was achieved after 15 min depending on frankfurter surface type. The time necessary to achieve a 3-log(10) reduction using post-processing pasteurization of frankfurters in a hot water-bath at 60 degrees C almost doubled for cells grown in TSBYE and heated in 23% fat frankfurter slurry (19.6 min) versus cells grown and heated in 8.5% fat frankfurter slurry (10.8 min).

  11. 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.

  12. 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

  13. How variable is a spontaneous mutation rate in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Boesen, Jan J.B.; Niericker, Matthieu J.; Dieteren, Nicole; Simons, Jo W.I.M. (MGC-Dept. of Radiation Genetics and Chemical Mutagenesis, State Univ. of Leiden (Netherlands))

    1994-05-01

    The Luria-Delbrueck fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrueck fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2x10[sup -6] to about 2x10[sup -7]/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7[+-]4.0x10[sup -7] compared to 4.0[+-]3.1x10[sup -7] for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7[+-]1.3x10[sup -7]. Thus altogether the mutation rate can vary from 2x10[sup -6] to 0.7x10[sup -7] (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

  14. A synthetic lymph node containing inactivated Treponema pallidum cells elicits strong, antigen-specific humoral and cellular immune responses in mice.

    Science.gov (United States)

    Stamm, Lola V; Drapp, Rebecca L

    2014-02-01

    The goal of this study was to investigate the use of a synthetic lymph node (SLN) for delivery of Treponema pallidum (Tp) antigens. Immune responses of C57BL/6 mice were analyzed at 4, 8, and 12 weeks after SLN implantation. Group 1 mice received SLN with no antigen; Group 2, SLN with formalin-inactivated Tp (f-Tp); and Group 3, SLN with f-Tp plus a CpG oligodeoxynucleotide. When tested by ELISA, sera from Group 2 and Group 3 mice showed stronger IgG antibody reactivity than sera from Group 1 mice to sonicates of f-Tp or untreated Tp, but not to sonicate of normal rabbit testicular extract at all times. The IgG1 level was higher than IgG2c level for Group 2 mice at all times and for Group 3 mice at 4 and 8 weeks. IgG1 and IgG2c levels were nearly equivalent for Group 3 mice at 12 weeks. Immunoblotting showed that IgG from Group 2 and Group 3 mice recognized several Tp proteins at all times. Supernatants of splenocytes from Group 2 and Group 3 mice contained significantly more IFNγ than those from Group 1 mice after stimulation with f-Tp at all times. A significant level of IL-4 was not detected in any supernatants. These data show that strong humoral and cellular immune responses to Tp can be elicited via a SLN. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  15. 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.

  16. Aberrant DNA methylation-induced gene inactivation is associated with the diagnosis and/or therapy of T-cell leukemias.

    Science.gov (United States)

    Kim, Sun Young; Shin, Dong-Yeop; Kim, Sang-Man; Lee, Minyoung; Kim, Eun Ju

    2016-08-01

    Aberrant hypermethylation of tumor suppressor genes is known to play an important role in the development of many tumors, and aberrant DNA hypermethylation was recently identified in hematologic malignancies, where it is thought to hold relevance in leukemogenesis. Here, we report that there are differences in the DNA methylation patterns seen in normal peripheral blood and two T-cell leukemia cell lines. We identify nine genes (CLEC4E, CR1, DBC1, EPO, HAL-DOA, IGF2, IL12B, ITGA1, and LMX1B) that are significantly hypermethylated in T-cell leukemias cell lines, and suggest that aberrant hypermethylation of these normally unmethylated genes may induce their transcriptional and expressional silencing. Furthermore, we observed that the expression levels of DNMT1 and DNMT3a were significantly decreased by 5-aza-2'-deoxycytidine (5-Aza-dC), which is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells and restore the expression levels of their target genes in Jurkat cells. This result suggests that the overexpression of DNMTs could contribute to the development of T-cell leukemias by inducing hypermethylation of the target genes. Together, our results show that aberrant hypermethylation is an important molecular mechanism in the progression of T-cell leukemias, and thus could prove useful as a prognostic and/or diagnostic marker. Moreover, 5-Aza-dC might be a promising candidate for the treatment of T-cell leukemia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. 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.

  18. 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

  19. Dengue virus photo-inactivated in presence of 1,5-iodonaphthylazide (INA) or AMT, a psoralen compound (4′-aminomethyl-trioxsalen) is highly immunogenic in mice

    Science.gov (United States)

    Raviprakash, Kanakatte; Sun, Peifang; Raviv, Yossef; Luke, Thomas; Martin, Nicholas; Kochel, Tadeusz

    2013-01-01

    Two novel methods of dengue virus inactivation using iodonaphthyl azide (INA) and aminomethyl trioxsalen (AMT) were compared with traditional virus inactivation by formaldehyde. The AMT inactivated dengue-2 virus retained its binding to a panel of 5 monoclonal antibodies specific for dengue-2 envelope protein, whereas inactivation by formaldehyde and INA led to 30–50% decrease in binding. All three inactivated viruses elicited high level virus neutralizing antibodies in vaccinated mice. However, only mice vaccinated with AMT inactivated virus mounted T cell responses similar to live, uninactivated virus. PMID:23835446

  20. 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.

  1. Cell tropism predicts long-term nucleotide substitution rates of mammalian RNA viruses.

    Directory of Open Access Journals (Sweden)

    Allison L Hicks

    2014-01-01

    Full Text Available The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001 as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively. These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.

  2. 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.

  3. Seasonal Inactivated Influenza Virus Vaccines

    OpenAIRE

    Couch, Robert B.

    2008-01-01

    Inactivated influenza virus vaccines are the primary modality used for prevention of influenza. A system of annual identification of new strains causing illnesses, selections for vaccines, chick embryo growth, inactivation, processing, packaging, distribution and usage has been in place for decades. Current vaccines contain 15 µg of the HA of an A/H1N1, A/H3N2 and B strain and are given parenterally to induce serum anti-HA antibody for prevention of subsequent infection and illness from natur...

  4. 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...

  5. From retroviral vector production to gene transfer: spontaneous inactivation is caused by loss of reverse transcription capacity.

    Science.gov (United States)

    Carmo, M; Panet, A; Carrondo, M J T; Alves, P M; Cruz, P E

    2008-04-01

    The loss of gene transfer capacity in retroviral vectors constitutes a major disadvantage in the development of retroviral vectors for gene therapy applications. In the present work the loss of a vector's capacity to perform reverse transcription was studied as a possible explanation for the low stability of retroviral vectors from the production stage to the target cell gene transfer event. Inactivation studies were performed with murine leukemia virus vectors at 37 degrees C and several residual activities were tested, including viral infectivity, reverse transcription capacity, reverse transcriptase (RT) activities and viral RNA stability. The results indicate a high correlation between loss of infectivity and the capacity of the virus to perform the initial steps of reverse transcription. To further understand the thermosensitivity of the reverse transcription process, the two enzyme activities of RT were investigated. The results indicate that, although the inactivation rate of the DNA polymerase is faster than that of RNase H, the decline of these two enzyme activities is significantly slower than that of reverse transcription. Also, viral RNA stability is not implicated in the loss of the virus capacity to perform reverse transcription as the rate of viral RNA degradation was very slow. Furthermore, it was observed that the amount of viral RNA that entered the cells decreased slowly due to viral inactivation at 37 degrees C. The reverse transcription process is thermolabile and this sensitivity determines the rate of retroviral inactivation. Strategies targeting stabilization of the reverse transcription complex should be pursued to improve the applicability of retroviral vectors in gene therapy studies. (c) 2008 John Wiley & Sons, Ltd.

  6. Selective inactivation of human immunodeficiency virus with subpicosecond near-infrared laser pulses

    International Nuclear Information System (INIS)

    Tsen, K T; Tsen, S-W D; Hung, C-F; Wu, T-C; Kiang, Juliann G

    2008-01-01

    We demonstrate for the first time that human immunodeficiency virus (HIV) can be inactivated by irradiation with subpicosecond near-infrared laser pulses at a moderate laser power density. By comparing the threshold laser power density for the inactivation of HIV with those of human red blood cells and mouse dendritic cells, we conclude that it is plausible to use the ultrashort pulsed laser to selectively inactivate blood-borne pathogens such as HIV while leaving sensitive materials like human red blood cells unharmed. This finding has important implications in the development of a new laser technology for disinfection of viral pathogens in blood products and in the clinic. (fast track communication)

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Inactivation of Herpes Simplex Viruses by Nonionic Surfactants

    Science.gov (United States)

    Asculai, Samuel S.; Weis, Margaret T.; Rancourt, Martha W.; Kupferberg, A. B.

