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Sample records for suspension cultures cold

  1. Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism.

    Directory of Open Access Journals (Sweden)

    Gesine Pless-Petig

    Full Text Available BACKGROUND & AIMS: Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells. METHODS: Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production of adherent cells were assessed. RESULTS: Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage. CONCLUSION: In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

  2. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    of growth regulators were observed to be 3 × 10−6M indoleacetic acid (JAA) combined with 3 × 10−6M benzylaminopurin (BAP) or 10−6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further....... Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  3. Regeneration of soybean via embryogenic suspension culture

    Directory of Open Access Journals (Sweden)

    Droste Annette

    2001-01-01

    Full Text Available In an attempt to establish an alternative plant regeneration system for soybean [Glycine max (L. Merrill] cultivars used in Brazilian breeding programs, ten genotypes were tested for their embryogenic potential. Cotyledons were removed as explants from immature seeds harvested from field-grown plants. After 45 days on induction medium, the number of responding cotyledons and the number of somatic embryos per immature cotyledon were evaluated. The percentage of explants that produced somatic embryos varied from 1 to 70% among cultivars. The average number of somatic embryos produced per cotyledon pair ranged from 0.01 to 10.3 with a mean of 3.4. Suspension cultures were initiated with three Agrobacterium tumefaciens susceptible cultivars. Suspensions were successfully developed from Bragg and IAS5 cultivars. The packed cell volume, in one-month growth, increased 8.1 fold for Bragg and 3.5 fold for IAS5 and the fresh weight increased 6.6 and 2.8 fold, respectively. The cultivars differed for the analysed parameters. All tissue from each cultivar was transferred to the maturation medium and subsequently to the germination medium. The germination frequency was 45.7 and 54.9% for Bragg and IAS5, respectively. Plants were gradually exposed to ambient humidity over one week and then planted in soil. All plants yielded seeds in the greenhouse.

  4. Phosphatidylinositol species of suspension cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Heim, S.; Wagner, K.G.

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with (/sup 3/H)inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different /sup 3/H-labeled species by autoradiography. The ratio of (/sup 3/H)inositol incorporation into PI, PIP and PIP/sub 2/ was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound.

  5. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. ... Key words: Sorghum, proteomics, callus, cell suspension cultures, total soluble protein, secretome. INTRODUCTION ..... system, are dynamic and heterogeneous, being com- posed of a ...

  6. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...

  7. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

    Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

    2000-01-01

    Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

  8. Stimulation of taxane production in suspension cultures of Taxus ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-06-17

    Jun 17, 2008 ... biosynthesis in transformed root cultures of Datura stramonium. Phytochemistry 50: 53-56. Zhang CH, Fevereiro PS, He G, Chen Z (2007). Enhanced paclitaxel productivity and release capacity of Taxus chinensis cell suspension cultures adapted to chitosan. Plant Sci. 172: 158-163. Zhang CH, Wu JY, He ...

  9. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... plant growth regulators on the callus induction and accumulation of condensed tannins, and (iii) determine the optimum medium and the hormone combination for cell suspension culture of E. angustifolia. This paper presents the feasibility of condensed tannins production in callus and cell culture of E.

  10. Abscisic acid induced freezing tolerance in chilling-sensitive suspension cultures and seedlings of rice

    Science.gov (United States)

    2013-01-01

    Background The role of abscisic acid (ABA) as a possible activator of cold acclimation process was postulated since endogenous levels of ABA increase temporarily or constitutively during cold-hardening. Exogenous application of ABA has been known to induce freezing tolerance at ambient temperatures in in vitro systems derived from cold hardy plants. Yet, some cell cultures acquired much greater freezing tolerance by ABA than by cold whilst maintaining active growth. This raises questions about the relationships among ABA, cold acclimation and growth cessation. To address this question, we attempted to 1) determine whether exogenous ABA can confer freezing tolerance in chilling-sensitive rice suspension cells and seedlings, which obviously lack the mechanisms to acquire freezing tolerance in response to cold; 2) characterize this phenomenon by optimizing the conditions and compare with the case of cold hardy bromegrass cells. Results Non-embryogenic suspension cells of rice suffered serious chilling injury when exposed to 4°C. When incubated with ABA at the optimal conditions (0.5-1 g cell inoculum, 75 μM ABA, 25-30°C, 7–10 days), they survived slow freezing (2°C/h) to −9.0 ~ −9.3°C (LT50: 50% killing temperature) while control cells were mostly injured at −3°C (LT50: -0.5 ~ −1.5°C). Ice-inoculation of the cell suspension at −3°C and survival determination by regrowth confirmed that ABA-treated rice cells survived extracellular freezing at −9°C. ABA-induced freezing tolerance did not require any exposure to cold and was best achieved at 25-30°C where the rice cells maintained high growth even in the presence of ABA. ABA treatment also increased tolerance to heat (43°C) as determined by regrowth. ABA-treated cells tended to have more augmented cytoplasm and/or reduced vacuole sizes compared to control cultures with a concomitant increase in osmolarity and a decrease in water content. ABA-treated (2–7 days) in vitro grown

  11. Biotransformation of Dydrogesterone by Cell Suspension Cultures of Azadirachta indica

    OpenAIRE

    KHAN, Saifullah; CHOUDHARY, Muhammad Iqbal

    2008-01-01

    Biotransformation of dydrogesterone (1) by using cell suspension cultures of Azadirachta indica yielded a metabolite 20R-hydroxy-9b ,10a-pregna-4,6-diene-3-one (2). The structure of this compound was deduced on the basis of various spectroscopic techniques.

  12. SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

    Science.gov (United States)

    This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

  13. Regeneration from embryogenic callus and suspension cultures of ...

    African Journals Online (AJOL)

    Somatic embryogenesis and plant regeneration from both callus and suspension cultures of the wild medicinal plant Cymbopogon schoenanthus subsp. proximus has been achieved. The species is rare and confined in its distribution to Africa. A range (0.5 to 8 mg/l) of 2,4-dichlorophenoxyacetic acid (2,4-D) for the induction ...

  14. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of E. angustifolia was ...

  15. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica. 113. J. Biosci. 33(1), March 2008. 1. Introduction. Chemical pesticides, once considered a boon for increasing the yield of food crops, have now become a bane owing to the numerous cases of pesticide poisoning. Alternative pesticides ...

  16. In vitro plant regeneration from embryogenic cell suspension culture ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... In vitro plant regeneration was achieved from embryogenic cell suspension culture of Astragalus chrysochlorus. When 30-day-old aseptically ... previous study, cytotoxic activities of stem and root ex-. *Corresponding author. E-mail: ... For callus induction, 30-day-old mesocotyl parts of seedlings were used.

  17. The characterisation of foaming in suspension cultures of morinda citrifolia

    OpenAIRE

    Cusack, Winifred (Úna)

    1998-01-01

    The characterisation of foaming in plant cell cultures was investigated using Monnda citnfoha, as a test organism Suspensions were cultivated in 250 ml shake flasks, over the course of 21 day batch growth cycles, and were characterised in terms of biomass concentration, conductivity, morphology, pH and metabolite production (extracellular proteins and extracellular polysaccharides (ECP)). The Theological and surface tension profiles of the cell-free broths were assessed, with a view to unders...

  18. Putting the spotlight back on plant suspension cultures

    Directory of Open Access Journals (Sweden)

    Rita B. Santos

    2016-03-01

    Full Text Available Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso® – the first licensed recombinant pharmaceutical protein derived from plants – is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plants cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

  19. Study on enzymatic browning in suspension cultures of licorice cells

    Directory of Open Access Journals (Sweden)

    Yali Li

    2016-03-01

    Full Text Available Enzymatic browning is one of the main obstacles encountered in the establishment of suspension systems of licorice cells. Browning of cells may result in decreased viability, poor growth and even death. The present study investigated the mechanism of browning reactions and the effective controlling methods. The results showed that the cell viability and membrane permeabilization obviously changed when the cells were transferred to liquid medium. The transformation caused rapid increase in the levels of polyphenol oxidase activity and in the production of polyphenols. Osmotic and hydrodynamic stresses arising from liquid culture were regarded as the major causes of enzymatic browning. Ascorbic acid and L-cysteine were found to be the most significant anti-browning agents that could decrease the degree of browning with 55.8% and 52.2%, respectively, at the end of the suspension culture's lag phase. When cultured with a cycle of 21 days, the maximum biomass of the cells cultured with ascorbic acid and L-cysteine increased with 31.1% and 26.5%, respectively, when compared to the control. These findings may be essential for the development of licorice cell cultures devoted to browning prevention and cell viability maintaining.

  20. Gas phase composition effects on suspension cultures of Taxus cuspidata

    Energy Technology Data Exchange (ETDEWEB)

    Mirjalili, N.; Linden, J.C. [Colorado State Univ., Fort Collins, CO (United States). Dept. of Chemical and Bioresources Engineering

    1995-10-20

    The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production of taxol. High carbon dioxide concentration (10% v/v) inhibited taxol production.The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown under ambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production.

  1. Isolation and culture of Celosia cristata L cell suspension protoplasts

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    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  2. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

  3. Multiple-dose pharmacokinetics and safety of an ibuprofen-pseudoephedrine cold suspension in children.

    Science.gov (United States)

    Gelotte, Cathy K; Prior, Mary Jane; Pendley, Charles; Zimmerman, Brenda; Lavins, Bernard J

    2010-07-01

    Two studies were conducted to characterize multiple-dose pharmacokinetics and potential drug interactions of ibuprofen and pseudoephedrine combined in a suspension and to evaluate safety of this combination in children with common cold, flu, or sinusitis. In the pharmacokinetic study, 24 healthy children aged 4-11 years were administered ibuprofen -pseudoephedrine suspension at 7.5 and 1.125 mg/kg, respectively, every 6 hours for 5 doses. Serial blood samples were drawn over 6 hours after final dose for assessment of steady-state pharmacokinetics. In the open-label, multicenter safety study, more than 100 children aged 2-11 years experiencing symptomatic rhinitis were enrolled. Ibuprofen -pseudoephedrine suspension was administered as needed at similar mg/kg doses every 6-8 hours for up to 3 days. Subjects enrolled in the pharmacokinetic study showed no accumulation of either drug; their weight-adjusted clearances were independent of age, and results were comparable with those from previous single-ingredient studies. For ibuprofen, oral clearance (Cl/F) was 77.5 + or - 16.4 mL/kg/h and volume of distribution (Vd/F) was 0.147 + or - 0.037 L/kg. For pseudoephedrine, Cl/F was 12.3 + or - 2.2 mL/kg/min and Vd/F was 2.52 + or - 0.47 L/kg. In the safety study, adverse events were reported for 18.4% of subjects; most were mild to moderate intensity. There was little difference in incidence of adverse events among different age and weight groups. In conclusion, administration of combined ibuprofen and pseudoephedrine in children demonstrated similar pharmacokinetics when compared with reports of the pharmacokinetics for the single-ingredient products, consistent with no apparent drug interactions. The combination suspension was generally well tolerated.

  4. Chemical stability of azacitidine suspensions for injection after cold-chain reconstitution of powder and storage.

    Science.gov (United States)

    Legeron, Rachel; Xuereb, Fabien; Djabarouti, Sarah; Saux, Marie-Claude; Breilh, Dominique

    2013-12-01

    The 72-hour chemical stability of refrigerated syringes of azacitidine suspension prepared via a cold-chain method is investigated. Three 25-mg/mL azacitidine suspensions were prepared from different lots of powdered drug. The suspensions were stored in 2-mL polypropylene syringes at 2-8 °C and protected from light. The concentrations of azacitidine and the mean area under the concentration-time curve (AUC) values for its degradation products were determined after 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours using high-performance liquid chromatography with ultraviolet detection. The degradation process was slow during the first 48 hours and then accelerated. During the first 48 hours of storage, 4.23% of the azacitidine was lost relative to the mean concentration measured at time zero, which complied with International Conference on Harmonisation (ICH) guidance specifying a maximum change of 5% from the initial measured value. Two degradation products were present immediately after syringe preparation; N-formylribosylguanylurea formed rapidly (as indicated by a 35.62% increase from the baseline AUC in the first 12 hours), whereas ribosylguanylurea formation occurred more slowly (a 7.69% mean increase from the baseline AUC at 12 hours) but then rapidly accelerated. The study results indicate that properly prepared azacitidine syringes for injection can be administered up to two days later while maintaining conformance with ICH stability standards. Azacitidine 25-mg/mL suspensions reconstituted with refrigerated water (2-8 °C) and stored in propylene syringes were chemically stable during the first 48 hours when stored protected from light at 2-8 °C.

  5. Physiological responses of suspension cultures of Catharanthus roseus to aluminum: changes in polyamines and inorganic ions

    Science.gov (United States)

    Xinhua Zhou; Rakesh Minocha; Subhash C. Minocha

    1995-01-01

    The effects of aluminum (Al) treatment on polyamines were studied using suspension cultures of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. The addition of A1 (0.2, 0.5, 1.0 mM) to the suspension cultures caused a significant increase in putrescine within 24h only in freshly transferred cells. By contrast, Al treatment reduced putrescine...

  6. Establishment of callus, cell suspension and shoot cultures of Leonurus cardiaca L. and diterpene analysis.

    Science.gov (United States)

    Knöss, W

    1995-10-01

    Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.

  7. Isolation and culture of suspension protoplasts of vetiver

    Directory of Open Access Journals (Sweden)

    Somporn Prasertsongskun

    2005-05-01

    Full Text Available In this research, protoplasts were isolated from cell suspension derived from inflorescence of vetiver (Vetiveria zizanioides Nash Surat Thani germplasm. The optimum condition for protoplast isolation was established by using 2% cellulase Onozuka R10, 2% macerozyme R10, 0.5% pectinase in 0.4 M mannitol and 7 mM CaCl2.2H2O at pH 5.8 and incubated for 10 hours in the dark on the rotary shaker at 50 rpm. Maximum protoplast yields were 8.4 × 104 protoplasts/ml PCV. Division of protoplasts was observed only in liquid medium. The first cell division was observed after 3 days of culture initiation, and the average division was 5.0% in the N6 medium supplemented with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid and 0.5 mg/l BA (Benzyladenine. An optimal density for culture division was 1 × 105 protoplasts/ml.

  8. [Effects of fungal elicitor on inophyllums production in suspension cultured cells of Calophyllum inophyllum L].

    Science.gov (United States)

    Luo, Huan-liang; Guo, Yong; Cui, Tang-bing; Dai, Jian-guo; Zhang, Jun-song; Xu, Bai-qiu

    2004-04-01

    To investigate the effects of fungal elicitors on inophyllums production in suspension cultured cell of Calophyllum inophyllum Linn. The pathogen of leaf spot disease of C. inophyllum L. was isolated and prepared as fungal elicitor. The fungal elicitor was added to the medium with different concentrations and culture period. Their effects on biomass and inophyllums content of the suspension of cultured cells were studied. The optimum effects of S-I fungal elicitor concentrations on inophyllums content was 60 mg GE x L(-1). Adding the fungi elicitor into the cell suspension culture system at stationary phase (being cultured for 18 days) resulted in a highest inophyllum content of 59.174 mg x L(-1) at the 3rd day with 27% higher than control. Fungal elicitor treatment promoted the inophyllums accumulation in medium. Adding the Stagonospora curtisii (Berk.) Sacc. to the medium was effective approaches to enhance inophyllums yield in the suspension of C. inophyllum L culture cell.

  9. Nitration of plant apoplastic proteins from cell suspension cultures.

    Science.gov (United States)

    Szuba, Agnieszka; Kasprowicz-Maluśki, Anna; Wojtaszek, Przemysław

    2015-04-29

    Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long

  10. Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

    Science.gov (United States)

    Natori, Tomomi; Fujiyoshi, Masachika; Uchida, Masashi; Abe, Natsuki; Kanaki, Tatsuro; Fukumoto, Yasunori; Ishii, Itsuko

    2017-03-01

    The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.

  11. Diacylglycerol Kinase from Suspension Cultured Plant Cells 1

    Science.gov (United States)

    Wissing, Josef B.; Wagner, Karl G.

    1992-01-01

    Diacylglycerol kinase (adenosine 5′-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5′-triphosphate and guanosine 5′-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane. PMID:16668739

  12. Metabolism of strobilurins by wheat cell suspension cultures.

    Science.gov (United States)

    Myung, Kyung; Williams, Daniel A; Xiong, Quanbo; Thornburgh, Scott

    2013-01-09

    Strobilurin fungicides are a leading class of antifungal chemicals used today in agricultural applications. Although degradation of some strobilurin fungicides has been assessed in plant residues, little information has appeared in the literature concerning the rates of metabolism of these fungicides in plants. In this study, we explored plant metabolism of three strobilurin fungicides, azoxystrobin, kresoxim-methyl, and trifloxystrobin, using wheat cell suspension cultures. Trifloxystrobin and kresoxim-methyl were completely metabolized within 24 h, whereas the metabolism of azoxystrobin was relatively slow with half-lives up to 48 h depending on specific experimental conditions. Metabolic rates of these fungicides were affected by the amounts of compound and cells added to the media. Structural analysis of metabolites of trifloxystrobin and kresoxim-methyl by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR) indicated that trifloxystrobin was first demethylated followed by subsequent hydroxylation, whereas kresoxim-methyl was largely demethylated. In contrast, a number of minor metabolites of azoxystrobin were present suggesting a differential metabolism of strobilurins by wheat cells.

  13. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...

  14. Soviet Cultural Diplomacy in Denmark during the Cold War

    DEFF Research Database (Denmark)

    Frederichsen, Kim

    2012-01-01

    The article discusses the Soviet apparatus for cultural diplomacy abroad during the Cold War period using the worlds oldest society for friendship with the Soviet Union as a case study. The article looks at question from 3 diffrent angels: 1: Organisation, planning and financing. 2: Activities. 3...

  15. Cold Storage of Plant Tissue Cultures – Effect of Sucrose

    Directory of Open Access Journals (Sweden)

    A.G. Enikeev

    2016-11-01

    Full Text Available Explants of normal (non-transformed carrot (Daucus carota L. tissue, scorzonera crown gall (Scorzonera hispanica L. and carrot tissues transformed with Agrobacterium tumefaciens (all cultured in vitro were planted in little glass jars with 6 cm3 of nutrition media containing 3, 5, 7 or 9% sucrose and maintained at +3 °C for 6, 8, 10 and 12 months. Viability of tissue cultures after cold storage was estimated by the degree of tissue necrosis, per cent of explants exhibiting growth resumption and intensity of the resumpted growth. Little or no growth was observed during cold storage of scorzonera and normal carrot tissues, whereas transformed carrot tissues showed significant growth. Dry matter content of tissues increased with an increase of sucrose concentration in the medium and failed to change during cold storage of scorzonera and normal carrot tissues. The dry matter content of the transformed carrot tissues fell down during cold storage due to the increase of fresh weight. The viability of all tissue cultures was preserved better and for a longer time with 7% sucrose, than with 3, 5 and 9%. Transformed carrot and tobacco callus tissues preserved cell viability better than non-transformed ones. Cycles consisting from long subcultures (several months at 3 °C interrupted by short subcultures (several weeks at 26 °C were applied in order to storage of tissue cultures for several years.

  16. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris

    NARCIS (Netherlands)

    Koulman, A; Kubbinga, M.E.; Batterman, S; Woerdenbag, H.J.; Pras, N.; Woolley, J.G.; Quax, Wim

    2003-01-01

    In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which

  17. Development of friable embryogenic callus and embryogenic suspension culture systems in cassava (Manihot esculenta Crantz)

    Science.gov (United States)

    Taylor, N J; Edwards, M; Kiernan, R J; Davey, C D; Blakesley, D; Henshaw, G G

    1996-06-01

    Procedures for the production of a new and highly prolific embryogenic culture system have been developed in cassava. The importance of the basal salts and type of auxin in controlling the development of cassava embryogenic tissues has been demonstrated, with culture on Gresshoff and Doy basal medium in the presence of 4-amino-3,5,6,trichloro-picolinic acid (picloram) inducing the formation of friable embryogenic callus from which highly totipotent embryogenic suspension cultures could be established. Plants have been regenerated from these cultures. The availability of embryogenic suspension cultures is considered to have important implications for the application of genetic transformation and other biotechnologies in the agronomic improvement of cassava.

  18. Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

    Directory of Open Access Journals (Sweden)

    Sutini Sutini

    2017-02-01

    Full Text Available This study was aimed to assess flavan-3-ol biomass in C. sinensis through callus cultures and suspension cultures derived from leaf explants. Callus initiation of both cultures were using Murashige and Skoog medium were enriched with plant growth regulators Naphtha-lene Acetic Acid 3.0 mg/L and kinetin 2.0 mg/L. The procedures in this study were: (1 callus initiation by cutting the leaves of C. sinen-sis shoots then planted on Murashige and Skoog medium that were enriched with plant growth regulators, (2 sub callus culture on fresh medium that enriched with the same growth regulators, (3 suspension culture initiation of liquid callus, (4 growth examination of callus and suspension cultures in week 12, (5 examination of qualitative-quantitative content of flavan-3-olin suspension cultures at week 4. The results show that suspension cultures contain biomass flavan-3-ol that increase in the same manner of the increase of callus age and weight

  19. Isolation and culture of protoplasts of Ma-phut (Garcinia dulcis derived from cell suspension culture

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2008-09-01

    Full Text Available Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS medium containing 3% sucrose,1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l benzyladenine (BA and 500 mg/l polyvinylpyrrolidone (PVP, was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4oC. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10 in combination with 1% macerozyme R-10 gave an optimum result in both yield and viability of protoplasts at 5.7x106/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x105/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%. The first division of the protoplasts was obtained after 2 days of culture and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callusformation and plantlet regeneration was not obtained.

  20. History of artificial cold, scientific, technological and cultural issues

    CERN Document Server

    2014-01-01

    The history of artificial cold has been a rather intriguing interdisciplinary subject (physics, chemistry, technology, sociology, economics, anthropology, consumer studies) which despite some excellent monographs and research papers, has not been systematically exploited. It is a subject with all kinds of scientific, technological as well as cultural dimensions. For example, the common home refrigerator has brought about unimaginably deep changes to our everyday lives changing drastically eating habits and shopping mentalities. From the end of the 19th century to the beginning of the 21st, issues related to the production and exploitation of artificial cold have never stopped to provide us with an incredibly interesting set of phenomena, novel theoretical explanations, amazing possibilities concerning technological applications and all encompassing cultural repercussions. The discovery of the unexpected and “bizarre” phenomena of superconductivity and superfluidity, the necessity to incorporate macroscopi...

  1. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate:ammonium showed that the ratio 4:1 revealed a profound effect, ...

  2. Establishment of embryogenic suspension cultures of Pinus radiata ...

    African Journals Online (AJOL)

    The development of embryonal suspensor mass (ESM) from immature embryos of Pinus radiata on a solidified growth medium containing 0, 5 mgl -1 benzyladenine, 3, 0 mgl -1 2, 4-dichlorophenoxyacetic acid, 500 mgl -1 casein hydrolysate and 250 mgl -1 L-glutamine was used as inoculum to establish cell suspension ...

  3. Ultrastructure of paraquat-treated pine cells (Pinus elliottii Engelm. ) in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Birchem, R.; Henk, W.G.; Brown, C.L.

    1979-01-01

    The ultrastructure of liquid suspension cultures of Pinus elliottii was studied, noting characteristics of dividing and senescent cells. The cultures were treated with 0.01, 0.1, 1.0 and 10.0 mg 1/sup -1/ paraquat, an herbicide which stimulates oleoresin synthesis and resinosis in the xylem of treated pine trees. The ultrastructural effects of the toxin were studied at each paraquat concentration over a period of 24 days. The ultrastructural observations are correlated with physiological studies in suspension culture and in living trees.

  4. The use of morphogenic suspension cultures for the development of a protoplast regeneration system in lily

    NARCIS (Netherlands)

    Famelaer, L.; Bordas, M.; Baliu', E.; Ennik, E.; Meijer, H.; Tuyl, van J.M.; Creemers-Molenaar, J.

    1997-01-01

    The present study reports data on the development of a protoplast regeneration procedure in lily. Established morphogenic suspension cultures were obtained from callus cultures induced on mature embryos from crosses between cultivars of L. longiflorum. The effect on the frequency of protoplast

  5. Growth characteristics and nutrient depletion of Miscanthus x ogiformis Honda 'Giganteus' suspension cultures

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted

    1998-01-01

    The growth characteristics and nutrient depletion in suspension cultures of Miscanthus ogiformis Honda ‘Giganteus' grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from...

  6. Production of secondary metabolites trimethyl xanthina by Camellia sinensis L suspension culture

    Science.gov (United States)

    Sutini, Sodiq, Mochamad; Muslihatin, Wirdhatul; Indra, Mochamad Rasjad

    2017-06-01

    Bioactive trimethyl xanthina can be obtained from the plant Camellia sinensis L. To obtain bioactive plant of which there are several hurdles for instance to wait up to five years to be harvested, also it needs land at a certain height from the sea level. Therefore, the production of secondary metabolites trimethyl xanthina need to be developed with suspense culture techniques. The purpose of this study obtained the production of bioactive trimethyl xanthina way culturally suspense in large scale with a relatively short time, potentially as anti-oxidants. Research methods include: (1) initiation of callus from pieces of leaves, shoots the youngest of the plant Camellia sinensis L in the media MS with the optimization of the addition of growth regulators, (2) the subculture of callus on media and plant growth regulator that is equal to the stage of initiation, (3) initiation of suspension culture using explants of callus Camellia sinensis L, (4) Analysis of secondary metabolites trimethyl xanthina growth in suspension culture, (5) the isolation and identification of trimethyl xanthina qualitatively and quantitatively using thin layer chromatography/high performance chromatography column. The results of the study suspension cultures containing bioactive trimethyl xanthina candidates that can be used as an antioxidant.

  7. Study on enzymatic browning in suspension cultures of licorice cells

    OpenAIRE

    Yali Li; Tingting Meng; Yuxi Wang; Xiaoli Zhang

    2016-01-01

    Enzymatic browning is one of the main obstacles encountered in the establishment of suspension systems of licorice cells. Browning of cells may result in decreased viability, poor growth and even death. The present study investigated the mechanism of browning reactions and the effective controlling methods. The results showed that the cell viability and membrane permeabilization obviously changed when the cells were transferred to liquid medium. The transformation caused rapid increase in the...

  8. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  9. Extraction and Estimation of Secondary Metabolites from Date Palm Cell Suspension Cultures.

    Science.gov (United States)

    Naik, Poornananda M; Al-Khayri, Jameel M

    2017-01-01

    The health benefits of dates arise from their content of phytochemicals, known for having pharmacological properties, including flavonoids, carotenoids, phenolic acids, sterols, procyanidins, and anthocyanins. In vitro cell culture technology has become an attractive means for the production of biomass and bioactive compounds. This chapter describes step-by-step procedures for the induction and proliferation of callus from date palm offshoots on Murashige and Skoog (MS) medium supplemented with plant growth regulators. Subsequently cell suspension cultures are established for optimum biomass accumulation, based on the growth curve developed by packed cell volume as well as fresh and dry weights. The highest production of biomass occurs at the 11th week after culturing. Moreover, this chapter describes methodologies for the extraction and analysis of secondary metabolites of date palm cell suspension cultures using high-performance liquid chromatography (HPLC). The optimum level of catechin, caffeic acid, apigenin, and kaempferol from the cell suspension cultures establishes after the 11th and 12th weeks of culture. This protocol is useful for scale-up production of secondary metabolites from date palm cell suspension cultures.

  10. Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism.

    Science.gov (United States)

    Travert, S.; Valerio, L.; Fouraste, I.; Boudet, A. M.; Teulieres, C.

    1997-08-01

    A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.

  11. Induction of pistil-like structures in suspension-derived callus cultures of wheat (Triticum aestivum).

    Science.gov (United States)

    Hunsinger, H; Schauz, K

    1987-10-01

    In the following a method for the induction of pistil-like structures in wheat suspension cultures (Triticum aestivum L.) is described. In young influorescences of plants, which were artificially infected with the wheat bunt fungi (Tilletia controversa), organogenic calli with pistil-like structures could be induced on loblolly pine medium + 3 mg/l 3,6-dichloro-2-methoxy benzoic acid. The yield of these structures in calli from a five-month-old suspension culture was up to 100 per gram of callus fresh mass.

  12. Regeneration from embryogenic callus and suspension cultures of ...

    African Journals Online (AJOL)

    ehab

    2012-04-25

    Apr 25, 2012 ... D) for the induction of embryogenic callus from seed cultures were used. Results show that .... ii) Seed culture. Sterile seeds were cultured on MS media supplemented with different 2,4-D concentrations (0.5, 1.0, 2.0, 4.0, 8.0 mg/l) and BA. (0.0, 0.2, 0.5 ...... bioassay with tobacco tissue culture. Plant Physiol.

  13. The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures.

    Science.gov (United States)

    Cetin, Emine Sema; Babalik, Zehra; Hallac-Turk, Filiz; Gokturk-Baydar, Nilgun

    2014-09-23

    Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6

  14. The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Emine Sema Cetin

    2014-01-01

    Full Text Available BACKGROUND: Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols and dry cell weights were determined in the harvested cells. RESULTS: Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g, total flavanol (15.94 mg/100 g, total flavonol (14.73 mg/100 g and trans-resveratrol (490.76 µg/100 g were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 µg/100 g were detected in the cell

  15. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

    Science.gov (United States)

    Nath, Suman Chandra; Nagamori, Eiji; Horie, Masanobu; Kino-Oka, Masahiro

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 106 cells mL-1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

  16. Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

    National Research Council Canada - National Science Library

    Zofia Chirek

    2014-01-01

    The auxin level in the cell mass and culture medium was determined by means of the Avena straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour...

  17. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different ...

  18. Surfactant-induced non-lethal release of anthraquinones from suspension culture of Morinda citrifolia.

    NARCIS (Netherlands)

    Bassetti, L.; Hagendoorn, M.J.M.; Tramper, J.

    1995-01-01

    A new approach based on the use of the surfactant Pluronic F-68 to obtain non-lethal release of plant cell intracellular products was investigated. Suspension cultures of Morinda citrifolia (Rubiaceae), producing anthraquinones as secondary metabolites, were selected as model system. By

  19. Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

    Science.gov (United States)

    R. Minocha; S.C. Minocha; A. Komamine; W.C. Shortle

    1991-01-01

    Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL α-difluoromethylarginine inhibited ADC activity, cellular...

  20. Cytological changes associated with induction of anthraquinone synthesis in photoautotrophic cell suspension cultures of Morinda lucida.

    Science.gov (United States)

    Yamamoto, H; Tabata, M; Leistner, E

    1987-06-01

    Differences in subcellular structures between anthraquinone-producing and non-producing cells were investigated using photoautotrophic and photoheterotrophic cell suspension cultures of Morinda lucida. Irregular or distorted plastids containing starch grains were observed in the anthraquinone-producing cells, together with a highly elongated rough endoplasmic reticulum. The possible participation of plastids and rough endoplasmic reticulum in the anthraquinone biosynthesis is discussed.

  1. CALLUS INDUCTION AND PHYTOCHEMICAL CHARACTERIZATION OF Cannabis sativa CELL SUSPENSION CULTURES

    Directory of Open Access Journals (Sweden)

    Tri Joko Raharjo

    2010-06-01

    Full Text Available Callus of Cannabis sativa has been successfully induced from C. sativa explants and seedings. It seems that flowers are the best explant for callus induction and induction under light also give better results than induction in dark. Four cell culture lines were established from flower induced callus. Phytochemical profiles of C. sativa suspension cell cultures were investigated using HPLC and 1H-NMR. Cannabinoids and phenolic compounds related to cannabinoids such as flavonoids could not be found in the cell suspension cultures and there is no major chemical difference between the cell lines though they can visually be distinguished by their colors. Only in one cell line some aromatic compounds in the water/methanol extract could be observed in the 1H-NMR. Further investigations showed that none of these compounds are flavonoids. It seems that lack of cannabinoids in the cell cultures is related to lack of polyketide synthase activity.   Keywords: Callus, Cannabis, phytochemical

  2. Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae

    Directory of Open Access Journals (Sweden)

    Laura Yesenia Solís-Ramos

    2013-09-01

    Full Text Available J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.

  3. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Science.gov (United States)

    2011-01-01

    Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within

  4. Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Mahanom Jalil

    2015-01-01

    Full Text Available Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

  5. Out-of-School Suspensions of Black Youths: Culture, Ability, Disability, Gender, and Perspective.

    Science.gov (United States)

    Haight, Wendy; Kayama, Misa; Gibson, Priscilla Ann

    2016-07-01

    Racial disproportionality in out-of-school suspensions is a persistent social justice issue in public schools. This article examines out-of-school suspensions of four black youths from the perspectives of the youths, their caregivers, and educators. The case involving David, a 14-year-old African American with a learning disability, illustrates the challenges of students experiencing the intersection of disability and race. The case involving George, a 14-year-old Liberian immigrant, illustrates how parents and teachers may form alliances around shared goals and values despite profound cultural differences in understanding of youths' misbehavior. The case involving Nina, a 12-year-old African American, illustrates how educators' failure to consider the context of her misbehaviors as responses to sexual harassment, along with their subsequent harsh punishment and failure to protect her, led to her disengagement from school. The case involving Craig, a 16-year-old African American, provides a glimpse into how the use of criminal justice language to refer to youths' misbehaviors can support the development of a criminalized self- and social identity. These cases illustrate the diversity of black students--including ability, disability, culture, and gender--and how events surrounding suspensions are interpreted by students, caregivers, and educators. Understanding such diversity will undergird implementation of effective alternatives to suspensions.

  6. Caffeine formation by suspension cultures of Coffea dewevrei

    Directory of Open Access Journals (Sweden)

    Rosana Mary Sartor

    2000-01-01

    Full Text Available The low caffeine content in leaves of C. dewevrei (~ 0.5 mg/g is due to a low biosynthesis associated with a fast degradation. On the other hand, high biosynthesis and low degradation confer a higher content (~ 8 mg/g in leaves of C. arabica. In this work it was observed that cell cultures of C. dewevrei recovered the ability to synthesize caffeine almost in similar levels of C. arabica cultures. Tracer experiments with labelled carbon dioxide showed a significant accumulation of radioactivity in caffeine and metabolites, indicating an active biosynthesis. When the cultures were fed with labelled caffeine most of the radioactivity was recovered in caffeine, indicating that although active, degradation was not so efficient as in leaves, and therefore, contributing for the alkaloid accumulation in the cell cultures.O baixo conteúdo de cafeína em folhas de C. dewevrei (~ 0.5 mg/g é devido a uma menor biossíntese associada a uma rápida degradação. Por outro lado, alta taxa de biossíntese e baixa degradação confere o maior conteúdo (~ 8 mg/g em folhas de C. arabica. Neste trabalho estudou-se a produção de cafeína em culturas de células em suspensão de C. dewevrei. Observou-se que culturas desta espécie de café recuperaram a habilidade em sintetizar cafeína, em níveis semelhantes aos de culturas de C. arabica. Em experimento em que carbonato de bário contendo carbono marcado foi adicionado ao meio de cultura observou-se expressivo acúmulo de radioatividade em cafeína e seus metabólitos, indicando ativa biossíntese. Quando culturas receberam cafeína marcada, a maior parte da radioatividade recuperada estava neste alcalóide, indicando que a via de degradação não era tão ativa como em tecidos intactos (folhas para reduzir o teor de cafeína, levando portanto ao seu acúmulo.

  7. Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture.

    Science.gov (United States)

    Meng, Guoliang; Liu, Shiying; Poon, Anna; Rancourt, Derrick Emile

    2017-10-10

    Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self-renew and differentiate into cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups including ours have developed methodologies for scaled-up hiPSC production in stirred bioreactors in chemically defined medium. Here, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred suspension cultures including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers.

  8. Fetal calf serum-free suspension culture of Chinese hamster ovary cells employing fish serum.

    Science.gov (United States)

    Fujiwara, Masashi; Aizu, Yu; Shioya, Itaru; Takagi, Mutsumi

    2010-03-01

    The effects of heat treatment and concentration of fish serum (FS) on cell growth in a suspension culture of recombinant Chinese hamster ovary (CHO) 1-15(500) (ATCC CRL-9606) cells were investigated. An increase in FS concentration from 1% to 4% markedly increased cell density. On the other hand, heat treatment of FS showed nearly no effect on cell density. Copyright 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

    Science.gov (United States)

    Bertolini, Alberto; Petrussa, Elisa; Patui, Sonia; Zancani, Marco; Peresson, Carlo; Casolo, Valentino; Vianello, Angelo; Braidot, Enrico

    2016-10-26

    Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

  10. Dialysis buffer with different ionic strength affects the antigenicity of cultured nervous necrosis virus (NNV) suspensions.

    Science.gov (United States)

    Gye, Hyun Jung; Nishizawa, Toyohiko

    2016-09-02

    Nervous necrosis virus (NNV) belongs to the genus Betanodavirus (Nodaviridae). It is highly pathogenic to various marine fishes. Here, we investigated the antigenicity changes of cultured NNV suspensions during 14days of dialyses using a dialysis tube at 1.4×10(4) molecular weight cut off (MWCO) in three different buffers (Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), and deionized water (DIW)). Total NNV antigen titers of cultured NNV suspension varied depending on different dialysis buffers. For example, total NNV antigen titer during D-PBS dialysis was increased once but then decreased. During Tris-HCl dialysis, it was relatively stable. During dialysis in DIW, total NNV antigen titer was increased gradually. These antigenicity changes in NNV suspension might be due to changes in the aggregation state of NNV particles and/or coat proteins (CPs). ELISA values of NNV suspension changed due to changing aggregates state of NNV antigens. NNV particles in suspension were aggregated at a certain level. These aggregates were progressive after D-PBS dialysis, but regressive after Tris-HCl dialysis. The purified NNV particles self-aggregated after dialysis in D-PBS or in Tris-HCl containing 600mM NaCl, but not after dialysis in Tris-HCl or DIW. Quantitative analysis is merited to determine NNV antigens in the highly purified NNV particles suspended in buffer at low salt condition. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Is the common cold a clinical entity or a cultural concept?

    Science.gov (United States)

    Eccles, R

    2013-03-01

    Common cold is the most common infectious disease of mankind and the term is widely used in the clinical literature as though it were a defined clinical syndrome. Clinical studies on this syndrome often use elaborate symptom scoring systems to diagnose a common cold. The symptom scores are based on a study conducted over 50 years ago to retrospectively diagnose experimental cold and this method cannot be applied to diagnosis of common cold in the community. Diagnosis of the common cold by virology is not feasible because of the number of viruses and the variability in the disease states caused by the viruses. Because of the familiarity of subjects with common cold and the variability in symptomatology it seems a more reasonable approach to use self-diagnosis of common cold for clinical research studies and accept that the common cold is a cultural concept and not a clinical entity.

  12. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45......% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied...... by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  13. Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

    Science.gov (United States)

    Nampe, Daniel; Joshi, Ronak; Keller, Kevin; Zur Nieden, Nicole I; Tsutsui, Hideaki

    2017-09-01

    The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Angelo Luis Caron

    2015-12-01

    Full Text Available ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI, reaching a total viral yield of 2.48x108 particles.

  15. Non-cultured epidermal suspension in vitiligo: From laboratory to clinic

    Directory of Open Access Journals (Sweden)

    Yvon Gauthier

    2012-01-01

    Full Text Available Background: Medical treatments are ineffective in many patients and surgical methods have therefore been developed. Objective: A review of autologous non-cultured melanocyte grafting techniques is proposed to obtain a successful repigmentation of vitiligo macules. Methods: Initially in 1992, we had developed a simplified grafting method which was carried out in the following two steps: production of blisters on the depigmented lesions by freezing with liquid nitrogen and injection in each blister of a non-cultured suspension of epidermal cells. The cellular suspension was obtained from samples of skin of the hair scalp after trypsinization. This very simple technique could be used at the dermatologist′s clinic. Since 1998 (Olsson MJ, Juhlin L, quite comparable but improved and more sophisticated techniques have been proposed for the surgical treatment of vitiligo. These techniques require a laboratory set up to perform the melanocyte transplantation. The donor zone was usually taken on the gluteal region. The time of trypsinization was reduced to 60 minutes at 37΀C and the centrifuged cellular suspension added with hyaluronic acid (Van Geel was directly applied on a dermabraded or laser abraded vitiligo lesions. Results: Whatever the technique chosen, repigmentation was evident within 25 to 30 days. Coalescence of the pigmented areas was spontaneously observed or obtained after UVB radiation. It is obvious that the complete repigmentation occurred more rapidly with the recent techniques compared with the initial method, but the efficiency was quite similar. Conclusion: The use of non-cultured epidermal suspension appears to be an effective, safe, and simple method for treating patients with achromic areas lacking melanocytes.

  16. Jasmonic and salicylic acids enhanced phytochemical production and biological activities in cell suspension cultures of spine gourd (Momordica dioica Roxb).

    Science.gov (United States)

    Chung, Ill-Min; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Thiruvengadam, Muthu

    2017-03-01

    In vitro cell suspension culture was established for the production of commercially valuable phytochemicals in Momordica dioica. The influence of elicitors in jasmonic acid (JA) and salicylic acid (SA) increased their effect on phytochemical production and biomass accumulation in M. dioica. The results indicate that compared with non-elicited cultures, JA- and SA-elicited cell suspension cultures had significantly enhanced phenolic, flavonoid, and carotenoid production, as well as antioxidant, antimicrobial, and antiproliferative activities. Furthermore, elicited cultures produced 22 phenolic compounds, such as flavonols, hydroxycinnamic acids, and hydroxybenzoic acids. Greater biomass production, phytochemical accumulation, and biological activity occurred in JA- than in SA-elicited cell cultures. This study is the first to successfully establish M. dioica cell suspension cultures for the production of phenolic compounds and carotenoids, as well as for biomass accumulation.

  17. Improvement of catechin productivity in suspension cultures of tea callus cells.

    Science.gov (United States)

    Shibasaki-Kitakawa, Naomi; Takeishi, Junna; Yonemoto, Toshikuni

    2003-01-01

    In the suspension cultures of tea callus cells, C.sinensis cv. Yabukita, the effects of the culture conditions, such as culture period and light irradiation, on cell growth and catechin production were investigated. The production of flavonoids (catechins + proanthocyanidins) was promoted by inoculating the cells into the fresh medium at the culture period giving the maximum flavonoid content in the cells. The cultivation under light irradiation was repeated several times by inoculating the cells with the maximum flavonoid content. The flavonoid production was significantly increased without inhibiting the cell growth. We obtained the maximum flavonoid production, 1.5 g/dm(3) medium, and the maximum content, 150 mg/(g of dry cell weight (DCW)). The latter value was larger than that in the leaves of the tea plant.