    1978-01-01

    Nonionic surface-active agents possessing ether or amide linkages between the hydrophillic and hydrophobic portions of the molecule rapidly inactivated the infectivity of herpes simplex viruses. The activity stemmed from the ability of nonionic surfactants to dissolve lipid-containing membranes. This was confirmed by observing surfactant destruction of mammalian cell plasma membranes and herpes simplex virus envelopes. Proprietary vaginal contraceptive formulations containing nonionic surfactants also inactivated herpes simplex virus infectivity. This observation suggests that nonionic surfactants in appropriate formulation could effectively prevent herpes simplex virus transmission. Images PMID:208460

  12. Kinetic parameters for thermal inactivation of soluble peroxidase from needles of Serbian spruce Picea omorika (Pancić) Purkyne.

    Science.gov (United States)

    Laketa, Danijela; Bogdanović, Jelena; Kalauzi, Aleksandar; Radotić, Ksenija

    2009-03-01

    Thermal inactivation of peroxidase (POD) in an extract of Picea omorika (Pancić) Purkyne needles initiated by heat treatment was studied. This is the first study of this kind on a conifer species. Non-linear regression analysis was applied on the inactivation rate data, combining Mitscherlich and Arrhenius equations, treating time and temperature simultaneously as explaining variables. We determined the inactivation rate constant k, the Arrhenius energy of inactivation E and the remaining activity C(min) for the crude extract and for separated acidic and basic enzyme fractions, as well as for individual isoenzymes separated electrophoretically. A comparison of inactivation parameters for acidic and basic fractions shows that the thermal inactivation rate of the basic fraction is higher. The obtained value of inactivation energy for crude extract was between the values for acidic and basic isoenzyme fractions. One of the three analysed individual isoenzymes was characterised by a lower inactivation rate constant and higher inactivation energy. Another isoenzyme showed considerably higher level of remaining activity compared to the others, which identified it as the most resistant to high temperatures. The acquired values of Arrhenius energy of inactivation for POD in crude extract were intermediate, considering a range of POD values for various other plant species.

  13. Differences in cell division rates drive the evolution of terminal differentiation in microbes.

    Directory of Open Access Journals (Sweden)

    João F Matias Rodrigues

    Full Text Available Multicellular differentiated organisms are composed of cells that begin by developing from a single pluripotent germ cell. In many organisms, a proportion of cells differentiate into specialized somatic cells. Whether these cells lose their pluripotency or are able to reverse their differentiated state has important consequences. Reversibly differentiated cells can potentially regenerate parts of an organism and allow reproduction through fragmentation. In many organisms, however, somatic differentiation is terminal, thereby restricting the developmental paths to reproduction. The reason why terminal differentiation is a common developmental strategy remains unexplored. To understand the conditions that affect the evolution of terminal versus reversible differentiation, we developed a computational model inspired by differentiating cyanobacteria. We simulated the evolution of a population of two cell types -nitrogen fixing or photosynthetic- that exchange resources. The traits that control differentiation rates between cell types are allowed to evolve in the model. Although the topology of cell interactions and differentiation costs play a role in the evolution of terminal and reversible differentiation, the most important factor is the difference in division rates between cell types. Faster dividing cells always evolve to become the germ line. Our results explain why most multicellular differentiated cyanobacteria have terminally differentiated cells, while some have reversibly differentiated cells. We further observed that symbioses involving two cooperating lineages can evolve under conditions where aggregate size, connectivity, and differentiation costs are high. This may explain why plants engage in symbiotic interactions with diazotrophic bacteria.

  14. 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...

  15. 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

  16. 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...

  17. Mitotically inactivated embryonic stem cells can be used as an in vivo feeder layer to nurse damaged myocardium after acute myocardial infarction: a preclinical study.

    Science.gov (United States)

    Burt, Richard K; Chen, You-hong; Verda, Larissa; Lucena, Carolina; Navale, Shankararao; Johnson, Jesse; Han, Xiaoqiang; Lomasney, Jon; Baker, Jessa M; Ngai, Ka-Leung; Kino, Aya; Carr, James; Kajstura, Jan; Anversa, Piero

    2012-10-26

    Various types of viable stem cells have been reported to result in modest improvement in cardiac function after acute myocardial infarction. The mechanisms for improvement from different stem cell populations remain unknown. To determine whether irradiated (nonviable) embryonic stem cells (iESCs) improve postischemic cardiac function without adverse consequences. After coronary artery ligation-induced cardiac infarction, either conditioned media or male murine or male human iESCs were injected into the penumbra of ischemic myocardial tissue of female mice or female rhesus macaque monkeys, respectively. Murine and human iESCs, despite irradiation doses that prevented proliferation and induced cell death, significantly improved cardiac function and decreased infarct size compared with untreated or media-treated controls. Fluorescent in situ hybridization of the Y chromosome revealed disappearance of iESCs within the myocardium, whereas 5-bromo-2'-deoxyuridine assays revealed de novo in vivo cardiomyocyte DNA synthesis. Microarray gene expression profiling demonstrated an early increase in metabolism, DNA proliferation, and chromatin remodeling pathways, and a decrease in fibrosis and inflammatory gene expression compared with media-treated controls. As a result of irradiation before injection, ex vivo and in vivo iESC existence is transient, yet iESCs provide a significant improvement in cardiac function after acute myocardial infarction. The mechanism(s) of action of iESCs seems to be related to cell-cell exchange, paracrine factors, and a scaffolding effect between iESCs and neighboring host cardiomyocytes.

  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. 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.

  20. 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.

  1. 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.

  2. Catalysis and inactivation of tyrosinase in its action on hydroxyhydroquinone.

    Science.gov (United States)

    del Mar Garcia-Molina, Maria; Muñoz-Muñoz, Jose Luis; Berna, Jose; García-Ruiz, Pedro Antonio; Rodriguez-Lopez, Jose Neptuno; Garcia-Canovas, Francisco

    2014-02-01

    Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mechanism is proposed, in which HHQ is considered as a monophenol or as an o-diphenol, depending on the part of the molecule that interacts with the enzyme. The kinetic parameters obtained from an analysis of the measurements of the initial steady state rate of 2-hydroxy p-benzoquinone formation were kcatapp=229.0±7.7 s(-1) and KMapp,HHQ=0.40±0.05 mM. Furthermore, the action of tyrosinase on HHQ led to the enzyme's inactivation through a suicide inactivation mechanism. This suicide inactivation process was characterized kinetically by λmaxapp (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of λmaxapp and r were (8.2±0.1)×10(-3) s(-1) and 35,740±2,548, respectively. © 2014 International Union of Biochemistry and Molecular Biology.

  3. 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).

  4. 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.

  5. 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

  6. 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.

  7. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......R. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  8. Inactivation of PI3K/Akt signaling mediates proliferation inhibition and G2/M phase arrest induced by andrographolide in human glioblastoma cells.

    Science.gov (United States)

    Li, Yanchun; Zhang, Pengfei; Qiu, Feng; Chen, Lixia; Miao, Caixia; Li, Jianchun; Xiao, Wei; Ma, Enlong

    2012-06-27

    Andrographolide, a principal diterpenoid lactone isolated from the traditional herbal medicine Andrographis paniculata, has been reported to show anti-tumor activity. Since the high lipid solubility of andrographolide permits it to penetrate the blood-brain barrier and concentrate in the brain, we hypothesized that andrographolide may be a potential chemotherapeutic agent for the treatment of glioblastomas. To clarify this point, we investigated the growth inhibitory effect and mechanisms of actions of andrographolide on human glioblastoma U251 and U87 cells. MTT assay and trypan blue exclusion assay were used to investigate the proliferation inhibitory and cytotoxic effects of andrographolide, respectively. Cell cycle distribution was analyzed using flow cytometry. Apoptosis analysis proceeded by detecting the cleavage of caspase-3. The levels of proteins were probed by Western blotting. The results showed that non-toxic concentrations of andrographolide inhibited the proliferation of human glioblastoma cells through induction of G2/M arrest, which was accompanied by down-regulating Cdk1 and Cdc25C proteins. Additionally, andrographolide decreased the activity of PI3K/Akt signaling, as demonstrated by down-regulation of the expression of phos-PI3K, phos-Akt, phos-mTOR and phos-p70s6k in U251 and U87 cells. Furthermore, additive effects on the proliferation inhibition, G2/M arrest and down-regulation of G2/M phase-related proteins were observed, when a combined treatment of andrographolide with PI3K inhibitor LY294002 was used in U251 and U87 cells. We prove that andrographolide inhibits the proliferation of human glioblastoma cells via inducing G2/M arrest, which is mediated by inhibiting the activity of PI3K/Akt signaling. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. 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

  10. 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

  11. Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines

    Directory of Open Access Journals (Sweden)

    RODRIGUES da SILVA Andréa de Cássia

    2000-01-01

    Full Text Available Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac and an inactivated-adjuvanted PV (IPVvac vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87% of the sera showed virus-neutralizing antibody titers (VNA higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.

  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. 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.