  18. Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

    Science.gov (United States)

    Alwael, Hussain A; Naik, Poornananda M; Al-Khayri, Jameel M

    2017-01-01

    Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 μM), the best synchronized growth was obtained in response to 50-100 μM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

  19. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  20. Comparison of Cuminaldehyde Contents from Cell Suspension Cultures and Seeds of [Bunium persicum (Boiss. B. Fedtsch.

    Directory of Open Access Journals (Sweden)

    Sara KHOSRAVINIA

    2012-11-01

    Full Text Available The cell suspension culture and seed samples of Bunium persicum were extracted by supercritical fluid, hydrodistillation and solvent methods and analyzed by Gas Chromatography. In this study to compare the different methods of extractions, cuminaldehyde was targeted as one of the Black zira essential oil constitute. For callus induction the germinated seeds were cultured as explants on Murashige and Skoog medium supplemented with 2 mg/l 2,4-dichlorophenoxy acetic acid and 0.5 mg/l kinetin (treatment A as well as 2 mg/l ?-naphthalene acetic acid and 0.5 mg/l 6-benzyl aminopurine (treatment B and followed by cells suspension cultures establishment for the first time. The results of cell culture showed that cells from treatment B have a growth rate higher than A. All extracts were dissolved in 1 ml hexane and analyzed by Gas Chromatography. According to the Gas Chromatography analysis, cuminaldehyde was not detected in the supercritical fluid samples, while it was present in hydrodistillation and solvent extract. Cuminaldehyde percentage in cell and seed solvent extracts was 4.65% and 18.61% respectively. Gas Chromatography results also showed that no cuminaldehyde is present in media extracts, means no cuminaldehyde has been secreted into the medium.

  1. Cell line selection combined with jasmonic acid elicitation enhance camptothecin production in cell suspension cultures of Ophiorrhiza mungos L.

    Science.gov (United States)

    Deepthi, S; Satheeshkumar, K

    2017-01-01

    Ophiorrhiza mungos is a herbaceous medicinal plant which contains a quinoline alkaloid, camptothecin (CPT), an anticancer compound. A high-yielding cell line, O. mungos cell line-3 (OMC3) was selected from cell suspension cultures of O. mungos using cell aggregate cloning method and established cell suspension culture. OMC3 cell suspension produced significantly high biomass (9.25 ± 1.3 g/flask fresh weight (FW)) and CPT yield (0.095 ± 0.002 mg g(-1) dry weight (DW)) compared with the original cell suspension. Inoculum size of OMC3 cell suspension culture was optimised as 14 g L(-1). Media optimisation has shown that 5 % (w/v) sucrose and an increased ammonium/nitrate concentration of 40/20 mM favoured CPT production, whereas 3 % (w/v) sucrose, an ammonium/nitrate concentration of 20/40 mM and 1.25 mM of phosphate favoured biomass accumulation. Jasmonic acid, chitin and salicylic acid was used to elicit CPT production in the original cell suspension culture and achieved significantly high CPT production with jasmonic acid (JA) elicitation. Further, OMC3 cell suspension culture was elicited with JA (50 μM) and obtained 1.12 ± 0.08 mg g(-1) DW CPT and 9.52 ± 1.4 g/flask FW (190.4 g L(-1) FW). The combination of cell line selection and elicitation has produced 18.66-fold increases in CPT production together with significantly high biomass yield. The study is helpful in the scale-up studies of O. mungos cell suspension culture in suitable bioreactor systems for the production of CPT.

  2. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

    Directory of Open Access Journals (Sweden)

    Muthu Thiruvengadam

    2016-11-01

    Full Text Available Anthraquinones (AQs and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs, media, sucrose, l-glutamine, jasmonic acid (JA, and salicylic acid (SA for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM; 3 and 2.93 g dry mass (DM and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds from cell suspension cultures, and the phytochemicals can be used for various biological activities.

  3. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum.

    Science.gov (United States)

    Thiruvengadam, Muthu; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Lee, Taek-Jun; Kim, Seung-Hyun; Chung, Ill-Min

    2016-11-16

    Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum . We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum . The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum . This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities.

  4. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

    Science.gov (United States)

    Thiruvengadam, Muthu; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Lee, Taek-Jun; Kim, Seung-Hyun; Chung, Ill-Min

    2016-01-01

    Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities. PMID:27854330

  5. Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines.

    Science.gov (United States)

    Huang, Ding; Peng, Wen-Juan; Ye, Qian; Liu, Xu-Ping; Zhao, Liang; Fan, Li; Xia-Hou, Kang; Jia, Han-Jing; Luo, Jian; Zhou, Lin-Ting; Li, Bei-Bei; Wang, Shi-Lei; Xu, Wen-Ting; Chen, Ze; Tan, Wen-Song

    2015-01-01

    Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

  6. Uptake and metabolism of sugars by suspension-cultured catharanthus roseus cells

    Energy Technology Data Exchange (ETDEWEB)

    Ashihara, Hiroshi; Sagishima, Kyoko; Kubota, Kaoru (Ochanomizu Univ., Tokyo (Japan))

    1989-04-01

    The Uptake and metabolism of sugars by suspension-cultured Catharanthus roseus cells were investigated. Substantially all the sucrose in the culture medium was hydrolyzed to glucose and fructose before being taken up by the cells. The activity of invertase bound to cell walls, determined in situ, was high at the early stage of culture. Glucose was more easily taken up by the cells than was fructose. Tracer experiments using (U-{sup 14}C)glucose and (U-{sup 14}C)fructose indicated that glucose is a better precursor for respiration than fructose, while fructose is preferentially utilized for the synthesis of sucrose, especially in the early phase of cell growth. These results suggest that fructose is utilized for the synthesis of sucrose via the reaction catalyzed by sucrose synthase, prior to the phosphorylation by hexokinase or fructokinase.

  7. Histology of embryoid development in oil palm (Elaeis guineensis Jacq. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Songrat Tinnongjig

    2001-11-01

    Full Text Available Embryos of oil palm (Elaeis guineensis Jacq. variety tenera were cultured on Eeuwens or Y3 (1976; 1978 medium supplemented with 2 mg/l 2,4-D. Calluses were initiated from these embryos. The eight-weekold calluses derived from embryos were transferred to modified Y3 liquid medium devoid of 2,4-D and supplemented with NAA, BA and coconut water to establish cell suspension culture. After a period of culture,these cells were then subcultured to the same medium without plant growth regulators to induce embryoid formation. The calluses and embryoids were harvested at various times, fixed, sectioned, stained and examined microscopically. Histological study revealed that embryoid occurred from meristematic cells with dense cytoplasm along the callus clumps.

  8. Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

    Science.gov (United States)

    Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

    2012-12-20

    Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis - total protein content, thiols - reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

  9. Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Rene Kizek

    2012-12-01

    Full Text Available Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles, electronics (high-resolution imaging, logical circuits on the molecular level and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases or imaging (contrast agents. Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs and modified magnetic nanoparticles (MNPs on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis — total protein content, thiols — reduced (GSH and oxidized (GSSG glutathione, phytochelatins PC2-5, glutathione S-transferase (GST activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension

  10. Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

    Science.gov (United States)

    Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

    2012-01-01

    Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis—total protein content, thiols—reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

  11. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    Science.gov (United States)

    Seifertová, Daniela; Skůpa, Petr; Rychtář, Jan; Laňková, Martina; Pařezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells. Copyright © 2013 Elsevier GmbH. All rights reserved.

  12. Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

    Science.gov (United States)

    Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

    2017-02-01

    Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

    Science.gov (United States)

    Johanson, Kelly; Allen, Patricia L.; Lewis, Fawn; Cubano, Luis A.; Hyman, Linda E.; Hammond, Timothy G.

    2002-01-01

    This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

  14. [The production of gastrodin through biotransformation of p-hydroxybenzaldehyde by cell suspension culture of Datura stramonium].

    Science.gov (United States)

    Gong, Jia-Shun; Ma, Wei-Peng; Pu, Jun-Xue; Xu, Shu-Guan; Zheng, Shuang-Qing; Xiao, Chun-Jie

    2006-10-01

    To investigate the production of p-hydroxymethylphenol-beta-D-glucoside (gastrodin) through biotransformation by plant cell suspension cultures. Using cell suspension cultures of Datura stramonium to convert the exogenous p-hydroxybenzaldehyde into gastrodin was conducted and the converted compounds were separated with a combination of multi-chromatography. Their chemical structures were determined on the basis of spectral analysis and chemical evidence. The conversion procedure of p-hydroxybenzaldehyde into gastrodin by Datura stramonium cell suspension cultures was established. The synthesized gastrodin (II) was isolated from the fermental liquor and identified by spectral analysis. At the same time, the p-hydroxybenzyl alcohol (I) converted through biotransformation of p-hydroxybenzaldehyde by cell suspension cultures of Datura stramonium was also isolated and identified. Two compounds were also isolated from the cell cultures and they were identified as beta-D-furanoallulose (III) and n-butyloxystyryl-beta-D-pyranoallulose (IV). Datura stramonium grown in suspension cultures can convert exogenous p-hydroxybenzaldehyde into the corresponding gastrodin.

  15. [Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims].

    Science.gov (United States)

    Cristovam, Priscila Cardoso; Glória, Maria Aparecida da; Melo, Gustavo Barreto; Gomes, José Alvaro Pereira

    2008-01-01

    To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

  16. Changes in cellulase and polygalacturonase activity in Rosa glauca suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Joseleau, J.P.; Chambat, G.

    The levels of cellulase and polygalacturonase activities in Rosa glauca suspension cultures were measured at 6, 10, 14 and 28 days of growth. Cellulase and polygalacturonase showed the highest activity in the 14-day-old cells. The changes in the activities followed closely the changes in the corresponding levels of cellulose and galacturonic acid-containing polysaccharides in the cell wall. The maximum in both activities coincided with the end of the exponential growth of the cells. These hydrolases are believed to play a role in the cell expansion and the polysaccharides synthesis.

  17. The establishment of suspension and meristem cultures for the development of a protoplast regeneration and fusion system in Lily

    NARCIS (Netherlands)

    Famelaer, I.; Ennik, E.; Tuyl, van J.M.; Meijer, H.; Creemers-Molenaar, J.

    1996-01-01

    The present results indicate that established morphogenic suspension cultures can be obtained from crosses between cultivars of L. longiflorum and from a cross between Asiatic hybrid 'Orlito' x 'Connecticut King'. Meristem cultures were obtained from L. longiflorum 'Gelria' and Oriental hybrid 'Star

  18. Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

    NARCIS (Netherlands)

    Woerdenbag, H J; van Uden, W; Frijlink, H W; Lerk, C F; Pras, N; Malingré, T M

    Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of β-cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with β-cyclodextrin, a

  19. Evaluation of Antioxidant and Antibacterial Potentials of Nigella sativa L. Suspension Cultures under Elicitation

    Directory of Open Access Journals (Sweden)

    Hera Chaudhry

    2015-01-01

    Full Text Available Nigella sativa L. (family Ranunculaceae is an annual herb of immense medicinal properties because of its major active components (i.e., thymoquinone (TQ, thymohydroquinone (THQ, and thymol (THY. Plant tissue culture techniques like elicitation, Agrobacterium mediated transformation, hairy root culture, and so on, are applied for substantial metabolite production. This study enumerates the antibacterial and antioxidant potentials of N. sativa epicotyl suspension cultures under biotic and abiotic elicitation along with concentration optimization of the elicitors for enhanced TQ and THY production. Cultures under different concentrations of pectin and manganese chloride (MnCl2 elicitation (i.e., 5 mg/L, 10 mg/L, and 15 mg/L showed that the control, MnCl2 10 mg/L, and pectin 15 mg/L suspension extracts greatly inhibited the growth of E. coli, S. typhimurium, and S. aureus (MIC against E. coli, i.e., 2.35±0.8, 2.4±0.2, and 2.46±0.5, resp.. Elicitation decreased SOD enzyme activity whereas CAT enzyme activity increased remarkably under MnCl2 elicitation. MnCl2 10 mg/L and pectin 15 mg/L elicitation enhanced the DPPH radical inhibition ability, but ferric scavenging activity was comparable to the control. TQ and THY were quantified by LC-MS/MS in the cultures with high bioactive properties revealing maximum content under MnCl2 10 mg/L elicitation. Therefore, MnCl2 elicitation can be undertaken on large scale for sustainable metabolite production.

  20. Impact of UV-B radiation on some biochemical changes and growth parameters in Echinacea purpurea callus and suspension culture

    Directory of Open Access Journals (Sweden)

    Hossam H. Manaf

    2016-12-01

    Full Text Available The effects of UV-B light force, exposure time and incubation period on producing caffeic acid derivatives and growth parameters in Echinacea purpurea callus and suspension culture were assessed. UV-B led to an increment of all growth parameters and antioxidant activity in callus and cell suspension and caffeic acid derivatives in cell suspension by increasing incubation period. The reverse was true for G-POD activity in cell suspension and PAL activity in both types of cultures. Incubation period 2 weeks was more effective in caffeic acid, total phenols and G-POD activity in callus cells and incubation period one week only for total phenols in cell suspension. The two exposure times 2 and 4 h increased antioxidant activity in the two types of cultures. Exposure time 2 h led to increase caffeic acid and total phenols in callus cells. The maximum increase in caffeic acid, total phenols and PAL activity in cell suspension was achieved by 4 h exposure time. Likewise, using 2 UV-B lamps for 2 h was the most effective in creating more biochemical components than the other treatments.

  1. Seasonal dynamics of trophic relationships among co-occurring suspension-feeders in two shellfish culture dominated ecosystems

    Science.gov (United States)

    Lefebvre, Sébastien; Marín Leal, Julio César; Dubois, Stanislas; Orvain, Francis; Blin, Jean-Louis; Bataillé, Marie-Paule; Ourry, Alain; Galois, Robert

    2009-04-01

    The temporal dynamics of carbon and nitrogen isotope values of co-occurring suspension-feeders in two shellfish culture areas (Normandy, France) were investigated over two years to evaluate the inter-specific trophic partitioning and relative contributions of organic matter sources to benthic suspension-feeders' diet. Oysters ( Crassostrea gigas), mussels ( Mytilus edulis), cockles ( Cerastoderma edule), slipper limpets ( Crepidula fornicata), and sand-mason worms ( Lanice conchilega) were sampled in an estuarine environment (Baie des Veys, east Cotentin, Normandy), while oysters, mussels, slipper limpets, and honeycomb worms ( Sabellaria alveolata) were sampled in an open-marine environment (Lingreville-sur-mer, west Cotentin, Normandy). Whatever the sampling period, the bivalves, C. gigas and M. edulis, exhibited the lowest values of δ13C and δ15N compared with the other species. Feeding relationships among suspension-feeders in both C. gigas culture areas exhibited temporal variations due to the marine/estuarine influence and seasonal changes in food supply. In the open-marine ecosystem, the contribution of phytoplankton remained the most important for all species except S. alveolata, while in the estuarine ecosystem, microphytobenthos and/or macroalgae detritus contributed a larger extent to the organisms' diets. During phytoplankton bloom periods (e.g. May and July) suspension-feeders, except for S. alveolata, relied strongly on phytoplankton; however, the majority of suspension-feeders exhibited different opportunistic behaviour in winter when phytoplankton biomass might be a limiting factor. We hypothesized that differences in particle capture and selection by the suspension-feeders influenced their isotopic values. Feeding ecology of suspension-feeders partly explained why competition was limited and why ecosystems can often support unexpectedly large numbers of suspension-feeders. We also showed that understanding ecosystem characteristics of the organic

  2. Five 2-(2-Phenylethylchromones from Sodium Chloride-Elicited Aquilaria sinensis Cell Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Zhongxiu Zhang

    2016-04-01

    Full Text Available Five 2-(2-phenylethylchromones including a new one, (5S,6R,7S,8R-5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (1, and four known ones (2–5, were isolated from 150 mM NaCl-elicited Aquilaria sinensis cell suspension cultures. In addition, three feruloyl amides (6–8, six nucleosides (9–14, (+-syringaresinol (15, indole-3-carboxaldehyde (16, and two glycosides (17–18 were also obtained. The structures were unambiguously identified by analysis of their UV, IR, NMR, and HRESIMS data. The absolute configuration of the new 2-(2-phenylethylchromone (1 was established by a dimolybdenum tetraacetate-induced circular dichroism experiment. Compared to un-elicited cell lines, the appearance of 2-(2-phenylethylchromones in NaCl-treated cells occurred on the 3rd and 5th days of their treatment. 2-(2-Phenylethylchromones, feruloyl amides, nucleosides, and lignins have been reported to be closely related to plant defense; therefore, the identification of these compounds from NaCl-elicited A. sinensis cell suspension cultures would be useful for further exploring the mechanism of agarwood formation.

  3. Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani.

    Science.gov (United States)

    Venugopalan, Aarthi; Srivastava, Smita

    2015-01-01

    Ethanolic extract of a non-camptothecin producing plant, Catharanthus roseus when added in the suspension culture of the endophyte Fusarium solani known to produce camptothecin, resulted in enhanced production of camptothecin by 10.6-fold in comparison to that in control (2.8 μg/L). Interestingly, addition of pure ethanol (up to 5% v/v) in the suspension culture of F. solani resulted in maximum enhancement in camptothecin production (up to 15.5-fold) from that obtained in control. In the presence of ethanol, a reduced glucose uptake (by ∼ 40%) and simultaneous ethanol consumption (up to 9.43 g/L) was observed during the cultivation period (14 days). Also, the total NAD level and the protein content in the biomass increased by 3.7- and 1.9-fold, respectively, in comparison to that in control. The study indicates a dual role of ethanol, presumably as an elicitor and also as a carbon/energy source, leading to enhanced biomass and camptothecin production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Evaluation of Antioxidant Enzyme Activity and Biochemical Characterization from Static and Suspension Culture of Withania somnifera L.

    Directory of Open Access Journals (Sweden)

    Satyajit Kanungo

    2015-03-01

    Full Text Available Withania somnifera (L. Dunal, is an erect evergreen shrub commonly known as Ashwagandha. It is widely used in Ayurvedic and in the traditional pharmacopeia system of India. It is one of the major ingredients in many formulations prescribed for a variety of musculoskeletal conditions including arthritis and rheumatism. In the present study the variation in quality and quantity of protein and antioxidant enzymes were evaluated biochemically and enzymatically from the static and suspension cultures. The nodal segments had provided maximum callusing of 90.25±0.06 % with (1mg/l of BAP and Kn with (2mg/l of 2, 4-D. The static and suspension cultures were taken for the analysis of total soluble protein and screened for antioxidant enzyme activity [catalase (CAT, superoxide dismutase (SOD and guaiacol peroxidase (GPX]. The protein content (1.2016 µg/µl was found to be higher in static culture samples (0.870 µg/µl than the protein obtained from the suspension culture. The antioxidant enzyme activity (CAT, SOD and GPX was higher in static culture samples (301.01± 0.42, 198.92 ± 0.29, 103.75 ± 0.11 nkat/ mg of protein than that of suspension culture. Specific activity staining of isozyme pattern exhibited three isoforms (CAT 1, CAT 2 and CAT 3 in static culture samples but CAT 1 was absent in the sample extracted from suspension cultures.  In case of SOD, four bands (SOD 1, SOD 2, SOD 3 and SOD 4 were found in both the samples whereas intensity of GPX activity was found to be more in static culture but both the samples exhibited three isoforms such as  (GPX 1, GPX 2 and GPX 3. The supplementation of required nutrients along with the phytohormones under in vitro condition might be an enhancing factor to yield antioxidant enzymes in the static culture samples. 

  5. Production and elicitation of benzalacetone and the raspberry ketone in cell suspension cultures of Rubus idaeus.

    Science.gov (United States)

    Pedapudi, S; Chin, C K; Pedersen, H

    2000-01-01

    Production levels of p-coumaric acid (p-CA), p-hydroxyphenylbut-3-ene-2-one (benzalacetone), and p-hydroxyphenyl-2-butanone (raspberry ketone) were measured in raspberry cell suspension cultures to investigate metabolite dynamics in a short (two-step) pathway. Intracellular concentrations of benzalacetone and the raspberry ketone fluctuated during the time course of a normal batch culture cycle but showed higher levels during periods of rapid growth. Cells elicited with the signal coupler methyl jasmonate yielded a 2- to 3-fold increase in metabolite concentrations after 24 h. The results suggest that raspberry ketone production is rapidly inducible during periods of high carbohydrate utilization. It is not an end product, however, and undergoes conversion to subsequent metabolites.

  6. Induction of linalool as a pharmaceutical and medicinal metabolite via cell suspension culture of cumin (Cuminum cyminum L.).

    Science.gov (United States)

    Kazemi, N; Kahrizi, D; Mansouri, M; Karim, H; Vaziri, S; Zargooshi, J; Khanahmadi, M; Shokrinia, M; Mohammadi, N

    2016-05-30

    Cumin is an important medicinal plant in Iran. Plant cell suspension culture is a method for the production of medicinal and secondary metabolites. The linalool is a plant secondary metabolite that has been recognized as a neuroprotective agent. The purpose of this study was to evaluate the effects of salicylic acid elicitor on induction of linalool in cell suspension culture of cumin. For this purpose, the cumin seeds were prepared, to obtain sterile seedling, were disinfected with sodium hypochlorite and alcohol, and were cultured on MS basal medium. This research was conducted in two separate experiments including callus induction and suspension cultures. Leaf explants were prepared from sterile seedlings and used to produce callus on MS medium supplemented with 1 mg/l NAA and 0.5 mg/l BAP. In order to establish suspension culture, the appropriate calli were transferred to liquid medium. Then cell cultures were treated with elicitors. The effects of elicitor on the production of linalool secondary metabolite and cell viability were assessed by GC-Mass and tetrazolium test respectively. For this purpose, the salicylic acid (at concentrations of 0, 1, 2, 4 and 8 mg/l) was used. The experimental design was a completely randomized design with five treatments and three replications. The results of cell culture and GC-Mass analysis showed that salicylic acid had significant effects on the linalool production (cumin. It is necessary to determine the best combination of medium and elicitor.

  7. Differential protein expression following low temperature culture of suspension CHO-K1 cells

    Directory of Open Access Journals (Sweden)

    Henry Michael

    2008-04-01

    Full Text Available Abstract Background To ensure maximal productivity of recombinant proteins (rP during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. Results In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis and mass spectrometry (MS. There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC, cap-independent translation (EIF4A, apoptosis (importin-α, the cytoskeleton (vimentin and glycoprotein quality control (alpha glucosidase 2. Conclusion These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key

  8. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

    2003-01-01

    this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield......Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  9. Production and glycosylation of recombinant beta-interferon in suspension and cytopore microcarrier cultures of CHO cells.

    Science.gov (United States)

    Spearman, Maureen; Rodriguez, Jose; Huzel, Norm; Butler, Michael

    2005-01-01

    Microcarriers are suitable for high-density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human beta-interferon (beta-IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the beta-IFN yield was up to 3-fold greater as a result of an almost 5-fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. Beta-IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N-linked glycans from beta-IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.

  10. Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida Benth.

    Science.gov (United States)

    Igbavboa, U; Sieweke, H J; Leistner, E; Röwer, I; Hüsemann, W; Barz, W

    1985-12-01

    Photoheterotrophic and photoautotrophic cell suspension cultures were raised from a callus tissue derived from a Morinda lucida Benth. plant (Rubiaceae). The cultures were characterized with regard to fresh weight, dry weight, cell number, pH, chlorophyll and quinoid natural products. The amount of lipoquinones (phylloquinone, α-tocopherol, plastoquinone, ubiquinone) isolated from the photoautotrophic cultures matched the amount detected in an intact leaf. Anthraquinone glycosides which are found in the roots of Morinda plants were not present in the photoautotrophic culture. The photoheterotrophic culture contained only trace amounts of these pigments. Abundant anthraquinone synthesis was observed when photoautotrophic and photoheterotrophic suspension cultures were transferred into darkness, provided sucrose was present in the medium. Induction of synthesis of anthraquinone pigments coincided with a rapid disappearance of lipoquinones from the culture. Thus, in the suspension culture, photoautotrophy correlates with lipoquinone synthesis and heterotrophy correlates with anthraquinone synthesis. This reflects the situation in the intact plants where lipoquinones are chloroplast-associated whereas anthraquinones occur in the roots.

  11. Divided dreamworlds? The cultural cold war in East and West

    NARCIS (Netherlands)

    Romijn, P.; Scott-Smith, G.; Segal, J.

    2012-01-01

    While the divide between capitalism and communism, embodied in the image of the Iron Curtain, seemed to be as wide and definitive as any cultural rift, Giles Scott-Smith, Joes Segal, and Peter Romijn have compiled a selection of essays on how culture contributed to the blurring of ideological

  12. [Adherent and single-cell suspension culture of Madin-Darby canine kidney cells in serum-free medium].

    Science.gov (United States)

    Huang, Ding; Zhao, Liang; Tan, Wensong

    2011-04-01

    In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture, serum-free adherent culture and serum-free single-cell suspension culture. We found that less metabolic by-products were produced in both serum-free cultures. In serum-free single-cell suspension batch culture, the viable cell density was highest. These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.

  13. Serum-free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential.

    Science.gov (United States)

    Alimperti, Stella; Lei, Pedro; Wen, Yuan; Tian, Jun; Campbell, Andrew M; Andreadis, Stelios T

    2014-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft-versus-host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC-based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In the present study, we developed and optimized serum-free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum-free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum-containing adherent MSC cultures. Our results suggest that serum-free media for MSC spheroids may pave the way for scale-up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors. © 2014 American Institute of Chemical Engineers.

  14. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

  15. Abscisic acid is involved in brassinosteroids-induced chilling tolerance in the suspension cultured cells from Chorispora bungeana.

    Science.gov (United States)

    Liu, Yajie; Jiang, Haifeng; Zhao, Zhiguang; An, Lizhe

    2011-06-15

    The objective of this study was to investigate whether abscisic acid (ABA), a second messenger in chilling stress responses, is involved in brassinosteroids (BRs)-induced chilling tolerance in suspension cultured cells from Chorispora bungeana. The suspension cells were treated with 24-epibrassinolide (EBR), ABA, ABA biosynthesis inhibitor fluridone (Flu) and EBR in combination with Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels and antioxidant defense system were analyzed. The results showed that EBR treatment markedly alleviated the decrease of cell viability and the increases of ion leakage and lipid peroxidation induced by chilling stress, suggesting that application of EBR could improve the chilling tolerance of C. bungeana suspension cultures. In addition, similar results were observed when exogenous ABA was applied. Treatment with Flu alone and in combination with EBR significantly suppressed cell viability and increased ion leakage and lipid peroxidation under low temperature conditions, indicating that the inhibition of ABA biosynthesis could decrease the chilling tolerance of C. bungeana suspension cultures and the EBR-enhanced chilling tolerance. Further analyses showed that EBR and ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. However, Flu application differentially blocked these protective effects of EBR. Moreover, EBR was able to mimic the effect of ABA by markedly increasing ABA content in the suspension cells under chilling conditions, whereas the EBR-induced ABA accumulation was inhibited by the addition of Flu. Taken together, these results demonstrate that EBR may confer chilling tolerance to C. bungeana suspension cultured cells by enhancing the antioxidant defense system, which is partially mediated by ABA, resulting in preventing the overproduction of ROS to alleviate oxidative injury induced by chilling. Copyright © 2011 Elsevier GmbH. All rights reserved.

  16. Dynamics of indole-3-acetic acid oxidase activity in suspension culture of sunflower crown-gall

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-01-01

    Full Text Available IAA oxidase activity was determined in several growth phases of a suspension culture of sunflower crown-gall. During the short phase of intensive growth (zero passage - PO a negative correlation was noted between enzymatic activity and the rate of growth. IAA oxidase activity increased to a certain level is not a factor limiting cell division. For protraction of the phase of intensive growth (first passage - P1, however, a decrease in the activity of this enzyme seems indispensable. IAA oxidase activity in the tested culture is under the control of inhibitors present in the cells and medium. High enzyme inhibition was observed in PO cells during the phase, of intensive growth and in P1 at the beginning and in the middle part of this phase. These results suggest' that the -auxin level determined in earlier studies in sunflower crown-gall culture is controlled by the IAA oxidase set. During the long phase of intensive growth (P1 this control is of negative feedback type.

  17. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Henning Kempf

    2014-12-01

    Full Text Available To harness the potential of human pluripotent stem cells (hPSCs, an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion. Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity was enabled that were directly applicable to bioartificial cardiac tissue formation.

  18. Guggulsterone production in cell suspension cultures of the guggul tree, Commiphora wightii, grown in shake-flasks and bioreactors.

    Science.gov (United States)

    Mathur, Meeta; Ramawat, K G

    2007-06-01

    Cell suspension cultures of Commiphora wightii, grown in modified MS medium containing 2,4-dichlorophenoxyacetic acid (0.5 mg l(-1)) and kinetin (0.25 mg l(-1)), produced approximately 5 microg guggulsterone g(-1) dry wt. In a 2 l stirred tank bioreactor, the biomass was 5.5 g l(-1) and total guggulsterone was 36 microg l(-1).

  19. Effect of culture conditions on synthesis of triterpenoids in suspension cultures of Lantana camara L.

    Science.gov (United States)

    Srivastava, Priyanka; Sisodia, Vikash; Chaturvedi, Rakhi

    2011-01-01

    Present report is aimed to study the batch kinetics of Lantana camara. Dynamic changes of parameters, such as pH, conductivity, wet and dry cell concentrations, consumption of major nutrients, carbon source and agitation speeds were investigated to understand the culture characteristics of suspended cells grown on MS + BAP + 2,4-D + NAA in shake flasks. Results indicated that the consumption of phosphate resulted in the onset of stationary phase in cultures. Maltose as carbon source resulted in production of maximum triterpenoid content (31.08 mg/L) while the least was found on glucose (10.69 mg/L). Notably, both did not support accumulation of betulinic acid. Sucrose, although stood second in terms of quantity (21.6 mg/L), supported the production of all the three triterpenoids-oleanolic, ursolic and betulinic acids. Maximum viable cultures were obtained at a rotation speed of 120 rpm. The present finding will form a background for further scale-up related studies.

  20. Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

    Science.gov (United States)

    Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

    1992-01-01

    The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687

  1. Effects of mercury (II) species on cell suspension cultures of catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, L. (Hangzhou Univ. (China)); Cullen, W.R. (Univ. of British Columbia, Vancouver, British Columbia (Canada))

    1994-11-01

    Mercury has received considerable attention because of its high toxicity. Widespread contamination with mercury poses severe environmental problems despite our extensive knowledge of its toxicity in living systems. It is generally accepted that the toxicity of mercury is related to its oxidation states and species, the organic forms being more toxic than the inorganic forms. In the aquatic environment, the toxicity of mercury depends on the aqueous speciation of the mercuric ion (Hg[sup 2+]). Because of the complex coordination chemistry of mercury in aqueous systems, the nature of the Hg[sup 2+] species present in aquatic environments is influenced greatly by water chemistry (e. g, pH, inorganic ion composition, and dissolved organics). Consequently, the influence of environmental factors on the aqueous speciation of mercury has been the focus of much attention. However, there is very little information available regarding the effects of the species and speciation on Hg (II) toxicity in plant-tissue cultures. Catharanthus roseus (C. roseus), commonly called the Madagascar Periwinkle, is a member of the alkaloid rich family Apocynaceae. The present investigation was concerned with the toxicity of mercury on the growth of C. roseus cell suspension cultures as influenced by mercury (II) species and speciation. The specific objectives of the study were to (a) study the effects of mercury species on the growth of C. roseus cultures from the point of view of environmental biology and toxicology; (b) evaluate the effects of selenate, selenite and selected ligands such as chloride, 1-cysteine in the media on the acute toxicity of mercuric oxide; (c) determine the impact of the initial pH of the culture media on the toxicities of mercuric compounds; (d) discuss the dependence of the toxicity on the chemical species and speciation of Hg (II). 11 refs., 7 figs., 2 tabs.

  2. High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

    Directory of Open Access Journals (Sweden)

    Hubbard Kyle S

    2012-10-01

    Full Text Available Abstract Background Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. Methods With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC lines (R1, C57BL/6 and D3 adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX, and botulinum neurotoxin (BoNT, were also evaluated. Results Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons

  3. Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures.

    Science.gov (United States)

    Fox, Emily P; Cowley, Elise S; Nobile, Clarissa J; Hartooni, Nairi; Newman, Dianne K; Johnson, Alexander D

    2014-10-20

    The human microbiome contains diverse microorganisms, which share and compete for the same environmental niches. A major microbial growth form in the human body is the biofilm state, where tightly packed bacterial, archaeal, and fungal cells must cooperate and/or compete for resources in order to survive. We examined mixed biofilms composed of the major fungal species of the gut microbiome, Candida albicans, and each of five prevalent bacterial gastrointestinal inhabitants: Bacteroides fragilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. We observed that biofilms formed by C. albicans provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found that coculture with bacteria in biofilms induces massive gene expression changes in C. albicans, including upregulation of WOR1, which encodes a transcription regulator that controls a phenotypic switch in C. albicans, from the "white" cell type to the "opaque" cell type. Finally, we observed that in suspension cultures, C. perfringens induces aggregation of C. albicans into "mini-biofilms," which allow C. perfringens cells to survive in a normally toxic environment. This work indicates that bacteria and C. albicans interactions modulate the local chemistry of their environment in multiple ways to create niches favorable to their growth and survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    , calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most......Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7...... days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...

  5. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  6. Swedish Military Bases of the Cold War The Making of a New Cultural Heritage

    Directory of Open Access Journals (Sweden)

    Per Strömberg

    2010-12-01

    Full Text Available The fall of the Berlin Wall and the dissolution of the Soviet Union completely transformed the military-political situation in the Nordic countries. The movement from invasion defence to input defence in Sweden has made many of the subter-ranean modern fortresses and permanent defence systems of the Cold War unnec-essary. The current problem is what the administration authorities will do with the superfluous military buildings: let them fall into decay, preserve or reuse them – and for what purpose? The aim of this article is to describe and analyze the cultural as well as spatial foundation of a new genre of heritage industry in Sweden – the cultural heritage of the Cold War – whose value is negotiated through a range of processes by the different stakeholders involved – emotional, social and cultural processes as well as legal and economic processes. The subterranean fortresses of Hemsö and Aspö are used as empirical case studies in the article. They both describe the making of a cultural heritage and illustrate the problems related to the ambitions of convert-ing cultural heritage into tourist attractions. One of the conclusions is that the previous making of the industrial cultural heritage in the 1980s and 1990s has many things in common with the one of the Cold War. The “post-military” landscape of bunkers and rusting barbed wires is regarded with the same romanticism and with similar preservation ideologies and economic interests as the post-industrial landscape was earlier. Similar negotiation issues appear, and these negotiations are carried out by similar stakeholders. The difference is that the military culture heritage of the Cold War was developed through a deeply centralized selection process directed by administration authori-ties, but was also influenced by certain persuasion campaigns and preservation actions made by local stakeholders such as retired officers and municipality ad-ministrations.

  7. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  8. Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2015-06-01

    Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

  9. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Chelliah Jayabaskaran

    2007-11-01

    Full Text Available Abstract Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc and strictosidine synthase (Str. In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s, Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

  10. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Science.gov (United States)

    Ramani, Shilpa; Chelliah, Jayabaskaran

    2007-01-01

    Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. PMID:17988378

  11. Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

    Science.gov (United States)

    Khurshid, Madiha; Mulet-Sierra, Aillette; Adesida, Adetola; Sen, Arindom

    2017-07-28

    Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Effects of boron deficiency in cell suspension cultures of Populus alba L.

    Science.gov (United States)

    Kakegawa, Koichi; Ishii, Tadashi; Matsunaga, Toshiro

    2005-01-01

    Cell suspension cultures of Populus alba L. (original cells) require at least 10 microM boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 microM). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 microM boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.

  13. Flow cytometry and phytochemical analysis of a sunflower cell suspension culture in a 5-L bioreactor.

    Science.gov (United States)

    Haas, Christiane; Weber, Jost; Ludwig-Müller, Jutta; Deponte, Sandra; Bley, Thomas; Georgiev, Milen

    2008-01-01

    A cell suspension culture of sunflower (Helianthus annuus), a producer of immunologically active polysaccharides, was cultivated in a 5-L stirred tank bioreactor, operated in batch mode. After some changes in the internal bioreactor design a stable growth of Helianthus cells was achieved and the accumulated biomass reached 15.2 g/L (only approximately 5% lower compared to the accumulated biomass in shake-flasks). Flow cytometry used for measuring the cell cycle parameters of suspended Helianthus cells did not reveal significant differences between shake-flasks and bioreactor cultivation modes. For both cultivation methods significant enhancement of the percentage of S-phase cells was observed at the beginning of the cultivation process. Concerning the metabolite production the maximum in exopolysaccharides was reached at day 9 of the cultivation period (1.9 g/L), while the highest amounts of alpha-tocopherol were accumulated at the beginning of the cultivation process (day 2 of the cultivation). These finding were related to the respective stress levels caused by the inoculation procedure. The kinetic parameters of growth and polysaccharide production as well as the time course of carbon source utilization were monitored and discussed.

  14. Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Thu V. Vuong

    2014-06-01

    Full Text Available Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA, salicylic acid, β-glucan (GLU, and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

  15. Adaption of FMDV Asia-1 to Suspension Culture: Cell Resistance Is Overcome by Virus Capsid Alterations

    Science.gov (United States)

    Dill, Veronika; Hoffmann, Bernd; Beer, Martin

    2017-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease with catastrophic economic impact for affected countries. BHK21 suspension cells are preferred for the industrial production of FMDV vaccine antigen, but not all virus strains can be successfully propagated in these cells. Serotype Asia-1 is often affected by this phenomenon. In this study, the Asia-1 strain Shamir was used to examine viral, cellular and environmental factors that contribute to resistance to cell culture infection. Cell media composition, pH and ammonium chloride concentration did not affect Asia-1 differently than other serotypes. Virus replication after transfection of viral genome was not impaired, but the adhesion to the cells was markedly reduced for Asia-1 in comparison to serotype A. The Asia-1 Shamir virus was successfully adapted to grow in the resistant cells by using a closely related but susceptible cell line. Sequence analysis of the adapted virus revealed two distinct mutations in the capsid protein VP1 that might mediate cell attachment and entry. PMID:28820470

  16. Quercetin-induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis.

    Science.gov (United States)

    Mahady, G B; Beecher, C W

    1994-12-01

    Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds: baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Of the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with doses of 100 microM increasing alkaloid production over 375% as compared to negative controls. Quercetin's inductive effects were similar to that of an elicitor derived from fungus Penicillium expansum (PE-elicitor). Suppression of quercetin and PE-induced alkaloid biosynthesis by low doses of actinomycin D (5 micrograms/ml, alpha-amanitin (20 micrograms/ml), or cycloheximide (1 microgram/ml) demonstrate a requirement for both RNA and de novo cytoplasmic protein synthesis and suggest that alterations in gene expression are involved in the inductive mechanism. Furthermore, quercetin-induced alkaloid biosynthesis was significantly reduced by pretreatment of the cells with the calcium chelator, EGTA (3 mM), or the calcium channel inhibitor, verapamil (100 microM), suggesting that this process was calcium dependent.

  17. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

    Science.gov (United States)

    Singh, Harmeet; Mok, Pamela; Balakrishnan, Thavamalar; Rahmat, Siti Norfiza Binte; Zweigerdt, Robert

    2010-05-01

    We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. Copyright 2010 Elsevier B.V. All rights reserved.

  18. Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco1

    Science.gov (United States)

    Fujita, Satoshi; Uchimura, Seiichi; Noguchi, Masahiro; Demura, Taku

    2016-01-01

    Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures. PMID:26747285

  19. Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Dipto

    2012-05-01

    Full Text Available Abstract Background Podophyllotoxin (PTOX, the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA elicitation. High-resolution two-dimensional gel electrophoresis (2-DE followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell’s proteome. Result The HPLC analysis showed approximately 7–8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed. Conclusions Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.

  20. A novel terpenoid indole alkaloid derived from catharanthine via biotransformation by suspension-cultured cells of Catharanthus roseus.

    Science.gov (United States)

    He, Shuijie; Zhu, Jianhua; Zi, Jiachen; Zhou, Pengfei; Liang, Jincai; Yu, Rongmin

    2015-12-01

    Although catharanthine (1) is well known as a biosynthetic precursor of the anticancer alkaloid, vinblastine, its alternative metabolic pathways are unclear. Biotransformation of 1 by suspension-cultured cells of Catharanthus roseus gave a new oxidative-cleavage product (2). The structure of 2 was determined as 3-hydroxy-4-imino-catharanthine by spectroscopic methods. Maximum conversion (9.75 %) of 2 was observed after 120 h adding 6 mg of 1/100 ml to 12-day-old suspension-cultured cells of C. roseus. Furthermore, qRT-PCR experiment was performed to reveal the effect of 1 on the expression of the genes in the biosynthetic pathway of TIA 1 up-regulated the transcript level of D4H whilst down-regulating the transcript levels of G10H, LAMT, GES, and IRS. A new metabolite of catharanthine, 3-hydroxy-4-imino-catharanthine, is reported.

  1. Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

    2015-04-01

    Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

  2. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

    Science.gov (United States)

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid

    2013-01-01

    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction. PMID:19609626

  3. Changes of Respiration Activities in Cells of Winter Wheat and Sugar Cane Suspension Cultures During Programmed Cell Death Process

    Directory of Open Access Journals (Sweden)

    I.V. Lyubushkina

    2015-09-01

    Full Text Available Process of cell death in suspension cultures of winter wheat and sugar cane under high (50 °С and negative (-8 °С temperature treatment has been studied. It has been shown, that programmed cell death (PCD process caused by the negative temperature in the culture of winter wheat was noted for slow rate of realization and it was carried out for 10 days. It has been state that rate of cell respiration was significantly higher than in the control culture. At the same time PCD processes induced by the high temperature in the culture of sugar cane and winter wheat and by the negative temperature in the culture of sugar cane realized for 24-48 h and was accompanied by graduate decrease of respiration activities. We can conclude that the main reason of PCD processes realization differences was a different level of respiration metabolism resistance to high and negative temperatures action.

  4. Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

    Science.gov (United States)

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2014-01-01

    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period. PMID:25089711

  5. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L. Dunal in shake-flask culture and bioreactor.

    Directory of Open Access Journals (Sweden)

    Ganeshan Sivanandhan

    Full Text Available The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg, withanolide B (4826.05 mg, withaferin A (3732.81 mg, withanone (6538.65 mg, 12 deoxy withanstramonolide (3176.63 mg, withanoside IV (2623.21 mg and withanoside V (2861.18 mg] were achieved in the combined treatment of chitosan (100 mg/l and squalene (6 mM along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold as well as bioreactor (1.66-fold when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

  6. "Noi Donne" and "Famiglia Cristiana": Communists, Catholics, and American Female Culture in Cold War Italy

    OpenAIRE

    Harris, Jessica L

    2017-01-01

    Italy's Cold War cultural contest for the hearts and minds of Italian women was a three way struggle between the Catholic Church, the Italian Communists, and the United States. The arrival of American consumer products and models in postwar Italy and their growing influence on upper to middle-class, and eventually working-class women, provided the two domestic groups, who previously had been engaged in a bipolar struggle with each other, with a common enemy - the materialistic, immoral, and a...