  14. Stress Altered Stem Cells with Decellularized Allograft to Improve Rate of Nerve Regeneration

    Science.gov (United States)

    2015-12-01

    of the cellular elements normally present in peripheral nerve . 2. KEYWORDS: peripheral nerve repair , nerve injury , decellularized nerve ... nerve regeneration. The slow rate of nerve re generation in limbs results in poor prognosis for patients suffering from severe injuries , leading to...allograft, neural regeneration, stem cells, stress altered cells, peripheral nerve injury model, nerve graft 3 This comprehensive final report summarizes

  15. Analysis of enhancement at small and large radiation doses for effectiveness of inactivation in cultured cells by combining two agents with radiation

    NARCIS (Netherlands)

    Franken, Nicolaas A. P.; Kok, H. Petra; Crezee, Johannes; Barendsen, Gerrit W.

    2016-01-01

    To evaluate the enhancement effect of two combined radiation-sensitizing agents in mammalian cells at small doses as compared to large doses using the linear-quadratic (LQ) mathematical model. Data on clonogenic assays concerning the radio-enhancement effects of combined halogenated pyrimidines and

  16. 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

  17. Effects of gemcitabine on cell survival and chromosome aberrations after pulsed low dose-rate irradiation

    NARCIS (Netherlands)

    Castro Kreder, Natasja; van Bree, Chris; Franken, Nicolaas A. P.; Haveman, Jaap

    2004-01-01

    The radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) was studied in combination with pulsed low dose-rate irradiation. The experiments were carried out with a human lung carcinoma cell line SW 1573. These were irradiated at pulsed low dose rate (p-LDR); the average dose

  18. 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 ...

  19. Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells

    International Nuclear Information System (INIS)

    Wilkins, David E.; Cheng, E. Ng; Raaphorst, G. Peter

    1996-01-01

    Purpose: Human glioma cell lines resistant (U373MG CP ) and sensitive (U373MG) to cisplatin were used to evaluate the effect of cisplatin as a sensitizer to low dose rate irradiation (LDRI). Methods and Materials: A cisplatin resistant glioma cell line U373MG CP was developed by chronic exposure of parental U373MG cells to cisplatin. Plateau phase cells were treated with cisplatin, high dose rate (HDR) irradiation (1.12 Gy/min), LDRI (0.0088 Gy/min), or cisplatin concurrent with LDRI. Cell survival was determined by the colony forming assay. Results: Both cell lines showed increased resistance to radiation at LDR compared with HDR, with Dose Modifying Factors (DMF at 10% survival level) of 1.7 for U373MG and 2.5 for U373MG CP . The increased LDR sparing effect in the cisplatin resistant U373MG CP cells indicates increased repair proficiency. The resistant cell line showed a fourfold increase in resistance to cisplatin cytotoxicity at the 10% survival level compared with the parental U373MG cells. Cisplatin enhanced the response of both cell lines to LDRI. The DMFs were 1.2, 1.2, and 1.7, respectively, for the sensitive U373MG cell line given 1 μg/ml, and the resistant cell line given 3 or 6 μg/ml cisplatin treatments concurrent with LDRI. Conclusions: These data show that cisplatin can be an effective sensitizer to LDRI in both cisplatin resistant and sensitive glioma cell lines. However, in the resistant cell line, higher concentrations of cisplatin were necessary to achieve the same level of sensitization as in the sensitive cell line

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

    OpenAIRE

    Ceuppens Siele; Uyttendaele Mieke; Hamelink Stefanie; Boon Nico; Van de Wiele Tom

    2012-01-01

    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 t...

  1. 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

  2. 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

    International Nuclear Information System (INIS)

    Sunaoshi, Masaaki; Amasaki, Yoshiko; Hirano-Sakairi, Shinobu; Blyth, Benjamin J.; Morioka, Takamitsu; Kaminishi, Mutsumi; Shang, Yi; Nishimura, Mayumi; Shimada, Yoshiya; Tachibana, Akira

    2015-01-01

    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

  3. 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

  4. 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.

  5. 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 137 Cs 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.

  6. SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Sabine Jägle

    2017-11-01

    Full Text Available Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining

  7. 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.

  8. 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.

  9. Inactivation of allergens and toxins.

    Science.gov (United States)

    Morandini, Piero

    2010-11-30

    Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Sucrose modulation of radiofrequency-induced heating rates and cell death.

    Science.gov (United States)

    Pulikkathara, Merlyn; Mark, Colette; Kumar, Natasha; Zaske, Ana Maria; Serda, Rita E

    2017-09-01

    Applied radiofrequency (RF) energy induces hyperthermia in tissues, facilitating vascular perfusion This study explores the impact of RF radiation on the integrity of the luminal endothelium, and then predominately explores the impact of altering the conductivity of biologically-relevant solutions on RF-induced heating rates and cell death. The ability of cells to survive high sucrose (i.e. hyperosmotic conditions) to achieve lower conductivity as a mechanism for directing hyperthermia is evaluated. RF radiation was generated using a capacitively-coupled radiofrequency system operating at 13.56 MHz. Temperatures were recorded using a FLIR SC 6000 infrared camera. RF radiation reduced cell-to-cell connections among endothelial cells and altered cell morphology towards a more rounded appearance at temperatures reported to cause in vivo vessel deformation. Isotonic solutions containing high sucrose and low levels of NaCl displayed low conductivity and faster heating rates compared to high salt solutions. Heating rates were positively correlated with cell death. Addition of sucrose to serum similarly reduced conductivity and increased heating rates in a dose-dependent manner. Cellular proliferation was normal for cells grown in media supplemented with 125 mM sucrose for 24 hours or for cells grown in 750 mM sucrose for 10 minutes followed by a 24 h recovery period. Sucrose is known to form weak hydrogen bonds in fluids as opposed to ions, freeing water molecules to rotate in an oscillating field of electromagnetic radiation and contributing to heat induction. The ability of cells to survive temporal exposures to hyperosmotic (i.e. elevated sucrose) conditions creates an opportunity to use sucrose or other saccharides to selectively elevate heating in specific tissues upon exposure to a radiofrequency field.

  11. Tailoring the rate-sensitivity of low density polyurea foams through cell wall aperture size

    Science.gov (United States)

    Ramirez, B. J.; Kingstedt, O. T.; Crum, R.; Gamez, C.; Gupta, V.

    2017-06-01

    The plateau stress and energy absorption of low density (≤300 kg/m3) polyurea (PU) foams and expanded polystyrene (EPS) were measured at deformation rates ranging from 0.004 s-1 to 5000 s-1. Low (≤10-1 s-1) strain rate testing was performed using an Instron load frame, intermediate (101-102 s-1) strain rates using a drop-weight impact tower, and high (≥103 s-1) strain rate conditions using a modified split-Hopkinson pressure bar. The plateau stress and energy absorption of low density PU foams exhibit a strong rate dependence across all deformation rates. This result has been previously unreported for low density polymer foams under low and intermediate strain rates. The strain rate sensitivity of PU foams was found to be strongly dependent on cell size for low strain rates and cell wall aperture size for intermediate and high strain rates. EPS type foam, however, remained nearly insensitive to strain rate. At low and intermediate strain rates, the plastic crushing in the EPS and the high plateau stress yield a much higher energy absorption capability than the viscoelastic dissipation in the PU foams. However, PU foams were found to display similar energy absorption properties as EPS based foams under high strain rates. Thus, controlling the strain rate sensitivity of PU foams through aperture diameter can lead to an increase in energy absorption properties at high strain rates, while simultaneously maintaining the peak stress below certain injury thresholds. Additionally, unlike EPS, which undergo plastic crushing after first impact, flexible polyurea foams will recover fully after each impact and thus will have multiple hit capabilities. This will allow these materials to have a wide range of applications, in advance body armors and protective headgears to use in low-cost protection systems for a wide range of military platforms, civilian, and space applications.

  12. Component Cell-Based Restriction of Spectral Conditions and the Impact on CPV Module Power Rating

    Energy Technology Data Exchange (ETDEWEB)

    Muller, Matthew T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Steiner, Marc [Fraunhofer Institute for Solar Energy Systems ISE; Siefer, Gerald [Fraunhofer Institute for Solar Energy Systems ISE; Bett, Andreas W. [Fraunhofer Institute for Solar Energy Systems ISE

    2018-01-26

    One approach to consider the prevailing spectral conditions when performing CPV module power ratings according to the standard IEC 62670-3 is based on spectral matching ratios (SMRs) determined by the means of component cell sensors. In this work, an uncertainty analysis of the SMR approach is performed based on a dataset of spectral irradiances created with SMARTS2. Using these illumination spectra, the respective efficiencies of multijunction solar cells with different cell architectures are calculated. These efficiencies were used to analyze the influence of different component cell sensors and SMR filtering methods. The 3 main findings of this work are as follows. First, component cells based on the lattice-matched triple-junction (LM3J) cell are suitable for restricting spectral conditions and are qualified for the standardized power rating of CPV modules - even if the CPV module is using multijunction cells other than LM3J. Second, a filtering of all 3 SMRs with +/-3.0% of unity results in the worst case scenario in an underestimation of -1.7% and overestimation of +2.4% compared to AM1.5d efficiency. Third, there is no benefit in matching the component cells to the module cell in respect to the measurement uncertainty.