  7. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Kajani Abolghasem

    2012-10-01

    Full Text Available Abstract Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale. Methods We investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing. Results The yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  8. Induction and Analysis of the Alkaloid Mitragynine Content of a Mitragyna speciosa Suspension Culture System upon Elicitation and Precursor Feeding

    Directory of Open Access Journals (Sweden)

    Nor Nahazima Mohamad Zuldin

    2013-01-01

    Full Text Available This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D, kinetin, 6-benzylaminopurine (BAP, and 1-naphthaleneacetic acid (NAA on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L−1 2, 4-D (70.83%. Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L−1 2,4-D and 3% sucrose (9.47±0.4667 mL. The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L−1 yeast extract (9.275±0.082 mg L−1 that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 μM tryptophan and harvested at 6 days (13.226±1.98 mg L−1.

  9. Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension cultures of Decalepis hamiltonii Wight & Arn., in shake flask culture.

    Science.gov (United States)

    Matam, Pradeep; Parvatam, Giridhar; Shetty, Nandini P

    2017-12-01

    The flavour rich tuberous roots of Decalepis hamiltonii are known for its edible and medicinal use and have become endangered due to commercial over-exploitation. Besides 2-Hydroxy-4-methoxy benzaldehyde (2H4MB), other flavour metabolites in tuberous roots include vanillin, 4-Methoxy Cinnamic acid derivatives, aromatic alcohols etc. So far, there are no reports on the pathway of 2H4MB biosynthesis nor there is an organized work on biotransformation using normal and cell suspension cultures for obtaining these metabolites using precursors. The main aim of the study is to develop a method for enhanced production of flavour attributing metabolites through ferulic acid (FA) feeding to the D. hamiltonii callus culture medium. Biomass of D. hamiltonii cell suspension cultures was maximum (200.38 ± 1.56 g/l) by 4th week. Maximum production of 2H4MB was recorded on 4th week (0.08 ± 0.01 mg/100 g dry weight) as quantified by HPLC. Addition of 0.1-1.5 mM ferulic acid as precursor in the culture medium showed significant (p < 0.001) effect on suspension cultures biomass and respective phenylpropanoid metabolites content and 2H4MB accumulation. The maximum accumulation of vanillin, 2H4MB, vanillic acid, ferulic acid were of 0.1 ± 0.02 mg/100 g, 0.44 ± 0.01 mg/100 g, 0.52 ± 0.04 mg/100 g, 0.18 ± 0.02 mg/100 g DW respectively in 4 weeks of cultured cells supplemented with 1 mM ferulic acid as a precursor. The results indicate that, substantial increase in the levels of flavour metabolites in D. hamiltonii callus suspension culture was achieved. This would be having implications in biosynthesis of respective vanilla flavour attributing metabolites at very high levels for their large scale production.

  10. Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells.

    Science.gov (United States)

    Nikolay, Alexander; Castilho, Leda R; Reichl, Udo; Genzel, Yvonne

    2017-03-23

    The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21 SUS ) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV PE ) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21 SUS cells with ZIKV PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×10 3 PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×10 7 cells/mL, and virus titers of 3.9×10 7 PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards

  11. The Congress for Cultural Freedom seen from the dynamics of the Cold War

    Directory of Open Access Journals (Sweden)

    Francisco Javier Ruiz Durán

    2014-01-01

    Full Text Available The aim of this paper is to show how, at the height of the Cold War, the British intelligence services responded to the new spy system created by the Comintern developing a secret campaign of political and cultural propaganda, under the cover of prestigious foundations to channel intellectuals in defense of a liberal democracy. This spy system expected to carry out propaganda secret operations and manipulate the intellectuals from almost the very beginning of the Soviet Revolution. The keystone to win the battle of consciences was the Congress for Cultural Freedom and its editorials, magazines, exhibitions, scholarships, concerts, congresses and conferences. Finally, it will be noted how the Congress for Cultural Freedom implemented the political conception of "non-communist left" to sustain the social democracy in the Western bloc.

  12. Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species.

    Science.gov (United States)

    Kauss, H.; Jeblick, W.; Ziegler, J.; Krabler, W.

    1994-05-01

    Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species. Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f. sp. glycinea (Pmg elicitor) and by the polycation chitosan. Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 [mu]M JAME. Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion. These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants. It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion. Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.

  13. Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

    Science.gov (United States)

    dos Santos, André Luis Wendt; El Gueddari, Nour Eddine; Trombotto, Stéphane; Moerschbacher, Bruno Maria

    2008-12-01

    Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

  14. Soviet Cultural Diplomacy towards Denmark during the Cold War, 1945-1991

    DEFF Research Database (Denmark)

    Frederichsen, Kim

    This dissertation presents an analysis and discussion of the Soviet cultural diplomatic activities towards Denmark during the Cold War, 1945-1991. The overall aim of the dissertation is to answer three questions: 1.How were the activities organized and financed? 2.What forms of activities took......, and the so-called public organizations, the International level contains various international front organizations (e.g. the World Peace Council or Cominform), and the national level contains the national affiliates of international peace organizations (e.g.“Fredens Tilhængere”), the Danish-Soviet friendship...

  15. Factors influencing cucumber (Cucumis sativus L.) somatic embryogenesis. I. The crucial role of pH and nitrogen in suspension culture

    OpenAIRE

    Tadeusz Wróblewski; Marcin K. Filipecki; Stefan Malepszy

    2014-01-01

    A method of obtaining and the characteristics of an embryogenic stabilised cucumber (Cucumis sativus L.) suspension culture which has many similarities to the carrot model are presented. The Specific Type I cells and proembryogenic mass were present in such a suspension. The maintenance of the proembryogenic stage took place in medium containing 2,4-D as the sole growth regulator, subsequent stages of embryogenesis occurred in hormone-free medium. Embryonic structures were also observed in me...

  16. Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda 'Giganteus' as affected by proline

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Krogstrup, Peter; Hansen, Jürgen

    1997-01-01

    The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and ...

  17. Altered nitrogen metabolism associated with de-differentiated suspension cultures derived from root cultures of Datura stramonium studied by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy.

    Science.gov (United States)

    Fliniaux, Ophélie; Mesnard, François; Raynaud-Le Grandic, Sophie; Baltora-Rosset, Sylvie; Bienaimé, Christophe; Robins, Richard J; Fliniaux, Marc-André

    2004-05-01

    De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. (15)N-Labelled precursors [((15)NH(4))(2)SO(4) and K(15)NO(3)] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and gamma-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.

  18. Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of Artemisia absinthium L.

    Science.gov (United States)

    Ali, Mohammad; Abbasi, Bilal Haider; Ahmad, Nisar; Ali, Syed Shujait; Ali, Shahid; Ali, Gul Shad

    2016-12-01

    Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.

  19. The Congress for Cultural Freedom, "Minerva," and the Quest for Instituting "Science Studies" in the Age of Cold War

    Science.gov (United States)

    Aronova, Elena

    2012-01-01

    The Congress for Cultural Freedom is remembered as a paramount example of the "cultural cold wars." In this paper, I discuss the ways in which this powerful transnational organization sought to promote "science studies" as a distinct--and politically relevant--area of expertise, and part of the CCF broader agenda to offer a renewed framework for…

  20. Effect of heavy metal treatments on metallothionein expression profiles in white poplar (Populus alba L. cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Anca MACOVEI

    2010-11-01

    Full Text Available Populus species and hybrids are intensively cultivated as sources of woody biomass and are good candidates for phytoremediation because of their rapid growth rate, extensive root system and ease of propagation and transformation. To date, the molecular mechanisms that regulate heavy metal tolerance have not been fully investigated. In the present work, white poplar (Populus alba L. cell suspension cultures were used as model system to investigate the response to heavy metal treatments. The VFMT2 cDNA, encoding a type 2 metallothionein from P. alba, was isolated by RT-PCR approach. The expression profiles of the VFMT2 gene were then investigated by Quantitative Real Time Polymerase Chain Reaction (QRT-PCR under oxidative stress conditions. The latter were induced by exposing the cell suspension cultures to different doses of cadmium (75 and 150 μM CdSO4, copper (50 and 100 μM CuCl2 and zinc (1 and 2 mM ZnSO4. Cell death was evidenced by Evans blue staining. The VFMT2 gene was up-regulated in response to heavy metal treatments and the highest mRNA level (up to 5-fold was observed 4 h following exposure to 100 μM CuCl2.

  1. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  2. Elicitation of Jerusalem artichoke (Helianthus tuberosus L.) cell suspension culture for enhancement of inulin production and altered degree of polymerisation.

    Science.gov (United States)

    Ma, Chunquan; Zhou, Dong; Wang, Haitao; Han, Dongming; Wang, Yang; Yan, Xiufeng

    2017-01-01

    Plant cell suspension cultures have emerged as a potential source of secondary metabolites for food additives and pharmaceuticals. In this study inulin accumulation and its degree of polymerisation (DP) in the treated cells in the same medium were investigated after treatment with six types of elicitors. An in vitro cell suspension culture of Jerusalem artichoke (Helianthus tuberosus L.) was optimised by adding an extra nitrogen source. According to the growth kinetics, a maximum biomass of 5.48 g L -1 was obtained from the optimal cell suspension medium consisted of Murashige and Skoog basic medium (MS) + 1.0 mg L -1 α-naphthalene acetic acid (NAA) + 1.0 mg L -1 6-benzylaminopurine (6-BA) + 0.5 mg L -1 proline + 1.0 mg L -1 glutamine. Methyl jasmonate (MeJA, 250 µmol L -1 ) treatment for 15 days led to the highest levels of inulin (2955.27 ± 9.81 mg L -1 compared to control of 1217.46 ± 0.26 mg L -1 ). The elicited effect of five elicitors to the suspension cells of Jerusalem artichoke is as follows: AgNO 3 (Ag, 10 µmol L -1 ), salicylic acid (SA, 75 µmol L -1 ), chitosan (KJT, 40 mg L -1 ), Trichoderma viride (Tv, 90 mg L -1 ), yeast extract (YE, 0.25 mg L -1 ), and the corresponding content of inulin is increased by 2.05-, 1.93-, 1.76-, 1.44- and 1.18-fold compared to control, respectively. The obvious effect on the percentage of lower DP in inulin was observed in cells treated with 40 mg L -1 KJT, 0.25 mg L -1 YE and 10 µmol L -1 Ag. Among the six types of elicitors, the descending order of inulin content is MeJA > Ag > SA > KJT > Tv > YE. For the purpose inulin with lower DP and its application to prebiotic food, three elicitors, including KJT, YE and Ag, can be used for the elicitation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  3. Characterization of culturable bacteria isolated from the cold-water coral Lophelia pertusa

    Science.gov (United States)

    Galkiewicz, Julia P.; Pratte, Zoe A.; Gray, Michael A.; Kellogg, Christina A.

    2011-01-01

    Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial-coral associations. The Kirby-Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.

  4. Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea.

    Science.gov (United States)

    Kenta, Tanaka; Edwards, Jessica E M; Butlin, Roger K; Burke, Terry; Quick, W Paul; Urwin, Peter; Davey, Matthew P

    2016-12-07

    While genotype-environment interaction is increasingly receiving attention by ecologists and evolutionary biologists, such studies need genetically homogeneous replicates-a challenging hurdle in outcrossing plants. This could be potentially overcome by using tissue culture techniques. However, plants regenerated from tissue culture may show aberrant phenotypes and "somaclonal" variation. Here, we examined somaclonal variation due to tissue culturing using the response to cold treatment of photosynthetic efficiency (chlorophyll fluorescence measurements for Fv/Fm, Fv'/Fm', and ΦPSII, representing maximum efficiency of photosynthesis for dark- and light-adapted leaves, and the actual electron transport operating efficiency, respectively, which are reliable indicators of photoinhibition and damage to the photosynthetic electron transport system). We compared this to variation among half-sibling seedlings from three different families of Arabidopsis lyrata ssp. petraea Somaclonal variation was limited, and we could detect within-family variation in change in chlorophyll fluorescence due to cold shock successfully with the help of tissue-culture derived replicates. Icelandic and Norwegian families exhibited higher chlorophyll fluorescence, suggesting higher performance after cold shock, than a Swedish family. Although the main effect of tissue culture on Fv/Fm, Fv'/Fm', and ΦPSII was small, there were significant interactions between tissue culture and family, suggesting that the effect of tissue culture is genotype-specific. Tissue-cultured plantlets were less affected by cold treatment than seedlings, but to a different extent in each family. These interactive effects, however, were comparable to, or much smaller than the single effect of family. These results suggest that tissue culture is a useful method for obtaining genetically homogenous replicates for studying genotype-environment interaction related to adaptively-relevant phenotypes, such as cold response, in

  5. Influence of rare earth elements on metabolism and related enzyme activity and isozyme expression in Tetrastigma hemsleyanum cell suspension cultures.

    Science.gov (United States)

    Xin, Peng; Shuang-Lin, Zhou; Jun-Yao, He; Li, Ding

    2013-04-01

    The effects of rare earth elements (REEs) not only on cell growth and flavonoid accumulation of Tetrastigma hemsleyanum suspension cells but also on the isoenzyme patterns and activities of related enzymes were studied in this paper. There were no significant differences in enhancement of flavonoid accumulation in T. hemsleyanum suspension cells among La(3+), Ce(3+), and Nd(3+). Whereas their inductive effects on cell proliferation varied greatly. The most significant effects were achieved with 100 μM Ce(3+)and Nd(3+). Under treatment over a 25-day culture period, the maximal biomass levels reached 1.92- and 1.74-fold and the total flavonoid contents are 1.45- and 1.49-fold, than that of control, respectively. Catalase, phenylalanine ammonia-lyase (PAL), and peroxidase (POD) activity was activated significantly when the REE concentration range from 0 to 300 μM, whereas no significant changes were found in superoxide dismutase activity. Differences of esterase isozymes under REE treatment only laid in expression level, and there were no specific bands. The expression level of some POD isozymes strengthened with increasing concentration of REEs within the range of 50-200 μM. When REE concentration was higher than 300 μM, the expression of some POD isozymes was inhibited; meanwhile, some other new POD isozymes were induced. Our results also showed REEs did not directly influence PAL activity. So, we speculated that 50-200 μM REEs could activate some of antioxidant enzymes, adjust some isozymes expression, trigger the defense responses of T. hemsleyanum suspension cells, and stimulate flavonoid accumulation by inducing PAL activity.

  6. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  7. Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-01-01

    Full Text Available The auxin level in the cell mass and culture medium was determined by means of the Avena straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour. The investigations were performed in the course of the zero passage (PO and first one (Pl, differing in their time of duration of maximum growth and its intensity. In both passages the intra- and extra-cellular auxin levels reach values of the same order. At the beginning of the maximal growth phase the activity corresponding to IAA in the cells prevails over that of the other auxin-like compounds. This disproportion diminishes with further development of the culture, and with the beginning of the stationary phase the cellular IAA level is lower than that of the remaining auxin-like compounds. The short phase of maximal growth (PO occurs with an auxin level decreasing in the cell mass and increasing in the medium, and towards the end of the cycle these levels become equal. During the long phase of maximal growth (Pl the total amount of auxins in the cells increases and is 2-3 times higher than in the medium, whereas IAA in the cells remains at a constant level. These results suggest that the participation of IAA in the intracellular pool of auxin-like substances is decisive for the mitotic activity of the cells and maintenance of growth in the culture.

  8. Accumulation of ixerin F and activities of some terpenoid bisynthetic enzymes in a cell suspension culture of Lactuca virosa L.

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    Anna Stojakowska

    2014-01-01

    Full Text Available A cell suspension culture of Lactuca virosa L. (Asteraceae, tribe Lactuceae is capable of synthesizing sesquiterpene lactones of which ixerin F is the main compound. The culture was characterized on growth (by dissimilation rates, on ixerin F accumulation (by RP-HPLC and on some enzyme activities involved in early steps of terpenoid biosynthesis. Acetoacetyl-coenzyme A thiolase (AACT, E.C. 2.3.1.9 and 3S-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS, E.C. 4.13.5 activities of the cells were assayed spectrophotometrically. HMGS activity increased during the culture period and reached a maximum during the stationary phase (190 pkat/mg protein, while AACT showed relatively high level of activity throughout the growth cycle, with transient decrease at the logarithmic growth phase and the beginning of stationary phase. Ixerin F accumulated inside the cells and the maximum concentration of 0.08% (on dry weight basis was found in the early stationary phase of the growth cycle of the culture.

  9. The Effect of Plant Growth Regulators and Different Explants on the Response of Tissue Culture and Cell Suspension Cultures of German Chamomile (Matricaria chamomilla L.

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    L. Koohi,

    2014-07-01

    Full Text Available German chamomile (Matricaria chamomilla L. is one of the most important medicinal plants that its essential oils used in different medicinal industries. In this study which was carried out in 2013 growing season at the Faculty of Agricultural Sciences of the University of Mohaghegh Ardabili, the in vitro response of leaf and hypocotyl explants of German Chamomile in B5 medium supplemented with different levels of plant growth regulators including 2,4-D, naphthalene acetic acid (NAA, kinetin and 6-benzylaminopurine (BAP were investigated in a factorial experiment based on completely randomized design (CRD.In addition, cell suspension cultures were established and characterized. Hypocotyl and leaf explants exhibited cell proliferation and produced callus within 1-2 weeks. The highest fresh weight of the callus (264.1 mg was produced by leaf explants in the medium supplemented with 0.5 mg/l 2,4-D and 1 mg/l BAP. However, the leaf explants cultured on medium containing 1.5 mg/l 2,4-D showed the lowest cell proliferation and callus yield (40.42 mg. The highest percentage of root induction from leaf explants (58.73% was observed on the medium containing 4 mg/l 2,4-D and 1 mg/l Kin, and from hypocotyl explants (48.61% was observed on medium supplemented with 1.5 mg/l NAA. The 42.22% of calli derived from hypocotyl explants on B5 medium supplemented with 4 mg/l NAA and 3 mg/l BAP, were friable. Cell suspension cultures of German chamomile were established by transferring of hypocotyl-derived friable calli into the MS medium supplemented with 1.5 mg/l 2,4-D and 1 mg/l kinetin. The growth curve of cell proliferations started 4 days after culture and continued to grow until day 13th, where the cells entered stationary phase.

  10. Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch

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    Abinaya Manivannan

    2016-03-01

    Full Text Available Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS medium containing 3.0 mg·L−1 6-benzyladenine (BA in a combination with 2 mg·L−1 2,4-dichlorophenoxy acetic acid (2,4-D. From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa, salicylic acid (SA, and sodium nitroprusside (SNP on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

  11. Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch.

    Science.gov (United States)

    Manivannan, Abinaya; Soundararajan, Prabhakaran; Park, Yoo Gyeong; Jeong, Byoung Ryong

    2016-03-18

    Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS) medium containing 3.0 mg·L(-1) 6-benzyladenine (BA) in a combination with 2 mg·L(-1) 2,4-dichlorophenoxy acetic acid (2,4-D). From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa), salicylic acid (SA), and sodium nitroprusside (SNP) on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

  12. Mathematical models, rational choice, and the search for Cold War culture.

    Science.gov (United States)

    Erickson, Paul

    2010-06-01

    A key feature of the social, behavioral, and biological sciences after World War II has been the widespread adoption of new mathematical techniques drawn from cybernetics, information theory, and theories of rational choice. Historians of science have typically sought to explain this adoption either by reference to military patronage, or to a characteristic Cold War culture or discursive framework strongly shaped by the concerns of national security. This essay explores several episodes in the history of game theory--a mathematical theory of rational choice--that demonstrate the limits of such explanations. Military funding was indeed critical to game theory's early development in the 1940s. However, the theory's subsequent spread across disciplines ranging from political science to evolutionary biology was the result of a diverse collection of debates about the nature of "rationality" and "choice" that marked the Cold War era. These debates are not easily reduced to the national security imperatives that have been the focus of much historiography to date.

  13. Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L.

    Science.gov (United States)

    Ali, Mohammad; Abbasi, Bilal Haider

    2014-11-01

    Light is an important factor influencing plant morphogenesis and biochemical pathways, including biosynthesis of primary and secondary metabolites. In the present study, we investigated the differential effect of light on biomass accumulation and secondary metabolites production in cell suspension cultures of Artemisia absinthium L. A prolonged log phase of 21 days was followed by light-grown cultures. Light-grown cultures displayed 3.9-fold maximum increase (8.88 g/l) in dry biomass on day 30 of culture which was comparable to 3.7-fold maximum increase (9.2 g/l) on day 27 in dark-grown cultures. Compared to dark grown-cultures, enhanced levels of total phenolic content (5.32 mg/g DW), total phenolic production (42.96 mg/l) and total secondary metabolites (6.79 mg/g) were found in light-grown suspension cultures during the log phase of growth. Further, a positive correlation among maximum levels of antioxidant activity (63.8%), total phenolic production (42.96 mg/l) and total secondary metabolites (6.79 mg/g DW) was displayed by light-grown suspension cultures. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. [Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

    Science.gov (United States)

    Wei, Yue-Rong; Huang, Xue-Lin; Li, Jia; Huang, Xia; Li, Zhe; Li, Xiao-Ju

    2005-01-01

    Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV

  15. Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.

    Science.gov (United States)

    Poudel, Sher Bahadur; Bhattarai, Govinda; Kook, Sung-Ho; Shin, Yun-Ji; Kwon, Tae-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

    2017-10-01

    Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

  16. Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures.

    Science.gov (United States)

    Schöpke, C; Taylor, N; Cárcamo, R; Konan, N K; Marmey, P; Henshaw, G G; Beachy, R N; Fauquet, C

    1996-06-01

    A protocol was established for the introduction of DNA into embryogenic suspension-derived tissues of cassava via microparticle bombardment, for the selection of genetically transformed cells, and for the regeneration of fully transgenic plants from these cells. The plasmid DNA used for bombardment contained a gene encoding neomycin phosphotransferase (nptII) and a gene encoding beta-glucuronidase (uidA). Selection of bombarded tissue with paromomycin resulted in the establishment of putative transgenic embryogenic calli. In most of these calli, beta-glucuronidase was detected histochemically. Molecular analysis of paromomycin-resistant embryogenic calli and of plants regenerated from these calli, confirmed the stable integration of bombarded DNA into the cassava genome.

  17. Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins.

    Science.gov (United States)

    Balestrazzi, Alma; Agoni, Valentina; Tava, Aldo; Avato, Pinarosa; Biazzi, Elisa; Raimondi, Elena; Macovei, Anca; Carbonera, Daniela

    2011-03-01

    The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar 'Villafranca') cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml⁻¹, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml⁻¹ and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin-induced NO production was sensitive to sodium azide and N(G)-monomethyl-L-arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells. Copyright © Physiologia Plantarum 2010.

  18. Role of Changes in Cell Fatty Acids Composition in the Increasing of Frost Resistance of Winter Wheat Suspension Culture

    Directory of Open Access Journals (Sweden)

    I.V. Lyubushkina

    2013-11-01

    Full Text Available Influences of low temperatures (4 and 8 ° С on the frost tolerance and fatty acid compositions of cells in a winter wheat suspension culture have been studied. It has been found that treatment of the culture with 4 °C (7 days did not protect cells from subsequent freezing temperature action (-8 °С, 6 h and was not accompanied significant changes in the fatty acid composition. On the contrary, the treatment of the culture with the temperature 8 °C (7 days prevented the death caused by freezing temperature and the content of saturated fatty acids decreased: pentadecanoic acid (by 35,0%, palmitic acid (by 19,9% and stearic acid (by 65,4%, and the content of α-linolenic acid increased by 94%. That was the cause of the double bond index (DBI increase by 16%. The role of fatty acids composition changes in the process of increasing frost tolerance in plants are discussed.

  19. Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

    Science.gov (United States)

    Rakesh Minocha; Subhash C. Minocha; Stephanie L. Long; Walter C. Shortle

    1992-01-01

    Increased aluminum (Al) solubility in soil waters due to acid precipitation has aroused considerable interest in the problem of Al toxicity in plants. In the present study, an in vitro suspension culture system of Catharanthus roseus (L.) G. Don was used to analyze the effects of aluminum on several biochemical processes in these cells. The aliphatic...

  20. Two-stage culture for producing berberine by cell suspension and shoot cultures of Berberis buxifolia Lam.

    Science.gov (United States)

    Alvarez, María A; Eraso, Natalia Fernandez; Pitta-Alvarez, Sandra I; Marconi, Patricia L

    2009-03-01

    In vitro cultures of Berberis buxifolia were established using thidiazuron (4.5, 23 and 45 mM) or picloram (4 and 40 mM) as plant growth regulators for sustaining growth. For producing berberine, a two-stage culture was performed. In the first step, thidiazuron or picloram were used for biomass production followed by the production stage where benzylaminopurine (4.4 mM) was added as a plant growth regulator. Berberine yields (102 mg g(-1) DW) and in vitro shoot cultures (200 mg g(-1) DW) were significantly lower than those of whole plants in the field (416 mg g(-1) DW). The highest productivity (0.18 mg 1(-1) day(-1)) was attained using picloram (either 4 on 40 mM) in the first stage for producing biomass.

  1. Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.

    Science.gov (United States)

    Chahal, Parminder Singh; Schulze, Erica; Tran, Rosa; Montes, Johnny; Kamen, Amine A

    2014-02-01

    Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  2. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    Science.gov (United States)

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

  3. Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine.

    Science.gov (United States)

    Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Márquez, Ascensión; Bru, Roque; Pedreño, María A

    2015-12-01

    In the present work the effect of cyclodextrin and coronatine on both trans-resveratrol production and the expression of stilbene biosynthetic genes in Vitis vinifera L. cv Monastrell suspension cultured cells were evaluated. The results showed the maximum level of trans-resveratrol produced by cells and secreted to the culture medium with 50 mM cyclodextrins and 1 μM coronatine. Since the levels of trans-resveratrol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. In addition, all the analysed genes were induced by cyclodextrins and/or coronatine. The expression of the phenylalanine ammonia lyase and stilbene synthase genes was greatly enhanced by coronatine although an increase in the amount of trans-resveratrol in the spent medium was not detected. Therefore, despite the fact that trans-resveratrol production is related with the expression of genes involved in the biosynthetic process, other factors may be involved, such as post-transcriptional and post-traductional regulation. The expression maximal levels of cinnamate 4-hydroxylase and 4-coumarate-CoA ligase genes were found with cyclodextrins alone or in combination with coronatine suggesting that the activity of these enzymes could be not only important for the formation of intermediates of trans-R biosynthesis but also for those intermediates involved in the biosynthesis of lignins and/or flavonoids. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  4. Interaction between abscisic acid and nitric oxide in PB90-induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures.

    Science.gov (United States)

    Chen, Qian; Chen, Zunwei; Lu, Li; Jin, Haihong; Sun, Lina; Yu, Qin; Xu, Hongke; Yang, Fengxia; Fu, Mengna; Li, Shengchao; Wang, Huizhong; Xu, Maojun

    2013-01-01

    Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers.

  5. The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies.

    Science.gov (United States)

    Sauter, M; Hager, A

    1989-08-01

    A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

  6. Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture.

    Science.gov (United States)

    Iranbakhsh, A R; Oshagi, M A; Ebadi, M

    2007-04-15

    Abstract: A number of physicochemical conditions such different concentration of glucose, sucrose, potassium nitrate, ammonium nitrate, calcium chloride and temperatures were tested to optimize growth and production of tropane alkaloids from Datura stramonium (Solanaceae) plants. Cell suspension from semi-clear calli of leave explants developed in MS medium containing kinetin (0.5 mg L(-1)) and NAA (2 mg L(-1)) hormones was used to measure biomass and total alkaloids and comparison of treatments. The results showed that 30 and 40 g L(-1) glucose led to the highest level of alkaloids and biomass productions, respectively. 20 and 40 g L(-1) sucrose concentrations resulted in order the most rates of alkaloids and biomass productions. The results showed that increasing of nitrate concentration led to the reduction of the alkaloids. The best concentration of potassium nitrate for the production of tropane alkaloids and biomass were in order 9.4 and 3.76 mM. Also it was evinced that the optimized concentration of ammonium nitrate for alkaloids production was 10.3 mM and for the biomass was 41.22 mM. The best concentration of calcium chloride for growth and production of the alkaloids was 7.92 mM. Testing different temperature specified that the best condition for production of the alkaloids was 20 degrees C whereas it was 25 degrees C for biomass production. The results of this study could be recommended to farmers involved in production of D. stramonium for tropain alkaloids at industrial and semi-industrial scales.

  7. Establishment of Cell Suspension Culture and Plant Regeneration in Abrus precatorius L., a Rare Medicinal Plant

    Directory of Open Access Journals (Sweden)

    Mohammad Serajur RAHMAN

    2012-02-01

    Full Text Available A new protocol has been developed for cell culture and in vitro regeneration of Abrus precatorius that holds enormous potentiality for preparation of medicines. In vitro grown calli were cultured in Murashige and Skoog (MS liquid media in agitated condition fortified with 0.5 mg/l 6-Benzylaminopurine. Growth curve of cells revealed that the cells continued to grow until 12 days of culture and got the highest peak from day 6-8. Isolated cell was found to produce highest 8.2% calli when suspended on MS medium supplemented with 0.5 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. Callus derived from single cell produced highest number of embryo (25-28% cultured on MS medium fortified with 2.0 mg/l 6-Benzylaminopurine and 0.2 mg/l 1-Naphthaleneacetic acid. The bipolar embryos were selected and optimum shoot formation was recorded on MS medium supplemented with 2.0 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. The optimum root induction was noticed in MS medium supplemented with 1.0 mg/l 3-Indolebutyric acid. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions.

  8. Role of polyamines in DNA synthesis of Catharanthus roseus cells grown in suspension culture

    Science.gov (United States)

    Rakesh Minocha; Subhash C. Minocha; Atsushi Komamine; Walter C. Shortle

    1990-01-01

    The requirement for polyamines in the proliferation of cells was first demonstrated in bacteria (3). While significant progress has been made in this field using animal cell cultures, only preliminary studies have been reported with plant tissues. Serafini-Fracassini et al. (9) showed a marked increase in polyamine synthesis early during the G 1 phase, concomitant with...

  9. Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for Steviol glycoside production.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2014-03-01

    Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of NaCl (0.05-0.20%) and Na2CO3 (0.0125-0.10%) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57% with 0.10% NaCl, and 0.025% Na2CO3, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na2CO3 enhanced the SGs content from 1.36 (control) to 2.61 and 5.14%, respectively, on the 10th day.

  10. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

    Science.gov (United States)

    2012-01-01

    Background Taxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation. PMID:22530557

  11. Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

    Science.gov (United States)

    Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

    2013-10-01

    In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

    Directory of Open Access Journals (Sweden)

    Lenka Sangram K

    2012-04-01

    Full Text Available Abstract Background Taxol® (paclitaxel promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

  13. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

    Directory of Open Access Journals (Sweden)

    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  14. Establishment of cell suspension culture in Marchantia linearis Lehm & Lindenb. for the optimum production of flavonoids.

    Science.gov (United States)

    Krishnan, Remya; Anil Kumar, V S; Murugan, K

    2014-02-01

    Bryophytes are the second largest group in the plant kingdom, but studies conducted to better understand their chemical composition are limited and scattered. Axenically grown bryophytes expressed potential in biotechnological processes. The present study was designed to investigate the in vitro cell growth, culture parameters and their effect on flavonoid synthesis. Chlorophyll-containing callus cells of Marchantia linearis Lehm & Lindenb. is able to grow under low light in the presence of organic carbon source and retain the ability to produce flavonoids. Highest flavonoid production was achieved using 2,4-dichlorophenoxyacetic acid as growth hormone. Inoculum size, light intensity, organic carbon source and cations are the culture parameters affecting flavonoid productivity. Maximum flavonoid productivity is observed under low light intensity, with a photon flux density ca. 20 μmol/m2/s. Optimal inoculum size and glucose concentration for flavonoid production are 10-14 and 2-3 %, respectively. Cations like ferrous trigger flavonoid synthesis by increasing its intracellular concentrations. Flavonoid production in the cell culture is shown to be significantly growth related. Osmotic stress is ineffective in triggering flavonoid synthesis. Methyl jasmonate and 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-carbothioamide elicitors showed positive effect on intracellular flavonoid content in cultured cells. Using the standard plot of quercetin (y = 0.0148x, R2 = 0.975), the flavonoid contents of in vitro samples were found ranging from 4.0 to 17.7 mg quercetin equivalent/g tissue. Flavonoids are fractionated by HPLC-PAD revealed the presence of quercetin (182.5 μg/g), luteolin (464.5 μg/g) and apigenin (297.5 μg/g). Further studies are warranted to analyze the therapeutic potentiality of the flavonoids in the liverwort.

  15. Effect of ultrasonic waves on crocin and safranal content and expression of their controlling genes in suspension culture of saffron (Crocus sativus L.).

    Science.gov (United States)

    Taherkhani, Tofigh; Asghari Zakaria, Rasool; Omidi, Mansoor; Zare, Naser

    2017-11-10

    The expression of biosynthesis controlling genes of crocin and safranal in saffron (Crocus sativus) can be influenced by ultrasonic waves. Sterilized saffron corms were cultured in a ½-MS medium supplemented by 2-4-D and BAP.  Saffron callus cells were treated with ultrasonic waves in a cellular suspension culture under optimal growth conditions. The samples were collected at 24 and 72 hours after treatment in three replications. The secondary metabolites were measured by high-performance liquid chromatography and the gene expression was analysed by the real-time polymerase chain reaction. Results indicate that this elicitor can influence the expressions of genes CsBCH, CsLYC and CsGT-2; the ultrasonic waves acted as an effective mechanical stimulus to the suspension cultures. The analysis of variance of the ultrasonically produced amounts of safranal and crocin indicates that there is a significant difference between once- and twice-treated samples in that the amount of safranal was the highest within the samples taken from the twice-treated suspension culture at 72 h after the ultrasound treatment, and the crocin was maximised after 24 h passed the twice-applied ultrasound treatment.

  16. White poplar (Populus alba L.) suspension cultures as a model system to study apoptosis induced by alfalfa saponins.

    Science.gov (United States)

    Balestrazzi, Alma; Carbonera, Daniela; Avato, Pinarosa; Tava, Aldo

    2014-01-01

    In animal cells, the anticancer function played by plant saponins involves a complex network of molecular processes that still deserves investigation and apoptosis seems to be the outstanding pathway. An intriguing aspect of the biological activity of saponins is related to their effects on genome integrity. As demonstrated by the studies carried out in white poplar (Populus alba L., cv Villafranca) cell suspension cultures, plant cells can as well be used as a model system to unravel the molecular mechanisms activated by plant saponins. These recent studies have evidenced that animal and plant cells share common features in their response to saponins, paving the way for novel opportunities for both basic and applied research. Indeed, there is a certain interest in replacing the animal models for pharmacological research, at least when preliminary large-scale cytotoxicity tests are performed on wide collections of natural extracts and/or purified compounds. The review provides an up-date of the molecular pathways (signal transduction, antioxidant response, DNA repair) associated with plant saponin bioactivity, with an emphasis on apoptosis induced by alfalfa (Medicago sativa L.) saponins. The comparison between animal and plant cells as tools for the study of saponin bioactivity is also discussed in view of the most recent literature and innovative future applications.

  17. Methyl Jasmonate and Salicylic Acid Induced Oxidative Stress and Accumulation of Phenolics in Panax ginseng Bioreactor Root Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Kee-Yoeup Paek

    2007-03-01

    Full Text Available To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 μM methyl jasmonate (MJ or salicylic acid (SA in a 5 L bioreactor suspension culture (working volume 4 L. Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2 contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH radical reducing activity were increased by MJ and SA. Fresh weight (FW and dry weight (DW decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%, DPPH activity (40%, flavonoids (88%, ascorbic acid (55%, NPSH (33%, and cysteine (62% contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and β-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

  18. Nitric Oxide Functions as a Signal in Ultraviolet-B-Induced Baicalin Accumulation in Scutellaria baicalensis Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Jin-Jie Zhang

    2014-03-01

    Full Text Available Stress induced by ultraviolet-B (UV-B irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP, led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, Nω-nitro-l-arginine (LNNA, and NO scavenger, 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation.

  19. Relationships between hydroxyproline-containing proteins secreted into the cell wall and medium by suspension-cultured Acer psedoplatanus cells

    Energy Technology Data Exchange (ETDEWEB)

    Pope, D.G.

    1977-05-01

    The pathway of hydroxyproline-containing proteins to the cell wall and to the growth medium in suspension-cultured Acer pseudoplatanus cells is traced by following the kinetics of the transfer of protein-bound /sup 14/C-hydroxyproline into various fractions, and by comparing the hydroxyproline-arabinoside profiles of these fractions after alkaline hydrolysis. Hydroxyproline-rich protein passes directly from a membrane-bound compartment in the cytoplasm to the cell wall, not via an intermediate salt-soluble pool in the wall. There are at least three hydroxyproline-containing glycoproteins in the cell wall. One which possesses mono-, tri-, and tetraarabinoside side chains accounts for over 90% of the total hydroxyproline. This glycoprotein is ''extensin.'' The hydroxyproline-containing proteins secreted into the medium have a glycosylation pattern markedly different from that of the major cell wall glycoprotein. It appears that there is little or no wall-like extensin in the medium. Approximately half of the protein-bound hydroxyproline secreted into the medium is linked to an arabinogalactan. This linkage is also found in a particulate wall protein precursor fraction from the cytoplasm, but only trace amounts can be detected in the cell wall.

  20. Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

    Directory of Open Access Journals (Sweden)

    Li Tan

    Full Text Available Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

  1. New Synthetic Pyridine Derivate as Potential Elicitor in Production of Isoflavonoids and Flavonoids in Trifolium pratense L. Suspension Culture

    Directory of Open Access Journals (Sweden)

    Marie Kašparová

    2012-01-01

    Full Text Available The production of secondary metabolites in Trifolium pratense L. suspension culture of the family of legume plants (Fabaceae is low, and therefore there was an attempt to increase it by elicitation. New synthetic substance, 2-(2-fluoro-6-nitrobenzylsulfanylpyridine-4-carbothioamide, was tested as elicitor—a substance that showed the best elicitation effect after 48-hour application of 1 μmol L−1 concentration. Maximum contents of genistin (11.60 mg g−1 DW, daidzein (8.31 mg g−1 DW, and genistein (1.50 mg g−1 DW were recorded, and the production of these isoflavonoids thus significantly increased, when compared with the control, by 152%, 151%, and 400%. The maximum content of flavonoids (5.78 mg g−1 DW and the increase in the production by 142%, when compared with the control, were induced by 6-hour application of 100 μmol L−1 concentration. The tested substance showed to be an effective elicitor of phenylpropane metabolism.

  2. Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

    Science.gov (United States)

    Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

    2012-03-01

    Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

  3. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Characterization of lentiviral vector production using microwell suspension cultures of HEK293T-derived producer cells.

    Science.gov (United States)

    Guy, Heather M; McCloskey, Laura; Lye, Gary J; Mitrophanous, Kyriacos A; Mukhopadhyay, Tarit K

    2013-04-01

    ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 μg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

  5. A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization).

    Science.gov (United States)

    Baldwin, T. C.; McCann, M. C.; Roberts, K.

    1993-09-01

    Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.

  6. Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid.

    Science.gov (United States)

    Sahraroo, Amir; Mirjalili, Mohammad Hossein; Corchete, Purificación; Babalar, Mesbah; Fattahi Moghadam, Mohammad Reza

    2016-08-01

    An in vitro approach to the production of rosmarinic acid (RA), a medicinally important caffeic acid ester, in a cell suspension culture (CSC) of Satureja khuzistanica Jamzad (Lamiaceae) has been investigated for the first time. The CSC was established from friable calli derived from shoot tip explants in Gamborg's B5 liquid medium supplemented with 30 g/L sucrose, 20 mg/L L-glutamine, 200 mg/L casein hydrolysate, 5 mg/L benzyladenine (BA) and 1 mg/L indole-3-butyric acid (IBA). The effect of nitrogen source (KNO3 and (NH4)2SO4) and their different concentrations on the fresh and dry weight (g/L), as well as RA content (mg/g dry weight) were measured. CSC growth measurements indicated a maximum specific cell growth rate of 1.5/day, a doubling time of 7.6 days and a high percentage of cell viability (96.4 %) throughout the growth cycle. Maximum cell fresh weight (353.5 g/L), dry weight (19.7 g/L) and RA production (180.0 mg/g) were attained at day 21 of culture. Cell growth and RA content were affected by nitrogen deficiency. Media containing 8.3 mM of total nitrogen (¼ of B5 standard medium) led to a minimum cell fresh weight (243.0 g/L), dry weight (17.4 g/L) and RA content (38.0 mg/g) after 21 days. The established CSC provided useful material for further optimization experiments aimed at a large-scale production of RA.

  7. Effect of Cold Stress on Fruiting Body Production by Medicinal Basidiomycetes in Submerged and Solid-phase Culture

    Directory of Open Access Journals (Sweden)

    E.P. Vetchinkina

    2017-05-01

    Full Text Available The ability of the medicinal xylotrophic basidiomycetes Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum and Grifola frondosa to produce typical and atypical fruiting bodies with viable basidiospores in submerged and solid-phase culture under stationary conditions in a beer wort-containing medium under cold stress was shown. The examined mushrooms, when not exposed to temperature stress, did not form fruiting bodies. In solid-phase culture in an agarized medium after cold treatment, the basidiome formation period was shortened by 1.5–2 times. Furthermore, the use of a mycelium subjected to temperature stress for inoculation induced and accelerated the formation of fruiting bodies on an industrial wood substrate, which is of great biotechnological importance.

  8. Improvement of growth parameters of prune callus cultures destined to initiate celi suspensions

    Directory of Open Access Journals (Sweden)

    Ewa Hanus-Fajerska

    2011-01-01

    Full Text Available Callus was inducted on wounded leaf explants from shoot tips of a particular Prunus domestica 'Węgierka Zwykła' clone cultivated in vitro. The improvement of Sweet Common Prune stock callus tissue parameters has been approached by experiments on culture protocols. Either for the induction or maintenance of tissue modified Murashige and Skoog medium, supplemented with different auxins and cytokinins at varying concentrations, was used. The goal was to obtain the highiest possible proliferative capacity of friable tissue without any signs of cell redifferentiation for about 10 weeks. The choice of auxin was an important factor regulating the rate and kind of tissue growth, and for the examined prune clone auxin alone brought a relatively small proportion of cells into division, so advantageous was to combine it with oxygenated cytokinin. Friable tissue was obtained on media supplemented with dicamba or with picloram, but not with 2.4-D neither alone nor combinated with IBA.