  13. Applicability of photodynamic antimicrobial chemotherapy as an alternative to inactivate fish pathogenic bacteria in aquaculture systems.

    Science.gov (United States)

    Arrojado, Cátia; Pereira, Carla; Tomé, João P C; Faustino, Maria A F; Neves, Maria G P M S; Tomé, Augusto C; Cavaleiro, José A S; Cunha, Angela; Calado, Ricardo; Gomes, Newton C M; Almeida, Adelaide

    2011-10-01

    Aquaculture activities are increasing worldwide, stimulated by the progressive reduction of natural fish stocks in the oceans. However, these activities also suffer heavy production and financial losses resulting from fish infections caused by microbial pathogens, including multidrug resistant bacteria. Therefore, strategies to control fish infections are urgently needed, in order to make aquaculture industry more sustainable. Antimicrobial photodynamic therapy (aPDT) has emerged as an alternative to treat diseases and prevent the development of antibiotic resistance by pathogenic bacteria. The aim of this work was to evaluate the applicability of aPDT to inactivate pathogenic fish bacteria. To reach this objective a cationic porphyrin Tri-Py(+)-Me-PF was tested against nine pathogenic bacteria isolated from a semi-intensive aquaculture system and against the cultivable bacteria of the aquaculture system. The ecological impact of aPDT in the aquatic environment was also tested on the natural bacterial community, using the overall bacterial community structure and the cultivable bacteria as indicators. Photodynamic inactivation of bacterial isolates and of cultivable bacteria was assessed counting the number of colonies. The impact of aPDT in the overall bacterial community structure of the aquaculture water was evaluated by denaturing gel gradient electrophoresis (DGGE). The results showed that, in the presence of Tri-Py(+)-Me-PF, the growth of bacterial isolates was inhibited, resulting in a decrease of ≈7-8 log after 60-270 min of irradiation. Cultivable bacteria were also considerably affected, showing decreases up to the detection limit (≈2 log decrease on cell survival), but the inactivation rate varied significantly with the sampling period. The DGGE fingerprint analyses revealed changes in the bacterial community structure caused by the combination of aPDT and light. The results indicate that aPDT can be regarded as a new approach to control fish

  14. 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.

  15. 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.

  16. 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 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.

  17. Simple and inexpensive technique for measuring oxygen consumption rate in adherent cultured cells.

    Science.gov (United States)

    Takahashi, Eiji; Yamaoka, Yoshihisa

    2017-11-01

    Measurement of cellular oxygen consumption rate (OCR) is essential in assessing roles of mitochondria in physiology and pathophysiology. Classical techniques, in which polarographic oxygen electrode measures the extracellular oxygen concentration in a closed measuring vessel, require isolation and suspension of the cell. Because cell functions depend on the extracellular milieu including the extracellular matrix, isolation of cultured cells prior to the measurement may significantly affect the OCR. More recent techniques utilize optical methods in which oxygen-dependent quenching of fluorophores determines oxygen concentration in the medium at a few microns above the surface of the cultured cells. These techniques allow the OCR measurement in cultured cells adhered to the culture dish. However, this technique requires special equipment such as a fluorescence lifetime microplate reader or specialized integrated system, which are usually quite expensive. Here, we introduce a simple and inexpensive technique for measuring OCR in adherent cultured cells that utilizes conventional fluorescence microscopy and a glassware called a gap cover glass.

  18. 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.

  19. 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.

  20. 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

  1. Lymphoid cell kinetics under continuous low dose-rate gamma irradiation: A comparison study

    Science.gov (United States)

    Foster, B. R.

    1975-01-01

    A comparison study was conducted of the effects of continuous low dose-rate gamma irradiation on cell population kinetics of lymphoid tissue (white pulp) of the mouse spleen with findings as they relate to the mouse thymus. Experimental techniques employed included autoradiography and specific labeling with tritiated thymidine (TdR-(h-3)). The problem studied involved the mechanism of cell proliferation of lymphoid tissue of the mouse spleen and thymus under the stress of continuous irradiation at a dose rate of 10 roentgens (R) per day for 105 days (15 weeks). The aim was to determine whether or not a steady state or near-steady state of cell population could be established for this period of time, and what compensatory mechanisms of cell population were involved.

  2. Dose rate effects of low-LET ionizing radiation on fish cells

    Energy Technology Data Exchange (ETDEWEB)

    Vo, Nguyen T.K. [McMaster University, Radiation Sciences Program, School of Graduate and Postdoctoral Studies, Hamilton, ON (Canada); Seymour, Colin B.; Mothersill, Carmel E. [McMaster University, Radiation Sciences Program, School of Graduate and Postdoctoral Studies, Hamilton, ON (Canada); McMaster University, Department of Biology, Hamilton, ON (Canada)

    2017-11-15

    Radiobiological responses of a highly clonogenic fish cell line, eelB, to low-LET ionizing radiation and effects of dose rates were studied. In acute exposure to 0.1-12 Gy of gamma rays, eelB's cell survival curve displayed a linear-quadratic (LQ) relationship. In the LQ model, α, β, and α/β ratio were 0.0024, 0.037, and 0.065, respectively; for the first time that these values were reported for fish cells. In the multi-target model, n, D{sub o}, and D{sub q} values were determined to be 4.42, 2.16, and 3.21 Gy, respectively, and were the smallest among fish cell lines being examined to date. The mitochondrial potential response to gamma radiation in eelB cells was at least biphasic: mitochondria hyperpolarized 2 h and then depolarized 5 h post-irradiation. Upon receiving gamma rays with a total dose of 5 Gy, dose rates (ranging between 83 and 1366 mGy/min) had different effects on the clonogenic survival but not the mitochondrial potential. The clonogenic survival was significantly higher at the lowest dose rate of 83 mGy/min than at the other higher dose rates. Upon continuous irradiation with beta particles from tritium at 0.5, 5, 50, and 500 mGy/day for 7 days, mitochondria significantly depolarized at the three higher dose rates. Clearly, dose rates had differential effects on the clonogenic survival of and mitochondrial membrane potential in fish cells. (orig.)

  3. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

    Science.gov (United States)

    Bhatia, Sujata K; Yetter, Ann B

    2008-08-01

    Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

  4. Status of Degradation Rates and Mechanisms in Nickel-Hydrogen Cells

    Science.gov (United States)

    1998-04-15

    Batteries , and Yardney Technical Products. The testing programs were supported by one of several different Air Force or NASA organizations and were...usable energy density of nickel- hydrogen cells and batteries with any degree of certainty. Newer configurations and cell chemistries with higher...requirement of the completed battery . 4. Conditions selected for the recharge step of the cycle that have been shown to accelerate the rate of

  5. Effect of radiation dose-rate on hematopoietic cell engraftment in adult zebrafish.

    Directory of Open Access Journals (Sweden)

    Tiffany J Glass

    Full Text Available Although exceptionally high radiation dose-rates are currently attaining clinical feasibility, there have been relatively few studies reporting the biological consequences of these dose-rates in hematopoietic cell transplant (HCT. In zebrafish models of HCT, preconditioning before transplant is typically achieved through radiation alone. We report the comparison of outcomes in adult zebrafish irradiated with 20 Gy at either 25 or 800 cGy/min in the context of experimental HCT. In non-transplanted irradiated fish we observed no substantial differences between dose-rate groups as assessed by fish mortality, cell death in the kidney, endogenous hematopoietic reconstitution, or gene expression levels of p53 and ddb2 (damage-specific DNA binding protein 2 in the kidney. However, following HCT, recipients conditioned with the higher dose rate showed significantly improved donor-derived engraftment at 9 days post transplant (p ≤ 0.0001, and improved engraftment persisted at 31 days post transplant. Analysis for sdf-1a expression, as well as transplant of hematopoietic cells from cxcr4b -/- zebrafish, (odysseus, cumulatively suggest that the sdf-1a/cxcr4b axis is not required of donor-derived cells for the observed dose-rate effect on engraftment. Overall, the adult zebrafish model of HCT indicates that exceptionally high radiation dose-rates can impact HCT outcome, and offers a new system for radiobiological and mechanistic interrogation of this phenomenon. Key words: Radiation dose rate, Total Marrow Irradiation (TMI, Total body irradiation (TBI, SDF-1, Zebrafish, hematopoietic cell transplant.

  6. Automated rotation rate tracking of pigmented cells by a customized block-matching algorithm.

    Science.gov (United States)

    Zhang, Guanglie; Ouyang, Mengxing; Mai, John; Li, Wen Jung; Liu, Wing Keung

    2013-04-01

    This article describes an automated rotation rate tracking algorithm for pigmented cells that undergo rotation in a dielectrophoretic (DEP) force field. In a completely automated process, we preprocess each frame of a video sequence, then analyze the sequence frame by frame using a rotating-circle template with a block-matching algorithm, and finally estimate the rotation rate of the pigmented cells using a pixel-patch correlation. The algorithm has been demonstrated to accurately calculate the DEP-induced rotation rate of the cell up to 250 rpm. Cell rotation rates in various DEP force fields (i.e., by varying the applied voltages, frequencies, and waveforms to induce different force fields) were analyzed using this automated algorithm and reported in this article. Most importantly, the algorithm is accurate even when the cells have simultaneous translational and rotational motions across the video image sequence. Also, the algorithm is capable of tracking changes in rotation speed over a long period of time (90 s) by stably analyzing a massive data set of video image frames.