  9. Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

    Science.gov (United States)

    Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

    2017-01-01

    Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

  10. Spy and Counterspy as a “Cultural Hero” in the Soviet Cinema of the Cold War

    Directory of Open Access Journals (Sweden)

    Viktoria Sukovataya

    2017-07-01

    Full Text Available This article aim to analyze the evolution of the Soviet spy cinema of the Cold War in the context of the cultural history and the social changes in the USA and the Soviet Union, and the relations with the political opponents. The public reception of the Soviet spy and spying was evolved in the Soviet Union and it was reflected in the cinema plots and characters transformations.

  11. Innovative, non-stirred bioreactors in scales from milliliters up to 1000 liters for suspension cultures of cells using disposable bags and containers--a Swiss contribution.

    Science.gov (United States)

    Werner, Sören; Eibl, Regine; Lettenbauer, Christine; Röll, Marcel; Eibl, Dieter; De Jesus, Maria; Zhang, Xiaowei; Stettler, Matthieu; Tissot, Stephanie; Bürki, Cedric; Broccard, Gilles; Kühner, Markus; Tanner, Rolf; Baldi, Lucia; Hacker, David; Wurm, Florian M

    2010-01-01

    Innovative mixing principles in bioreactors, for example using the rocking of a platform to induce a backwards and forwards 'wave', or using orbital shaking to generate a 'wave' that runs round in a cylindrical container, have proved to be successful for the suspension cultures of cells, especially when combined with disposable materials. This article presents an overview of the engineering characteristics when these new principles are applied in bioreactors, and case studies covering scales of operation from milliliters to 1000 liters.

  12. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Science.gov (United States)

    Ke, Liping; Liu, RuiE; Chu, Bijue; Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang

    2012-01-01

    Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 µmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  13. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Directory of Open Access Journals (Sweden)

    Liping Ke

    Full Text Available Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel. In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L and bentazon (4.2 µmol. A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  14. Bioreactor-Based Production of Glycoproteins in Plant Cell Suspension Cultures.

    Science.gov (United States)

    Holland, Tanja; Buyel, Johannes Felix

    2018-01-01

    Recombinant glycoproteins such as monoclonal antibodies have a major impact on modern healthcare systems, e.g., as the active pharmaceutical ingredients in anticancer drugs. A specific glycan profile is often necessary to achieve certain desirable activities, such as the effector functions of an antibody, receptor binding or a sufficient serum half-life. However, many expression systems produce glycan profiles that differ substantially from the preferred form (usually the form found in humans) or produce a diverse array of glycans with a range of in vivo activities, thus necessitating laborious and costly separation and purification processes. In contrast, protein glycosylation in plant cells is much more homogeneous than other systems, with only one or two dominant forms. Additionally, these glycan profiles tend to remain stable when the process and cultivation conditions are changed, making plant cells an ideal expression system to produce recombinant glycoproteins with uniform glycan profiles in a consistent manner. This chapter describes a protocol that uses fermentations using plant cell cultures to produce glycosylated proteins using two different types of bioreactors, a classical autoclavable STR 3-L and a wave reactor.

  15. Selection of bioprotective cultures for preventing cold-smoked salmon spoilage.

    Science.gov (United States)

    Leroi, Françoise; Cornet, Josiane; Chevalier, Frédérique; Cardinal, Mireille; Coeuret, Gwendoline; Chaillou, Stéphane; Joffraud, Jean-Jacques

    2015-11-20

    Biopreservation is a natural technology of food preservation, which consists of inoculating food with microorganisms selected for their antibacterial properties. The objective of this study was to select lactic acid bacteria (LAB) to improve the quality of cold-smoked salmon (CSS). In this work, different strains representative of the 4 dominant species, identified in a previous study by pyrosequencing the 16S rRNA gene, were isolated and their spoiling potential in CSS blocks, sterilized by ionization, was assessed by twelve trained panelists along the vacuum storage at 8°C. Photobacterium phosphoreum, Brochothrix thermosphacta and Serratia proteamaculans released strong off-odors whereas the spoiling potential of Carnobacterium divergens was weaker. The spoiling capacity of Lactococcus piscium EU2241, Leuconostoc gelidum EU2247, Lactobacillus sakei EU2885, Staphylococcus equorum S030674 and 4 commercial starters was tested by the same method and 2 strains were eliminated due to off-odor production. The effect of the 6 selected LAB against the 4 specific spoiling organisms (SSOs) selected was tested by challenge tests in sterile CSS blocks. The protective effect of the LAB differed from one SSO to another and no correlation could be established between the sensory improvement, SSO inhibition, and the implantation or acidification of protective cultures (PCs). All the PCs except L. piscium reduced the off-odors released by P. phosphoreum although some of them had no effect on its growth. S. equorum, which did not grow in CSS, favored the implantation of P. phosphoreum but prevented its off-odor formation. L. piscium was the only strain that prevented the spoilage of B. thermosphacta and S. proteamaculans although it did not grow very well and did not acidify the product. L. gelidum EU2247 inhibited the growth of these 2 SSOs and lowered the pH but had no effect on the sensory quality. Finally, L. piscium was tested in 2 naturally contaminated products, with a

  16. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Directory of Open Access Journals (Sweden)

    Natalie Bordag

    Full Text Available The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli as well as mammalian cells chinese hamster ovary (CHO and mouse myeloma cells (NS0.The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  17. Factors influencing cucumber (Cucumis sativus L. somatic embryogenesis. I. The crucial role of pH and nitrogen in suspension culture

    Directory of Open Access Journals (Sweden)

    Tadeusz Wróblewski

    2014-01-01

    Full Text Available A method of obtaining and the characteristics of an embryogenic stabilised cucumber (Cucumis sativus L. suspension culture which has many similarities to the carrot model are presented. The Specific Type I cells and proembryogenic mass were present in such a suspension. The maintenance of the proembryogenic stage took place in medium containing 2,4-D as the sole growth regulator, subsequent stages of embryogenesis occurred in hormone-free medium. Embryonic structures were also observed in medium with auxin in the late stages of growth, probably due to the depletion of 2,4-D in the medium during subculture. The choice of the proper inorganic nitrogen sources and the maintenance of correct proportions between them had a significant effect on the formation of these structures. We have shown that the pH of the medium with an embryogenic culture became stabilized regardless of the initial pH value and depended on the medium composition. The inoculum used for the initiation of subsequent subcultures of the stable suspension culture was 1 part tissue to 300 parts medium and was small in comparison to the systems described for the cucumber so far. From 1 ml of basic suspension 7 embryos were obtained on medium without growth regulators 10 days after inoculation, and this amount increased to 21 after 3 weeks. From 3.2% of the somatic embryos it was posible to regenerate plants. The high yield and synchronisation of the process and the development of embryos without passing through callus tissue create the possibility of using this system for molecular investigations and in the technology of somatic seed production.

  18. Jasmonic Acid Effect on the Fatty Acid and Terpenoid Indole Alkaloid Accumulation in Cell Suspension Cultures of Catharanthus roseus

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    Guitele Dalia Goldhaber-Pasillas

    2014-07-01

    Full Text Available The stress response after jasmonic acid (JA treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA and terpenoid indole alkaloids (TIA. According to multivariate data analyses (MVDA, three major time events were observed and characterized according to the variations of specific FA and TIA: after 0–30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90–360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system.

  19. Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

    Science.gov (United States)

    de la Rosa, Javier; Sáenz Antoñanzas, Ander; Shahi, Mehdi H; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2016-09-01

    Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

  20. Intensity of Cold Water and its effects on marine culturing farms along the southeast coast of Korea

    Science.gov (United States)

    Lee, Yong-Hwa; Shim, JeongHee; Choi, Yang-Ho; Kim, Sang-Woo; Shim, Jeong-Min

    2017-04-01

    To understand the characteristics and strength of the cold water that has caused damage to marine-culturing farms around Guryongpo, in the southeast coast of Korea, surface and water column temperatures were collected from temperature loggers deployed at a sea squirt farm during August-November 2007 and from a Real-time Information System for aquaculture environments operated by NIFS during July-August 2015 and 2016. During the study period, surface temperature at Guryongpo decreased sharply when south/southwestern winds prevailed (the 18-26th of August and 20-22nd of September 2007 and the 13-15th of July 2015) as a result of upwelling. However, the deep-water (20-30m) temperature increased during periods of strong north/northeasterly winds (the 5-7th and 16-18th of September 2007) as a result of downwelling. Among the cold water events that occurred at Guryongpo, the mass death of cultured fish followed strong cold water events (surface temperatures below 10℃) that were caused by more than two days of successive south/southeastern winds with maximum speeds higher than 5 m/s. A Cold Water Index (CWI) was defined and calculated using maximum wind speed and direction as measured daily at Pohang Meteorological Observatory. When the average CWI over two days (CWI2d) was higher than 100, mass fish mortality occurred. The four-day average CWI (CWI4d) showed a high negative correlation with surface temperature from July-August in the Guryongpo area (R2 = 0.5), suggesting that CWI is a good index for predicting strong cold water events and massive mortality. In October 2007, the sea temperature at a depth of 30 m showed a high fluctuation that ranged from 7-23℃, with frequency and spectrum coinciding with tidal levels at Ulsan, affected by the North Korean Cold Current. If temperature variations at the depth of fish cages also regularly fluctuate within this range, damage may be caused to the fish industry along the southeast coast of Korea.

  1. Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.

    Science.gov (United States)

    Horiguchi, Ikki; Sakai, Yasuyuki

    2016-07-08

    Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2- to 10-fold increase) without any influence on pluripotency. In addition, albumin-associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009-1016, 2016. © 2016 American Institute of Chemical Engineers.

  2. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell.

    Science.gov (United States)

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles

    2013-02-15

    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack. Copyright © 2012 Elsevier GmbH. All rights reserved.

  3. Positive selection of Wharton's jelly-derived CD105(+) cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells.

    Science.gov (United States)

    Amiri, Fatemeh; Halabian, Raheleh; Dehgan Harati, Mozhgan; Bahadori, Marzie; Mehdipour, Ahmad; Mohammadi Roushandeh, Amaneh; Habibi Roudkenar, Mehryar

    2015-05-01

    Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.

  4. Culture-dependent and independent techniques to monitor yeast species during cold soak carried out at different temperatures in winemaking.

    Science.gov (United States)

    Maturano, Y Paola; Mestre, M Victoria; Combina, Mariana; Toro, María Eugenia; Vazquez, Fabio; Esteve-Zarzoso, Braulio

    2016-11-21

    Transformation of grape must into wine is a process that may vary according to the consumers' requirements. Application of cold soak prior to alcoholic fermentation is a common practice in cellars in order to enhance flavor complexity and extraction of phenolic compounds. However, the effect of this step on wine yeast microbiota is not well-known. The current study simultaneously analyzed the effect of different cold soak temperatures on the microbiological population throughout the process and the use of culture-dependent and independent techniques to study this yeast ecology. The temperatures assayed were those normally applied in wineries: 2.5, 8 and 12°C. PCR-DGGE allowed detection of the most representative species such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae. As could be expected, highest diversity indices were obtained at the beginning of each process, and survival of H. uvarum or S. bacillaris depended on the temperature. Our results are in agreement with those obtained with culture independent methods, but qPCR showed higher precision and a different behavior was observed for each yeast species and at each temperature assayed. Comparison of both culture-independent techniques can provide a general overview of the whole process, although DGGE does not reveal the diversity expected due to the reported problems with the sensitivity of this technique. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Condition and biochemical profile of blue mussels (Mytilus edulis L.) cultured at different depths in a cold water coastal environment

    Science.gov (United States)

    Gallardi, Daria; Mills, Terry; Donnet, Sebastien; Parrish, Christopher C.; Murray, Harry M.

    2017-08-01

    The growth and health of cultured blue mussels (Mytilus edulis) are affected by environmental conditions. Typically, culture sites are situated in sheltered areas near shore (i.e., conflicts and environmental impact in coastal areas are concerns and interest in developing deep water (> 20 m depth) mussel culture has been growing. This study evaluated the effect of culture depth on blue mussels in a cold water coastal environment (Newfoundland, Canada). Culture depth was examined over two years from September 2012 to September 2014; mussels from three shallow water (5 m) and three deep water (15 m) sites were compared for growth and biochemical composition; culture depths were compared for temperature and chlorophyll a. Differences between the two years examined were noted, possibly due to harsh winter conditions in the second year of the experiment. In both years shallow and deep water mussels presented similar condition; in year 2 deep water mussels had a significantly better biochemical profile. Lipid and glycogen analyses showed seasonal variations, but no significant differences between shallow and deep water were noted. Fatty acid profiles showed a significantly higher content of omega-3 s (20:5ω3; EPA) and lower content of bacterial fatty acids in deep water sites in year 2. Everything considered, deep water appeared to provide a more favorable environment for mussel growth than shallow water under harsher weather conditions.

  6. Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells.

    Science.gov (United States)

    Kaushal, G P; Pastuszak, I; Hatanaka, K; Elbein, A D

    1990-09-25

    Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation

  7. On the Cultural Legacy of the Cold War: Sino-US Educational Exchange (1949-1990)

    Science.gov (United States)

    Gu, Ning

    2006-01-01

    The Cold War affected the Sino-US educational exchange between 1949 and 1990. During those years, preparation for educational exchanges, personal contact and cross-government relations characterized the three periods of the exchanges. However, even though the relationship had developed very fast, it was by no means smooth sailing. These exchanges…

  8. "Our Bruised Arms Hung Up as Monuments": Nuclear Iconography in Post-Cold War Culture.

    Science.gov (United States)

    Taylor, Bryan C.

    2003-01-01

    Notes that communication scholars have traditionally examined nuclear discourse at the expense of nuclear images. Develops a nuclear-critical iconology, one sensitive to the role of images in creating and disrupting popular consent to the production of nuclear weapons. Examines three aesthetics in post-Cold War iconography for their significance…

  9. Neither Acceptable Nor Certain - Cold War Antics for 21st Century Precautionary Culture

    NARCIS (Netherlands)

    J.C. Hanekamp (Jaap)

    2009-01-01

    textabstractPrecaution is regarded as the paradigmatic approach to uncertainty. Either proposed as a form of radical prevention or, as put forward recently, as an innovative normative procedure to handle uncertainties in advancing human activities, it nevertheless finds its source in Cold War

  10. An established Arabidopsis thaliana var. Landsberg erecta cell suspension culture accumulates chlorophyll and exhibits a stay-green phenotype in response to high external sucrose concentrations.

    Science.gov (United States)

    McCarthy, Avery; Chung, Michelle; Ivanov, Alexander G; Krol, Marianna; Inman, Michael; Maxwell, Denis P; Hüner, Norman P A

    2016-07-20

    An established cell suspension culture of Arabidopsis thaliana var. Landsberg erecta was grown in liquid media containing 0-15%(w/v) sucrose. Exponential growth rates of about 0.40d-1 were maintained between 1.5-6%(w/v) sucrose, which decreased to about 0.30d-1 between 6 and 15%(w/v) sucrose. Despite the presence of external sucrose, cells maintained a stay-green phenotype at 0-15% (w/v) sucrose. Sucrose stimulated transcript levels of genes involved in the chlorophyll biosynthetic pathway (ChlH, ChlI2, DVR). Although most of the genes associated with photosystem II and photosystem I reaction centers and light harvesting complexes as well as genes associated with the cytochrome b6f and the ATP synthase complexes were downregulated or remained unaffected by high sucrose, immunoblotting indicated that protein levels of PsaA, Lhcb2 and Rubisco per gram fresh weight changed minimallyon a Chl basis as a function of external sucrose concentration. The green cell culture was photosynthetically competent based on light-dependent, CO2-saturated rates of O2 evolution as well as Fv/Fm and P700 oxidation. Similar to Arabidopsis WT seedlings, the suspension cells etiolated in the dark and but remained green in the light. However, the exponential growth rate of the cell suspension cultures in the dark (0.45±0.07d-1) was comparable to that in the light (0.42±0.02d-1). High external sucrose levels induced feedback inhibition of photosynthesis as indicated by the increase in excitation pressure measured as a function of external sucrose concentration. Regardless, the cell suspension culture still maintained a stay-green phenotype in the light at sucrose concentrations from 0 to 15%(w/v) due, in part, to a stimulation of photoprotection through nonphotochemical quenching. The stay-green, sugar-insensitive phenotype of the cell suspension contrasted with the sugar-dependent, non-green phenotype of Arabidopsis Landsberg erecta WT seedlings grown at comparable external sucrose

  11. An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

    Science.gov (United States)

    Hasegawa, Ai; Oyanagi, Tomoya; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2014-11-01

    A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

  12. Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

    Science.gov (United States)

    Horiguchi, Ayumi; Yazaki, Koyuki; Aoyagi, Mami; Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

    2014-11-01

    Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Production of Limonoids with Insect Antifeedant Activity in a Two-Stage Bioreactor Process with Cell Suspension Culture of Azadirachta indica.

    Science.gov (United States)

    Vásquez-Rivera, Andrés; Chicaiza-Finley, Diego; Hoyos, Rodrigo A; Orozco-Sánchez, Fernando

    2015-09-01

    Neem tree (Azadirachta indica) cell suspension culture is an alternative for the production of limonoids for insect control that overcomes limitations related to the supply of neem seeds. To establish conditions for cell growth and azadiracthin-related limonoid production, the effect of different sucrose concentrations, nitrate and phosphate in Murashige and Skoog (MS) medium, and the addition of one precursor and three elicitors was evaluated in shake flasks. The process was scaled up to a 3-l stirred tank bioreactor in one- and two-stage batch cultivation. In shake flasks, more than fivefold increase in the production of limonoids with the modified MS medium was observed (increase from 0.77 to 4.52 mg limonoids/g dry cell weight, DCW), while an increase of more than fourfold was achieved by adding the elicitors chitosan, salicylic acid, and jasmonic acid together (increase from 1.03 to 4.32 mg limonoids/g DCW). In the bioreactor, the volumetric production of limonoids was increased more than threefold with a two-stage culture in day 18 (13.82 mg limonoids/l in control single-stage process and 41.44 mg/l in two-stage process). The cultivation and operating mode of the bioreactor reported in this study may be adapted and used in optimization and process plant development for production of insect antifeedant limonoids with A. indica cell suspension cultures.

  14. Diadenosine triphosphate is a novel factor which in combination with cyclodextrins synergistically enhances the biosynthesis of trans-resveratrol in Vitis vinifera cv. Monastrell suspension cultured cells.

    Science.gov (United States)

    Pietrowska-Borek, Małgorzata; Czekała, Łukasz; Belchí-Navarro, Sarai; Pedreño, María Angeles; Guranowski, Andrzej

    2014-11-01

    Dinucleoside polyphosphates are considered as signal molecules that may evoke response of plant cells to stress. Other compounds whose biological effects have been recognized are cyclodextrins. They are cyclic oligosaccharides that chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack, and they act as true elicitors, since, when added to plant cell culture, they induce the expression of genes involved in some secondary metabolism pathways. Previously, we demonstrated that some dinucleoside polyphosphates triggered the biosynthesis of enzymes involved in the phenylpropanoid pathway in Arabidopsis thaliana. In Vitis vinifera suspension cultured cells, cyclodextrins were shown to enhance the accumulation of trans-resveratrol, one of the basic units of the stilbenes derived from the phenylpropanoid pathway. Here, we show that diadenosine triphosphate, applied alone or in combination with cyclodextrins to the grapevine suspension-cultured cells, increased the transcript level of genes encoding key phenylpropanoid-pathway enzymes as well as the trans-resveratrol production inside cells and its secretion into the extracellular medium. In the latter case, these two compounds acted synergistically. However, the accumulation of trans-resveratrol and its glucoside trans-piceid inside cells were stimulated much better by diadenosine triphosphate than by cyclodextrins. Copyright © 2014. Published by Elsevier Masson SAS.

  15. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    Science.gov (United States)

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  16. Comparative proteomic analysis of cultured suspension cells of the halophyte Halogeton glomeratus by iTRAQ provides insights into response mechanisms to salt stress

    Directory of Open Access Journals (Sweden)

    Huajun eWang

    2016-02-01

    Full Text Available Soil salinity severely threatens land use capability and crop yields worldwide. An analysis of the molecular mechanisms of salt tolerance in halophytes will contribute to the development of salt-tolerant crops. In this study, a combination of physiological characteristics and iTRAQ-based proteomic approaches was conducted to investigate the molecular mechanisms underlying the salt response of suspension cell cultures of halophytic Halogeton glomeratus. These cells showed halophytic growth responses comparable to those of the whole plant. In total, 97 up-regulated proteins and 192 down-regulated proteins were identified as common to both 200 and 400 mM NaCl concentration treatments. Such salinity responsive proteins were mainly involved in energy, carbohydrate metabolism, stress defense, protein metabolism, signal transduction, cell growth, and cytoskeleton metabolism. Effective regulatory protein expression related to energy, stress defense, and carbohydrate metabolism play important roles in the salt-tolerance of H. glomeratus suspension cell cultures. However, known proteins regulating Na+ efflux from the cytoplasm and its compartmentalization into the vacuole did not change significantly under salinity stress suggesting our existing knowledge concerning Na+ extrusion and compartmentalization in halophytes needs to be evaluated further. Such data are discussed in the context of our current understandings of the mechanisms involved in the salinity response of the halophyte, H. glomeratus.