  7. A CMI (cell metabolic indicator)-based controller for achieving high growth rate Escherichia coli cultures.

    Science.gov (United States)

    Pepper, Matthew E; Wang, Li; Padmakumar, Ajay; Burg, Timothy C; Harcum, Sarah W; Groff, Richard E

    2014-01-01

    A large fraction of biopharmaceuticals are produced in Escherichia coli, where each new product and strain currently requires a high degree of growth characterization in benchtop and industrial bioreactors to achieve economical production protocols. The capability to use a standard set of sensors to characterize a system quickly without the need to conduct numerous experiments to determine stable growth rate for the strain would significantly decrease development time. This paper presents a cell metabolic indicator (CMI) which provides better insight into the E. coli metabolism than a growth rate value. The CMI is the ratio of the oxygen uptake rate (OUR) of the culture and the base addition rate (BAR) required to keep pH at a desired setpoint. The OUR and BAR are measured using a off-gas sensor and pH probe, respectively, and thus the CMI can be computed online. Experimental results demonstrate the relationship between CMI and the different cell metabolic states. A previously published model is augmented with acid production dynamics, allowing for comparison of the CMI-based controller with an open-loop controller in simulation. The CMI-based controller required little a priori knowledge about the E. coli strain in order to achieve a high growth rate. Since many different types of cells exhibit similar behaviors, the CMI concept can be extended to mammalian and stem cells.

  8. Case-positive versus case-negative designs for low-rate lithium thionyl chloride cells

    Science.gov (United States)

    Mahy, T. X.

    1982-03-01

    Case polarity design choices are discussed. Two examples of case-negative designs are presented. One battery is thionyl chloride limited and the other is lithium limited. The case-positive design is thionyl chloride limited. It is found that the case-positive/case-negative design consideration does not seem to have much bearing on storage. However, during low rate discharge, the case-negative cells show a steadily decreasing capacity as you go to lower and lower rates.

  9. Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture

    International Nuclear Information System (INIS)

    Esterman, A.L.; Cohen, B.I.; Javitt, N.B.

    1985-01-01

    Cholesterol synthesis in cell culture in the presence of D 2 O yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen. Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry. Mevinolin and 26-hydroxycholesterol both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity

  10. 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.

  11. Investigation of silica additive for high-rate sealed lead-acid cells

    International Nuclear Information System (INIS)

    Wang, L.-F.; Fang, B.-J.; Lin, Y.-K.; Chen, J.-S.

    2006-01-01

    This study investigated the effects of silica (SiO 2 ) additive in the positive electrode on the high-rate discharge performance of sealed lead-acid cells. X-ray diffraction studies reveal that the plate's chemical composition and crystal morphology is independent of the SiO 2 additive in the positive electrodes. Cells with SiO 2 additive in the positive plates decrease the active material, causing a lower initial capacity. However, the plates with SiO 2 additive exhibit a smaller pore size, which has a good mechanical strength and retains the electrolyte in the pore during high-rate discharge causing a lower capacity loss per cycle and a higher average capacity per cycle. Apparently, the SiO 2 additive has the beneficial effect of decreasing the capacity loss during the high-rate discharge cycles. Moreover, the discharge capacity loss rate does not correlate with the amount of SiO 2 additives. Adding more SiO 2 additives to the positive electrode decreases the active material appreciably causing a lower average capacity. The optimal amount of SiO 2 additive is about 3 wt%. The phase composition of the positive electrodes at different locations was determined after the cells completed the high-rate cycle test. The results reveal that the higher utilization of electrode plates at high-rate discharge occurred through the terminal and diagonal areas

  12. Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice.

    Science.gov (United States)

    Beuchat, L R; Brackett, R E; Hao, D Y; Conner, D E

    1986-10-01

    Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.

  13. Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Young-Kug Choo

    2012-12-01

    Full Text Available Gangliosides play important roles in the control of severalbiological processes, including proliferation and transmembranesignaling. In this study, we demonstrate the effect ofganglioside GM1 on the proliferation of mouse inducedpluripotent stem cells (miPSCs. The proliferation rate ofmiPSCs was lower than in mouse embryonic stem cells(mESCs. Fluorescence activated cell sorting analysis showedthat the percentage of cells in the G2/M phase in miPSCs waslower than that in mESCs. GM1 was expressed in mESCs, butnot miPSCs. To confirm the role of GM1 in miPSC proliferation,miPSCs were treated with GM1. GM1-treated miPSCsexhibited increased cell proliferation and a larger number ofcells in the G2/M phase. Furthermore, phosphorylation ofmitogen-activated protein kinases was increased in GM1-treated miPSCs.

  14. Acute inhalation toxicity of 3-methylfuran in the mouse: pathology, cell kinetics, and respiratory rate effects

    Energy Technology Data Exchange (ETDEWEB)

    Haschek, W.M.; Boyd, M.R.; Hakkinen, P.J.; Owenby, C.S.; Witschi, H.

    1984-01-01

    The acute inhalation toxicity of 3-methylfuran (3MF) was investigated in male BALB/c mice by morphologic examination of animals killed at varying timepoints following a 1-hr exposure to an initial chamber concentration of 14 to 37 mumol/liter (343 to 906 ppm). In addition, respiratory rate measurements and cell kinetics were used to assess quantitatively pulmonary damage and repair. Necrosis of nonciliated bronchiolar epithelial (Clara) cells was seen 1 day following exposure and was followed by regeneration, which was virtually complete, within 21 days. Cell kinetic studies showed peak bronchiolar cell proliferation at 3 days with a labeling index (LI) of 5.0% compared to 0.4% in controls. An increase in parenchymal cell proliferation was also noted coincident with a mild interstitial pneumonitis. This parenchymal proliferation, peaking at 10 days with an LI of 1.4% compared to 0.2% in controls, consisted primarily of type II epithelial and endothelial cell proliferation indicating possible delayed damage and repair of type I epithelial and endothelial cells. The respiratory rate showed an initial transient increase followed by a more prolonged decrease with eventual return to control levels. 3MF toxicity was also evidenced by a necrotizing suppurative rhinitis, centrilobular hepatic necrosis, lymphocyte necrosis in the thymus and spleen, sialoadenitis, and otitis media.

  15. Inactivation of Amphidinium sp. in ballast waters using UV/Ag-TiO2+O3 advanced oxidation treatment.

    Science.gov (United States)

    Wu, Donghai; You, Hong; Zhang, Ran; Chen, Chuan; Lee, Duu-Jong

    2011-11-01

    Ballast water poses a biological threat to the world's waterways by transferring aquatic species from one body of water to another. This study investigates the use of combined ultraviolet (UV)/Ag-TiO(2)+ozone (O(3)) processes for treating ballast water using Amphidinium sp. as an indicator microorganism. Sufficient Amphidinium sp. cells in ballast waters can be inactivated using O(3) alone, UV irradiation alone (with or without an Ag-TiO(2) coating), and combined treatments. For the low inactivation ratio (treatment produced excess hydroxyl radicals and total residual oxidants (TROs), and readily damaged cell membranes to release intracellular substances. The comparison tests revealed that the combined treatments synergistically inactivate Escherichia coli in ballast waters. However, the combined process did not synergistically inactivate Amphidinium sp. cells. Inactivating different aqua species in ballast waters needs distinct treatment methods and dosages. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. 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.

  17. Model cerebellar granule cells can faithfully transmit modulated firing rate signals

    Directory of Open Access Journals (Sweden)

    Christian eRössert

    2014-10-01

    Full Text Available A crucial assumption of many high-level system models of the cerebellum is that information in the granular layer is encoded in a linear manner. However, granule cells are known for their non-linear and resonant synaptic and intrinsic properties that could potentially impede linear signal transmission.In this modelling study we analyse how electrophysiological granule cell properties and spike sampling influence information coded by firing rate modulation, assuming no signal-related, i.e. uncorrelated inhibitory feedback (open-loop mode.A detailed one-compartment granule cell model was excited in simulation by either direct current or mossy-fibre synaptic inputs. Vestibular signals were represented as tonic inputs to the flocculus modulated at frequencies up to 20 Hz (approximate upper frequency limit of vestibular-ocular reflex, VOR. Model outputs were assessed using estimates of both the transfer function, and the fidelity of input-signal reconstruction measured as variance-accounted-for.The detailed granule cell model with realistic mossy-fibre synaptic inputs could transmit information faithfully and linearly in the frequency range of the vestibular-ocular reflex. This was achieved most simply if the model neurons had a firing rate at least twice the highest required frequency of modulation, but lower rates were also adequate provided a population of neurons was utilized, especially in combination with push-pull coding. The exact number of neurons required for faithful transmission depended on the precise values of firing rate and noise. The model neurons were also able to combine excitatory and inhibitory signals linearly, and could be replaced by a simpler (modified integrate-and-fire neuron in the case of high tonic firing rates.These findings suggest that granule cells can in principle code modulated firing-rate inputs in a linear manner, and are thus consistent with the high-level adaptive-filter model of the cerebellar microcircuit.

  18. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    Science.gov (United States)

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Optimization of the synthesis process of an iron oxide nanocatalyst supported on activated carbon for the inactivation of Ascaris eggs in water using the heterogeneous Fenton-like reaction.

    Science.gov (United States)

    Morales-Pérez, Ariadna A; Maravilla, Pablo; Solís-López, Myriam; Schouwenaars, Rafael; Durán-Moreno, Alfonso; Ramírez-Zamora, Rosa-María

    2016-01-01

    An experimental design methodology was used to optimize the synthesis of an iron-supported nanocatalyst as well as the inactivation process of Ascaris eggs (Ae) using this material. A factor screening design was used for identifying the significant experimental factors for nanocatalyst support (supported %Fe, (w/w), temperature and time of calcination) and for the inactivation process called the heterogeneous Fenton-like reaction (H2O2 dose, mass ratio Fe/H2O2, pH and reaction time). The optimization of the significant factors was carried out using a face-centered central composite design. The optimal operating conditions for both processes were estimated with a statistical model and implemented experimentally with five replicates. The predicted value of the Ae inactivation rate was close to the laboratory results. At the optimal operating conditions of the nanocatalyst production and Ae inactivation process, the Ascaris ova showed genomic damage to the point that no cell reparation was possible showing that this advanced oxidation process was highly efficient for inactivating this pathogen.

  20. The radiation inactivation of glutamate and isocitrate dehydrogenases

    International Nuclear Information System (INIS)

    El Failat, R.R.A.

    1980-12-01

    The reaction of free radicals produced by ionizing radiation with the enzymes glutamate dehydrogenase (GDH) and NADP + -specific isocitrate dehydrogenase (ICDH) have been studied by steady-state and pulse radiolysis techniques. In de-aerated GDH solutions, hydroxyl radicals have been found to be the most efficient of the primary radicals generated from water in causing inactivation. The effect of reaction with the enzyme of selective free radicals (SCN) 2 - , (Br) 2 - and (I) 2 - on its activity has also been studied. In neutral solutions, the order of inactivating effectiveness is (I) 2 - > (Br) 2 - > (SCN) 2 - . In the case of the thiocyanate radical anion (SCN) 2 - , the inactivation efficiency is found to depend on KSCN concentration. The radiation inactivation of GDH at both neutral and alkaline pH is accompanied by the loss of sulphydryl groups. Pulse radiolysis was also used to determine the rate constants and the transient absorption spectra following the reaction of the free radicals with GDH. 60 Co-γ-radiolysis and pulse radiolysis were also used to study the effect of ionizing radiation on the activity of ICDH. The results obtained were similar to those of GDH. (author)

  1. Effect of turbulent gas-liquid contact in a static mixer on Cryptosporidium parvum oocyst inactivation by ozone.

    Science.gov (United States)

    Craik, Stephen A; Smith, Daniel W; Chandrakanth, Mysore; Belosevic, Miodrag

    2003-09-01

    Static mixers may be used to dissolve gaseous ozone in water treatment facilities in order to provide protection against the waterborne parasite Cryptosporidium parvum. The objective of this study was to determine the effect of a brief exposure to turbulent gas-liquid mixing conditions in a static mixer on inactivation of C. parvum oocysts by ozone. Inactivation measured in an ozone contacting apparatus that employed a static mixer for ozone dissolution was compared to predictions based on a previously published kinetic model of C. parvum inactivation by dissolved ozone in gently stirred batch reactors. Although initial contact in the static mixer had no immediate effect on the oocysts, a 20% increase in the rate of inactivation during subsequent contact with dissolved ozone was observed. Increasing the degree of turbulence within the static mixer did not yield additional inactivation. Use of static mixers for dissolution of ozone in drinking water treatment systems may provide limited enhancement of C. parvum inactivation by dissolved ozone.

  2. Sunlight inactivation of human viruses and bacteriophages in coastal waters containing natural photosensitizers.

    Science.gov (United States)

    Silverman, Andrea I; Peterson, Britt M; Boehm, Alexandria B; McNeill, Kristopher; Nelson, Kara L

    2013-02-19

    Sunlight inactivation of poliovirus type 3 (PV3), adenovirus type 2 (HAdV2), and two bacteriophage (MS2 and PRD1) was investigated in an array of coastal waters to better understand solar inactivation mechanisms and the effect of natural water constituents on observed inactivation rates (k(obs)). Reactor scale inactivation experiments were conducted using a solar simulator, and k(obs) for each virus was measured in a sensitizer-free control and five unfiltered surface water samples collected from different sources. k(obs) values varied between viruses in the same water matrix, and for each virus in different matrices, with PV3 having the fastest and MS2 the slowest k(obs) in all waters. When exposed to full-spectrum sunlight, the presence of photosensitizers increased k(obs) of HAdV2, PRD1 and MS2, but not PV3, which provides evidence that the exogenous sunlight inactivation mechanism, involving damage by exogenously produced reactive intermediates, played a greater role for these viruses. While PV3 inactivation was observed to be dominated by endogenous mechanisms, this may be due to a masking of exogenous k(obs) by significantly faster endogenous k(obs). Results illustrate that differences in water composition can shift absolute and relative inactivation rates of viruses, which has important implications for natural wastewater treatment systems, solar disinfection (SODIS), and the use of indicator organisms for monitoring water quality.

  3. [Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions].

    Science.gov (United States)

    Tarun, E I; Kurchenko, V P; Metelitsa, D I

    2006-01-01

    Inactivation of soybean urease in aqueous solution at pH 5.4, 36 degrees C, and high-frequency sonication (2.64 MHz, 1.0 W/cm2) is substantially reduced in the presence of seven structurally different flavonoids. A comparative kinetic study of the effect of these flavonoids on the effective first-order rate constants that characterize the total (thermal and ultrasonic) inactivation k(i), thermal inactivation k(i)*, and ultrasonic inactivation k(i)(US) of 25 nM enzyme solution was carried out. The dependences of the three inactivation rate constants of the urease on the concentrations of flavonoids within the range from 10(-11) to 10(-4) M were obtained. The following order of the efficiency of the flavonoids used in respect of the urease protection from ultrasonic inactivation was found: astragalin > silybin > naringin > hesperidin > quercetin > kaempferol > morin. The results confirm a significant role in the inactivation of the urease of HO* and HO2*, free radicals, which are formed in the ultrasonic cavitation field.

  4. Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

    Science.gov (United States)

    Simon, B J; Klein, M G; Schneider, M F

    1991-03-01

    The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of

  5. Microenvironmental Stiffness of 3D Polymeric Structures to Study Invasive Rates of Cancer Cells.

    Science.gov (United States)

    Lemma, Enrico Domenico; Spagnolo, Barbara; Rizzi, Francesco; Corvaglia, Stefania; Pisanello, Marco; De Vittorio, Massimo; Pisanello, Ferruccio

    2017-11-01

    Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness "weak spots" is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell-structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential

    Directory of Open Access Journals (Sweden)

    Adriana Castillo-Villanueva

    2017-12-01

    Full Text Available Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222 by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug. Keywords: Giardiasis, Drug repurposing, Neglected disease, Recombinant protein, Enzyme inactivation

  7. Stable inheritable postradiation increase in the death-rate of yeast cells

    International Nuclear Information System (INIS)

    Bychkovskaya, I.B.; Stanzhevskaya, T.I.

    1980-01-01

    It was demonstrated by the use of serial equal dilution of irradiated and control cultures, which extended the logarithmic phase of growth, that irradiation of 0.5-2.5 krad, as well as the effect of a preirradiated medium, induce in Saccharomyces ellipsoideus Megri an inheritable stable increase in the death-rate of cells which was previously demonstrated on individually cultured protozoa

  8. Effects of proton radiation dose, dose rate and dose fractionation on hematopoietic cells in mice

    International Nuclear Information System (INIS)

    Ware, J.H.; Rusek, A.; Sanzari, J.; Avery, S.; Sayers, C.; Krigsfeld, G.; Nuth, M.; Wan, X.S.; Kennedy, A.R.

    2010-01-01

    The present study evaluated the acute effects of radiation dose, dose rate and fractionation as well as the energy of protons in hematopoietic cells of irradiated mice. The mice were irradiated with a single dose of 51.24 MeV protons at a dose of 2 Gy and a dose rate of 0.05-0.07 Gy/min or 1 GeV protons at doses of 0.1, 0.2, 0.5, 1, 1.5 and 2 Gy delivered in a single dose at dose rates of 0.05 or 0.5 Gy/min or in five daily dose fractions at a dose rate of 0.05 Gy/min. Sham-irradiated animals were used as controls. The results demonstrate a dose-dependent loss of white blood cells (WBCs) and lymphocytes by up to 61% and 72%, respectively, in mice irradiated with protons at doses up to 2 Gy. The results also demonstrate that the dose rate, fractionation pattern and energy of the proton radiation did not have significant effects on WBC and lymphocyte counts in the irradiated animals. These results suggest that the acute effects of proton radiation on WBC and lymphocyte counts are determined mainly by the radiation dose, with very little contribution from the dose rate (over the range of dose rates evaluated), fractionation and energy of the protons.

  9. Effects of proton radiation dose, dose rate and dose fractionation on hematopoietic cells in mice.

    Science.gov (United States)

    Ware, J H; Sanzari, J; Avery, S; Sayers, C; Krigsfeld, G; Nuth, M; Wan, X S; Rusek, A; Kennedy, A R

    2010-09-01

    The present study evaluated the acute effects of radiation dose, dose rate and fractionation as well as the energy of protons in hematopoietic cells of irradiated mice. The mice were irradiated with a single dose of 51.24 MeV protons at a dose of 2 Gy and a dose rate of 0.05-0.07 Gy/min or 1 GeV protons at doses of 0.1, 0.2, 0.5, 1, 1.5 and 2 Gy delivered in a single dose at dose rates of 0.05 or 0.5 Gy/min or in five daily dose fractions at a dose rate of 0.05 Gy/min. Sham-irradiated animals were used as controls. The results demonstrate a dose-dependent loss of white blood cells (WBCs) and lymphocytes by up to 61% and 72%, respectively, in mice irradiated with protons at doses up to 2 Gy. The results also demonstrate that the dose rate, fractionation pattern and energy of the proton radiation did not have significant effects on WBC and lymphocyte counts in the irradiated animals. These results suggest that the acute effects of proton radiation on WBC and lymphocyte counts are determined mainly by the radiation dose, with very little contribution from the dose rate (over the range of dose rates evaluated), fractionation and energy of the protons.

  10. Sunlight mediated inactivation mechanisms of Enterococcus faecalis and Escherichia coli in clear water versus waste stabilization pond water.

    Science.gov (United States)

    Kadir, Khalid; Nelson, Kara L

    2014-03-01

    Escherichia coli and enterococci have been previously reported to differ in the mechanisms and conditions that affect their sunlight-mediated inactivation in waste stabilization ponds. This study was undertaken to further characterize these mechanisms, using simulated sunlight and single strains of laboratory-grown E. coli and Enterococcus faecalis, with a focus on characterizing the contribution of exogenous reactive oxygen species to the inactivation process. We found that direct damage by UVB light (280-320 nm) was not a significant inactivation mechanism for either organism. E. coli inactivation was strongly dependent on dissolved oxygen concentrations and the presence of UVB wavelengths but E. coli were not susceptible to inactivation by exogenous sensitizers present in waste stabilization pond water. In contrast, E. faecalis inactivation in pond water occurred primarily through exogenous mechanisms, with strong evidence that singlet oxygen is an important transient reactive species. The exogenous mechanism could utilize wavelengths into the visible spectrum and sensitizers were mainly colloidal, distributed between 0.2 and ∼1 μm in size. Singlet oxygen is likely an important endogenous species in both E. faecalis and E. coli inactivation due to sunlight. Although the two organisms had similar inactivation rates in buffered, clear water, the inactivation rate of E. faecalis was 7 times greater than that of E. coli in air-saturated pond water at circumneutral pH due to its susceptibility to exogenous sensitizers and longer wavelengths. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Absolute Density Calibration Cell for Laser Induced Fluorescence Erosion Rate Measurements

    Science.gov (United States)

    Domonkos, Matthew T.; Stevens, Richard E.

    2001-01-01

    Flight qualification of ion thrusters typically requires testing on the order of 10,000 hours. Extensive knowledge of wear mechanisms and rates is necessary to establish design confidence prior to long duration tests. Consequently, real-time erosion rate measurements offer the potential both to reduce development costs and to enhance knowledge of the dependency of component wear on operating conditions. Several previous studies have used laser-induced fluorescence (LIF) to measure real-time, in situ erosion rates of ion thruster accelerator grids. Those studies provided only relative measurements of the erosion rate. In the present investigation, a molybdenum tube was resistively heated such that the evaporation rate yielded densities within the tube on the order of those expected from accelerator grid erosion. This work examines the suitability of the density cell as an absolute calibration source for LIF measurements, and the intrinsic error was evaluated.

  12. Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

    Science.gov (United States)

    George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

  13. Reactive hydroxyl radical-driven oral bacterial inactivation by radio frequency atmospheric plasma

    International Nuclear Information System (INIS)

    Kang, Sung Kil; Lee, Jae Koo; Choi, Myeong Yeol; Koo, Il Gyo; Kim, Paul Y.; Kim, Yoonsun; Kim, Gon Jun; Collins, George J.; Mohamed, Abdel-Aleam H.

    2011-01-01

    We demonstrated bacterial (Streptococcus mutans) inactivation by a radio frequency power driven atmospheric pressure plasma torch with H 2 O 2 entrained in the feedstock gas. Optical emission spectroscopy identified substantial excited state OH generation inside the plasma and relative OH formation was verified by optical absorption. The bacterial inactivation rate increased with increasing OH generation and reached a maximum 5-log 10 reduction with 0.6%H 2 O 2 vapor. Generation of large amounts of toxic ozone is drawback of plasma bacterial inactivation, thus it is significant that the ozone concentration falls within recommended safe allowable levels with addition of H 2 O 2 vapor to the plasma.

  14. Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.

    Science.gov (United States)

    Tham, Daniel Kai Long; Moukhles, Hakima

    2017-07-03

    Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

  15. Development of a moderate rate lithium/thionyl-chloride D'' cell

    Energy Technology Data Exchange (ETDEWEB)

    Cieslak, W.R.; Street, H.K.

    1990-01-01

    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 cm{sup 2} of active electrode area, 1.0 M LiAlCl{sub 4} electrolyte that may have SO{sub 2} 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{degree}C and 6.25 {Omega} has been 14.9 Ah (50 Wh). We have aged the cells at 30{degree}C and 50{degree}C, and measured complex impedance and microcalorimetry during the aging period. The cells have been discharged after the aging period at 25{degree}C and 0{degree}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 refs., 6 figs.

  16. Red Blood Cell Hematocrit Influences Platelet Adhesion Rate in a Microchannel

    Science.gov (United States)

    Spann, Andrew; Campbell, James; Fitzgibbon, Sean; Rodriguez, Armando; Shaqfeh, Eric

    2014-11-01

    The creation of a blood clot to stop bleeding involves platelets forming a plug at the site of injury. Red blood cells indirectly play a role in ensuring that the distribution of platelets across the height of the channel is not uniform - the contrast in deformability and size between platelets and red blood cells allows the platelets to preferentially marginate close to the walls. We perform 3D boundary integral simulations of a suspension of platelets and red blood cells in a periodic channel with a model that allows for platelet binding at the walls. The relative rate of platelet activity with varying hematocrit (volume fraction of red blood cells) is compared to experiments in which red blood cells and platelets flow through a channel coated with von Willebrand factor. In the simulations as well as the experiments, a decrease in hematocrit of red blood cells is found to reduce the rate at which platelets adhere to the channel wall in a manner that is both qualitatively and quantitatively similar. We conclude with a discussion of the tumbling and wobbling motions of platelets in 3D leading up to the time at which the platelets bind to the wall. Funded by Stanford Army High Performance Computing Research Center, experiments by US Army Institute of Surgical Research.

  17. Photodynamic inactivation of contaminated blood with Staphylococcus aureus

    Science.gov (United States)

    Corrêa, Thaila Q.; Inada, Natalia M.; Pratavieira, Sebastião.; Blanco, Kate C.; Kurachi, Cristina; Bagnato, Vanderlei S.

    2016-03-01

    The presence of bacteria in the bloodstream can trigger a serious systemic inflammation and lead to sepsis that cause septic shock and death. Studies have shown an increase in the incidence of sepsis over the years and it is mainly due to the increased resistance of microorganisms to antibiotics, since these drugs are still sold and used improperly. The bacterial contamination of blood is also a risk to blood transfusions. Thus, bacteria inactivation in blood is being studied in order to increase the security of the blood supply. The purpose of this study was to decontaminate the blood using the photodynamic inactivation (PDI). Human blood samples in the presence of Photogem® were illuminated at an intensity of 30 mW/cm2, and light doses of 10 and 15 J/cm2. Blood counts were carried out for the quantitative evaluation and blood smears were prepared for qualitative and morphological evaluation by microscopy. The results showed normal viability values for the blood cells analyzed. The light doses showed minimal morphological changes in the membrane of red blood cells, but the irradiation in the presence of the photosensitizer caused hemolysis in red blood cells at the higher concentrations of the photosensitizer. Experiments with Staphylococcus aureus, one of the responsible of sepsis, showed 7 logs10 of photodynamic inactivation with 50 μg/mL and 15 J/cm2 and 1 log10 of this microorganism in a co-culture with blood.

  18. Modeling the effect of marination and temperature on Salmonella inactivation during drying of beef jerky.

    Science.gov (United States)

    Yoon, Yohan; Geornaras, Ifigenia; Kendall, Patricia A; Sofos, John N

    2009-01-01

    This study modeled the effect of drying temperature in combination with predrying marination treatments to inactivate Salmonella on beef jerky. Beef inside round slices were inoculated with Salmonella and treated with (1) nothing (C), (2) traditional marinade (M), or (3) dipped into a 5% acetic acid solution for 10 min before exposure to M (AM). After 24 h of marination at 4 degrees C, samples were dehydrated at 52, 57, or 63 degrees C. Total counts (tryptic soy agar supplemented with 0.1% sodium pyruvate, TSAP) and Salmonella (XLD agar) were enumerated after inoculation and at 0, 2, 4, 6, 8, and 10 h during drying. For calculation of death rates (DR, log CFU/cm(2)/h), shoulder period (h), low asymptote, and upper asymptote, cell counts from TSAP were fitted to the Baranyi model. The DRs were then further expressed as a function of storage temperature. Inactivation occurred without an initial lag phase (shoulder period), while correlation (R(2)) values of fitted curves were >/= 0.861. The DRs of C (-0.29 to -0.62) and M (-0.36 to -0.63) treatments were similar, while DRs of the AM treatment were higher (-1.22 to -1.46). The DRs were then fitted to a polynomial equation as a function of temperature. After validation, good (C and M) or acceptable (AM) model performances were observed (R(2)= 0.954 to 0.987; bias factors: 1.03 [C], 1.01 [M], 0.71 [AM]; accuracy factors: 1.05 [C], 1.06 [M], 1.41 [AM]). The developed models may be useful in selecting drying temperatures and times in combination with predrying treatments for adequate inactivation of Salmonella in beef jerky.

  19. Inactivation Effect of Antibiotic-Resistant Gene Using Chlorine Disinfection

    Directory of Open Access Journals (Sweden)

    Takashi Furukawa

    2017-07-01

    Full Text Available The aim of this study was to elucidate the inactivation effects on the antibiotic-resistance gene (vanA of vancomycin-resistant enterococci (VRE using chlorination, a disinfection method widely used in various water treatment facilities. Suspensions of VRE were prepared by adding VRE to phosphate-buffered saline, or the sterilized secondary effluent of a wastewater treatment plant. The inactivation experiments were carried out at several chlorine concentrations and stirring time. Enterococci concentration and presence of vanA were determined. The enterococci concentration decreased as chlorine concentrations and stirring times increased, with more than 7.0 log reduction occurring under the following conditions: 40 min stirring at 0.5 mg Cl2/L, 20 min stirring at 1.0 mg Cl2/L, and 3 min stirring at 3.0 mg Cl2/L. In the inactivation experiment using VRE suspended in secondary effluent, the culturable enterococci required much higher chlorine concentration and longer treatment time for complete disinfection than the cases of suspension of VRE. However, vanA was detected in all chlorinated suspensions of VRE, even in samples where no enterococcal colonies were present on the medium agar plate. The chlorine disinfection was not able to destroy antibiotic-resistance genes, though it can inactivate and decrease bacterial counts of antibiotic-resistant bacteria (ARB. Therefore, it was suggested that remaining ARB and/or antibiotic-resistance gene in inactivated bacterial cells after chlorine disinfection tank could be discharged into water environments.

  20. Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds

    Science.gov (United States)

    Madani, Navid; Princiotto, Amy M.; Zhao, Connie; Jahanbakhshsefidi, Fatemeh; Mertens, Max; Herschhorn, Alon; Melillo, Bruno; Smith, Amos B.

    2016-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility. IMPORTANCE The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important

  1. Living with two X chromosomes: of mice and women : studies on the initiation mechanisms of X chromosome inactivation in stem cells and mouse models, and the role of RNF12 herein

    NARCIS (Netherlands)

    T.S. Barakat (Tahsin Stefan)

    2012-01-01

    markdownabstract__Abstract__ In this thesis work, we have investigated mechanisms involved in regulation of the initiation of X chromosome inactivation (XCI). Starting point was our earlier hypothesis that the initiation of XCI is a stochastic process, controlled in trans by autosomally-encoded

  2. How the growth rate of host cells affects cancer risk in a deterministic way

    Science.gov (United States)

    Draghi, Clément; Viger, Louise; Denis, Fabrice; Letellier, Christophe

    2017-09-01

    It is well known that cancers are significantly more often encountered in some tissues than in other ones. In this paper, by using a deterministic model describing the interactions between host, effector immune and tumor cells at the tissue level, we show that this can be explained by the dependency of tumor growth on parameter values characterizing the type as well as the state of the tissue considered due to the "way of life" (environmental factors, food consumption, drinking or smoking habits, etc.). Our approach is purely deterministic and, consequently, the strong correlation (r = 0.99) between the number of detectable growing tumors and the growth rate of cells from the nesting tissue can be explained without evoking random mutation arising during DNA replications in nonmalignant cells or "bad luck". Strategies to limit the mortality induced by cancer could therefore be well based on improving the way of life, that is, by better preserving the tissue where mutant cells randomly arise.

  3. Spontaneous mutation rate in Chinese hamster cell clones differing in UV-sensitivity

    International Nuclear Information System (INIS)

    Manuilova, E.S.; Bagrova, A.M.; Moskovskij Gosudarstvennyj Univ.

    1983-01-01

    The spontaneous rate of appearance of mutations to 6-mercaptopurine (6 MP) resistence in the cells of CHR2 and CHs2 clones dofferent in sensitivity to lethal and matagenous effect of UV-rays, is investigated. Increased UV-sensitivity of CHs2 clone is caused by the violation of postreplicative DNA reparation. It is established that the purity of spontaneously occuring mutations in both clones turns out to be similar, i.e. (1.5-1.8)x10 -5 for the cell pergeneration. It is shown that the effect of postreplicative DNA reparation in the cells of chinese hamster is not connected with the increase of spontaneous mutation ability. The problem on the possible role of reparation in the mechanism of appearance of spontaneous and induced mutations in the cells of Chinese hamster with increased UV-sensitivity is discussed

  4. Importance of dose-rate and cell proliferation in the evaluation of biological experimental results

    Science.gov (United States)

    Curtis, S. B.

    1994-01-01

    The nuclei of cells within the bodies of astronauts traveling on extended missions outside the geomagnetosphere will experience single traversals of particles with high Linear Energy Transfer (LET) (e.g., one iron ion per one hundred years, on average) superimposed on a background of tracks with low LET (approximately one proton every two to three days, and one helium ion per month). In addition, some cell populations within the body will be proliferating, thus possibly providing increasing numbers of cells with 'initiated' targets for subsequent radiation hits. These temporal characteristics are not generally reproduced in laboratory experimental protocols. Implications of the differences in the temporal patterns of radiation delivery between conventionally designed radiation biology experiments and the pattern to be experienced in space are examined and the importance of dose-rate and cell proliferation are pointed out in the context of radiation risk assessment on long mission in space.

  5. Viral inactivation in hemotherapy: systematic review on inactivators with action on nucleic acids

    Directory of Open Access Journals (Sweden)

    Patricia Marial Sobral

    2012-01-01

    Full Text Available The aim of this study was to conduct a systematic review on the photoinactivators used in hemotherapy, with action on viral genomes. The SciELO, Science Direct, PubMed and Lilacs databases were searched for articles. The inclusion criterion was that these should be articles on inactivators with action on genetic material that had been published between 2000 and 2010. The key words used in identifying such articles were "hemovigilance", "viral inactivation", "photodynamics", "chemoprevention" and "transfusion safety". Twenty-four articles on viral photoinactivation were found with the main photoinactivators covered being: methylene blue, amotosalen HCl, S-303 frangible anchor linker effector (FRALE, riboflavin and inactin. The results showed that methylene blue has currently been studied least, because it diminishes coagulation factors and fibrinogen. Riboflavin has been studied most because it is a photoinactivator of endogenous origin and has few collateral effects. Amotosalen HCl is effective for platelets and is also used on plasma, but may cause changes both to plasma and to platelets, although these are not significant for hemostasis. S-303 FRALE may lead to neoantigens in erythrocytes and is less indicated for red-cell treatment; in such cases, PEN 110 is recommended. Thus, none of the methods for pathogen reduction is effective for all classes of agents and for all blood components, but despite the high cost, these photoinactivators may diminish the risk of blood-transmitted diseases.

  6. Development of methods to measure virus inactivation in fresh waters.

    OpenAIRE

    Ward, R L; Winston, P E

    1985-01-01

    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