  17. Purification and characterization of three chitinases and one beta-1,3-glucanase accumulating in the medium of cell suspension cultures of barley (Hordeum vulgare L.)

    DEFF Research Database (Denmark)

    Kragh, K.M.; Jacobsen, S.; Dalgaard Mikkelsen, J.

    1991-01-01

    Three basic chitinases and one basic beta-1,3-glucanase were secreted into the medium when embryogenic cell suspensions of barley (Hordeum vulgare L.) cv. 'Igri' were cultured as undifferentiated aggregates in the presence of 2,4-D. The enzymes were purified by affinity and ion exchange chromatog......Three basic chitinases and one basic beta-1,3-glucanase were secreted into the medium when embryogenic cell suspensions of barley (Hordeum vulgare L.) cv. 'Igri' were cultured as undifferentiated aggregates in the presence of 2,4-D. The enzymes were purified by affinity and ion exchange...... chromatography. Two of the chitinases were identified as the previously described endochitinases T and C from barley grain. The third and novel chitinase, designated K, was the major basic chitinase in the medium constituting 4% of the soluble protein. Chitinase K was found to be a 33-kDa endochitinase with p......I at 8.7. Further analysis showed that this enzyme is also expressed in barley grain. The amino acid composition and five partial amino acid sequences covering 93 residues of chitinase K were determined. A high similarity was found between chitinase K and barley chitinase T and C as well as basic...

  18. The role of the focal adhesion protein PINCH1 for the radiosensitivity of adhesion and suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Veit Sandfort

    Full Text Available Focal adhesion (FA signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1(fl/fl and PINCH1(-/- mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130(CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions.

  19. Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

    Science.gov (United States)

    Nath, Suman C; Tokura, Tomohiro; Kim, Mee-Hae; Kino-Oka, Masahiro

    2018-04-01

    Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10 6 cells ml -1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture. © 2017 Wiley Periodicals, Inc.

  20. Immunogenicity of an adeno-vector vaccine expressing the F protein of a respiratory syncytial virus manufactured from serum-free suspension culture.

    Science.gov (United States)

    Shao, Hsiao-Yun; Hsu, Huai-Sheng; Yu, Shu-Ling; Wu, Shang-Rung; Hu, Kai-Chieh; Chang, Ching-Kun; Liu, Chia-Chyi; Chow, Yen-Hung

    2016-06-01

    We have developed an efficient cell culture process to scale up the production of a recombinant adenovirus that expresses the membrane-trunked fusion protein of respiratory syncytial virus (RSV; Ad-F0ΔTM). Adherent cells of human embryonic kidney (HEK) 293-derived cell, 293A, which supports the production of E1/E3-deleted Ad-F0ΔTM when cultured in the presence of fetal bovine serum (FBS), were adapted to suspension growth under serum-free medium. In doing so, we studied the immunogenicity of Ad-F0ΔTMsus, which propagated in a bioreactor that was cultured with serum-free suspension of 293A, in comparison with Ad-F0ΔTMadh, which was produced from parental 293A cells that were adherently cultured in medium containing FBS. The size and morphology of Ad-F0ΔTMsus and Ad-F0ΔTMadh virions were identical upon inspection with electron microscopy. The results showed that anti-F IgG and RSV-neutralizing titer were raised in the serum of both mice that were intranasally immunized twice with Ad-F0ΔTMsus or Ad-F0ΔTMadh at two-week injection intervals. Furthermore, the immune responses persisted for six months after vaccination. Activation of F protein-specific CD8(+) T cell's epitope associated IFN-ɣ and IL-4 was induced in both Ad-F0ΔTMsus- and Ad-F0ΔTMadh, but not in Ad-LacZsus, -immunized mouse splenocytes. No vaccine-enhanced lung inflammation, airway mucus occlusion or eosinophils infiltration were observed in Ad-immunized mice followed by RSV challenge; however, these symptoms were observed following immunization with formalin-inactivated RSV vaccine. These results indicate that the safety and potency of Ad-F0ΔTM produced from either adherent cells or suspension and serum-free cells are the same. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

    Science.gov (United States)

    Ahlawat, Seema; Saxena, Parul; Ali, Athar; Khan, Shazia; Abdin, Malik Z

    2017-05-01

    Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock 1

    Science.gov (United States)

    Salerno, Graciela L.; Pontis, Horacio G.

    1989-01-01

    Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24°C to 4°C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24°C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before. PMID:16666596

  3. Increasing anthraquinone production by overexpression of 1-deoxy-D: -xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia.

    Science.gov (United States)

    Quevedo, Carla; Perassolo, María; Alechine, Eugenia; Corach, Daniel; Giulietti, Ana María; Talou, Julián Rodriguez

    2010-07-01

    A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

  4. Identification of the human Lewis(a) carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.).

    Science.gov (United States)

    Melo, N S; Nimtz, M; Conradt, H S; Fevereiro, P S; Costa, J

    1997-09-29

    This paper reports for the first time the presence of the human Lewis(a) type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewis(a) [structure in text]. A corresponding fucosyltransferase activity catalyzing the formation of Lewis(a) type structures in vitro was identified in cellular extracts of the suspension cultures.

  5. Suramin inhibits initiation of defense signaling by systemin, chitosan, and a β-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells

    Science.gov (United States)

    Stratmann, Johannes; Scheer, Justin; Ryan, Clarence A.

    2000-01-01

    Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and β-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog 125I-Tyr-2,Ala-15-systemin to the systemin receptor with an IC50 of 160 μM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding. PMID:10922047

  6. Suramin inhibits initiation of defense signaling by systemin, chitosan, and a beta-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells.

    Science.gov (United States)

    Stratmann, J; Scheer, J; Ryan, C A

    2000-08-01

    Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and beta-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog (125)I-Tyr-2, Ala-15-systemin to the systemin receptor with an IC(50) of 160 microM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding.

  7. Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

    Science.gov (United States)

    Gilbert, Rénald; Guilbault, Claire; Gagnon, David; Bernier, Alice; Bourget, Lucie; Elahi, Seyyed Mehdy; Kamen, Amine; Massie, Bernard

    2014-11-01

    E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications. Copyright © 2014. Published by Elsevier B.V.

  8. Catalase and alternative oxidase cooperatively regulate programmed cell death induced by beta-glucan elicitor in potato suspension cultures.

    Science.gov (United States)

    Mizuno, Masashi; Tada, Yasuomi; Uchii, Kimitaka; Kawakami, Sachiko; Mayama, Shigeyuki

    2005-04-01

    In potato (Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H2O2 accumulation were induced by treatment with beta-glucan elicitor. The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H2O2, whereas the ascorbate peroxidase inhibitor did not have any effect on these responses. Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H2O2 accumulation, leading to the disruption of mitochondrial membrane potential (deltapsi(m)) and programmed cell death (PCD). The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial deltapsi(m) breakdown and PCD induced by beta-glucan elicitor.

  9. Experimental infection of New Zealand Merino sheep with a suspension of Mycobacterium avium subspecies paratuberculosis (Map) strain Telford: Kinetics of the immune response, histopathology and Map culture.

    Science.gov (United States)

    Dukkipati, Venkata S R; Ridler, Anne L; Thompson, Keith G; Buddle, Bryce M; Hedgespeth, Barry A; Price-Carter, Marian; Begg, Douglas J; Whittington, Richard J; Gicquel, Brigitte; Murray, Alan

    2016-11-15

    A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johne's disease in NZ Merino lambs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method.

    Science.gov (United States)

    Vasilev, Nikolay; Grömping, Ulrike; Lipperts, Anja; Raven, Nicole; Fischer, Rainer; Schillberg, Stefan

    2013-09-01

    We have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY-2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO₃, NH₄NO₃ and CaCl₂ and the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO₃, NH₄NO₃ and CaCl₂) and also individually between 2,4-D and three other components (KNO₃, NH₄NO₃ and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 μg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20-fold (day 7) and 31-fold (day 10) compared to the conventional MS medium. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Public science of the savage mind: contesting cultural anthropology in the Cold War classroom.

    Science.gov (United States)

    Milam, Erika Lorraine

    2013-01-01

    "What is human about human beings? How did they get that way? How can they be made more so?" These three questions formed the basis of a fifth-grade social studies curriculum project developed in the 1960s called Man: A Course of Study, or MACOS. In the years between the curriculum's development in the 1960s and its controversial implementation in the 1970s, two separate sets of concerns served to problematize the use of anthropological materials in public school classrooms. On the one hand, MACOS designers were wary of the possibly racist interpretations of exploring so-called "primitive" cultures in the classroom. On the other, conservative textbook reformers objected to claims that all cultural solutions to biological problems were morally equivalent. Once MACOS earned a place in national news, it came to embody both hopes for the redemption of American democratic society and fears about the violent nature of humans, depending on one's political perspective. These mixed messages eventually undermined the long-term success of the program as public science. © 2013 Wiley Periodicals, Inc.

  12. The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Kwiatkowska, Aleksandra, E-mail: A.Kwiatkows@gmail.com [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Zebrowski, Jacek [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Oklejewicz, Bernadetta [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland); Czarnik, Justyna [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Halibart-Puzio, Joanna [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Wnuk, Maciej [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland)

    2014-05-02

    Highlights: • A decrease in proliferation rate during long-term cultivation of Arabidopsis cells. • Age-dependent increase in senescence-associated gene expression in Arabidopsis cells. • Age-related increase in DNA methylation, H3K9me2, and H3K27me3 in Arabidopsis cells. • High potential of photosynthetic efficiency of long-term cultured Arabidopsis cells. - Abstract: Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.

  13. Highly efficient in vitro regeneration, establishment of callus and cell suspension cultures and RAPD analysis of regenerants of Swertia lawii Burkill

    Directory of Open Access Journals (Sweden)

    Parthraj R. Kshirsagar

    2015-06-01

    Full Text Available Highly efficient in vitro regeneration system has been developed for Swertia lawii Burkill, an important herb used as substitute for Swertia chirayita. Shoot tips explants were cultured on MS medium with various phytohormones for multiple shoot production. The best shoot production frequency (100% and maximum shoots (10.4 ± 0.8 were obtained on MS media containing TDZ (3.0 mg l−1 in combination with IBA (0.3 mg l−1. Maximum callus induction (95 ± 4.8% and callus growth (1.7 ± 0.4 gm was achieved on MS medium with 2, 4-D (3.0 mg l−1. Cell suspension cultures were established and studied for their growth kinetics. Shoots were rooted best (22.1 ± 2.5 in 1/2 MS medium with IAA (3.0 mg l−1. The genetic uniformity of the micropropagated clones was assessed using RAPD markers. Out of 405 bands, 400 (98.76% were monomorphic and rest 5 (1.24% were polymorphic. High multiplication frequency and low risk of genetic instability ensures the efficacy of this protocol.

  14. Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

    Science.gov (United States)

    Islam, Md Reyazul; Kim, Nan-Sun; Jung, Jae-Wan; Kim, Hyo-Boon; Han, So-Chon; Yang, Moon-Sik

    2018-01-01

    A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Diversity and cold adaptation of culturable endophytic fungi from bryophytes in the Fildes Region, King George Island, maritime Antarctica.

    Science.gov (United States)

    Zhang, Tao; Zhang, Yu-Qin; Liu, Hong-Yu; Wei, Yu-Zhen; Li, Hai-Long; Su, Jing; Zhao, Li-Xun; Yu, Li-Yan

    2013-04-01

    Endophytic fungi associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica, that is, the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata, were studied by culture-dependent method. A total of 128 endophytic fungi were isolated from 1329 tissue segments of 14 samples. The colonization rate of endophytic fungi in three bryophytes species were 12.3%, 12.1%, and 8.7%, respectively. These isolates were identified to 21 taxa, with 15 Ascomycota, 5 Basidiomycota, and 1 unidentified fungus, based on morphological characteristics and sequence analyses of ITS region and D1/D2 domain. The dominant fungal endophyte was Hyaloscyphaceae sp. in B. hatcheri, Rhizoscyphus sp. in C. aciphyllum, and one unidentified fungus in S. uncinata; and their relative frequencies were 33.3%, 32.1%, and 80.0%, respectively. Furthermore, different Shannon-Weiner diversity indices (0.91-1.99) for endophytic fungi and low endophytic fungal composition similarities (0.19-0.40) were found in three bryophyte species. Growth temperature tests indicated that 21 taxa belong to psychrophiles (9), psychrotrophs (11), and mesophile (1). The results herein demonstrate that the Antarctic bryophytes are an interesting source of fungal endophytes and the endophytic fungal composition is different among the bryophyte species, and suggest that these fungal endophytes are adapted to cold stress in Antarctica. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. (Univ. of Illinois, Urbana (USA))

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I{sub 50} values for growth, chlorophyll (Chl), {beta}-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in ({sup 14}C)clomazone uptake cannot account for selectivity since there were significantly greater levels of domazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  17. Independent suspension

    National Research Council Canada - National Science Library

    Chaikin, Don

    1992-01-01

    ... independent suspension. INDEPENDENCE! An independent system is simply one in which each of the vehicle's wheels is free to react totally separate from any of the other wheels. If the right rear wheel hits a bump, the left rear wheel is undisturbed. Since the whole car does not bounce and shake every time one of the wheels hits a potho...

  18. Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana.

    Science.gov (United States)

    Arias, J P; Zapata, K; Rojano, B; Arias, M

    2016-10-01

    Thevetia peruviana (T. peruviana) has been considered as a potentially important plant for industrial and pharmacological application. Among the number of compounds which are produced by T. peruviana, antioxidants and polyphenols are of particular interest due to their benefits on human health. Cell suspension cultures of T. peruviana were established under different conditions: 1) constant illumination (24h/day) at different light wavelengths (red, green, blue, yellow and white), 2) darkness and 3) control (12h/12h: day light/dark) to investigate their biomass, substrate uptake, polyphenols production and oxidizing activity. The results showed biomass concentrations between 17.1g dry weight (DW)/l (green light) and 18.2g DW/l (control) after 13days. The cultures that grew under green light conditions consumed completely all substrates after 10days, while other cultures required at least 13days or more. The total phenolic content was between 7.21 and 9.46mg gallic acid (GA)/g DW for all light conditions. In addition the ferric reducing antioxidant power and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid antioxidant activity ranged from 5.41-6.58mg ascorbic acid (AA)/g DW and 82.93-110.39μmol Trolox/g DW, respectively. Interestingly, the samples which grew under the darkness presented a higher phenolic content and antioxidant capacity when compared to the light conditions. All together, these results demonstrate the extraordinary effect of different lighting conditions on polyphenols production and antioxidant compounds by T. peruviana. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Minocha, R.; Shortle, W.C. (USDA Forest Service, Durham (US)); Minocha, S.C.; Long, S.L. (Dept. of Plant Biology, Univ. of New Hamshire, Durham (US))

    1992-01-01

    Increased aluminium (Al) solubility in soil waters due to acid precipitation has aroused considerable interest in the problem of Al toxicity in plants. In the present study, an in vitro suspension culture system of Catharanthus roseus (L.) G. Don was used to analyze the effects of aluminum on several biochemical processes in these cells. The aliphatic polyamines, spermine and spermidine, and their precusor, putrescine, have been implicated in a number of stress responses of plants. Addition of 0.2, 0.5 or 1.0 mM AlCl{sub 3} to cells cultured for 3 days caused a small but significant increase in cellular levels of putrescine at 4 h followed by a sharp decline by 16 h. There was no further decline in levels of putrescine during the next 32 h. Spermidine levels did not change appreciably compared to those in the control cultures. However, spermine levels increased by 2-3-fold at 24 and 48 h. Cellular activities of arginine decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) were both inhibited by 20-25% at 4 and 7 h. Ornithine decarboxylase (ODC; EC 4.1.1.17) was less than 10% of ADC activity at all times. Whereas all concentrations of Al caused a slight decrease in total cell number, cell viability was affected only by 1.0 mM Al. There was a decrease in the cellular levels of Ca, Mg, Na, K, Mn, P and Fe in the cells treated with Al at 4 h, but a significant increase by 16 and 24 h. The results presented here suggest that both the absolute amounts of Al and the length of exposure to it are important for cell toxicity. (au).

  20. Establishment of a fully automated microtiter plate-based system for suspension cell culture and its application for enhanced process optimization.

    Science.gov (United States)

    Markert, Sven; Joeris, Klaus

    2017-01-01

    We developed an automated microtiter plate (MTP)-based system for suspension cell culture to meet the increased demands for miniaturized high throughput applications in biopharmaceutical process development. The generic system is based on off-the-shelf commercial laboratory automation equipment and is able to utilize MTPs of different configurations (6-24 wells per plate) in orbital shaken mode. The shaking conditions were optimized by Computational Fluid Dynamics simulations. The fully automated system handles plate transport, seeding and feeding of cells, daily sampling, and preparation of analytical assays. The integration of all required analytical instrumentation into the system enables a hands-off operation which prevents bottlenecks in sample processing. The modular set-up makes the system flexible and adaptable for a continuous extension of analytical parameters and add-on components. The system proved suitable as screening tool for process development by verifying the comparability of results for the MTP-based system and bioreactors regarding profiles of viable cell density, lactate, and product concentration of CHO cell lines. These studies confirmed that 6 well MTPs as well as 24 deepwell MTPs were predictive for a scale up to a 1000 L stirred tank reactor (scale factor 1:200,000). Applying the established cell culture system for automated media blend screening in late stage development, a 22% increase in product yield was achieved in comparison to the reference process. The predicted product increase was subsequently confirmed in 2 L bioreactors. Thus, we demonstrated the feasibility of the automated MTP-based cell culture system for enhanced screening and optimization applications in process development and identified further application areas such as process robustness. The system offers a great potential to accelerate time-to-market for new biopharmaceuticals. Biotechnol. Bioeng. 2017;114: 113-121. © 2016 Wiley Periodicals, Inc. © 2016 Wiley

  1. Application of rice (Oryza sativa L.) suspension culture in studying senescence in vitro (II).: Changes in DNA integrity

    OpenAIRE

    Ramin,Hosseini; Mulligan,Bernard J.

    2002-01-01

    Changes in the DNA content and organisation of senescing rice cell cultures (Taipei 309) were studied, using PCR and Southern blot analyses. A mitochondrial gene (coxII), a plastid gene (psaA) and a nuclear DNA maker (RG64) were analysed. The amplification of mitochondrial (mt), plastid and nuclear DNA produced the expected fragments, indicating that there were still some intact organelles and nuclei in the senescing rice cells. However, in plastid and nuclear DNA, changes in the number and s...

  2. Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

    OpenAIRE

    Ikuo, Igaue; Masatoshi, Shimizu; Satoshi, Miyauchi; Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University

    1980-01-01

    A series of myo-inositol phosphates including myo-inositol mono- to hexa-phosphates was observed during growth of cultured rice plant cells. We also found that ^Pi and myo-[2-^3H] inositol were incorporated into all these myo-inositol phosphates. myo-inositol phosphorylating activity, which depended on ATP and Mg^ was detected in the soluble fraction from the cells, and the reaction product was identified as myo-inositol-2-phosphate.

  3. Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.

    Science.gov (United States)

    Miranda, Cláudia C; Fernandes, Tiago G; Diogo, M Margarida; Cabral, Joaquim M S

    2016-12-01

    The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Culturable and culture-independent bacterial diversity and the prevalence of cold-adapted enzymes from the Himalayan mountain ranges of India and Nepal.

    Science.gov (United States)

    Venkatachalam, Siddarthan; Gowdaman, Vasudevan; Prabagaran, Solai Ramatchandirane

    2015-04-01

    Bacterial diversity of soil samples collected from different geographical regions of Himalayan mountains was studied through culturable (13 samples) and culture-independent approaches (5 samples based on abundance of diversity indices in each ecological niche). Shannon-Wiener diversity index and total bacterial count ranged from 1.50 ± 0.1 to 2.57 ± 0.15 and 7.8 ± 1.6 × 10(5) to 30.9 ± 1.7 × 10(5) cfu ml(-1) of soil, respectively. Based on morphology and pigmentation, 406 isolates were selected by culturing in different cultivable media at various strengths and concentrations. All the strains were subjected to amplified ribosomal DNA restriction analysis and the representative isolates from each cluster were chosen for 16S rRNA gene sequence-based identification. Soil habitat in Himalayan foot hills was dominated by the genera Arthrobacter, Exiguobacterium, Bacillus, Cedecea, Erwinia, and Pseudomonas. Five 16S rRNA gene libraries from the selected five samples yielded 268 clones and were grouped into 53 phylotypes covering 25 genera including the genus of Ferribacterium, Rothia, and Wautersiella, which were reported for the first time in Himalayan tracks. Principal coordinates analysis indicates that all the clone libraries were clearly separated and found to be significantly different from each other. Further, extracellular investigation of cold-active enzymes showed activity of cellulase (23.71%), pectinase (20.24%), amylase (17.32%), phytase (13.87%), protease (12.72%), and lipase (23.71%) among the isolates. Four isolates namely Exiguobacterium mexicanum (BSa14), Exiguobacterium sibiricum (BZa11), Micrococcus antarcticus (BSb10), and Bacillus simplex (BZb3) showed multiple enzyme activity for five different types of enzymes. In addition, various genera like Exiguobacterium, Erwinia, Mycetecola, Cedecea, Pantoea, and Trichococcus have also shown novel hydrolytic enzyme activity in the Himalayan foothills.

  5. Enhancement of 20-hydroxyecdysone production in cell suspension ...

    African Journals Online (AJOL)

    SERVER

    2007-07-18

    hydroxyecdysone production of. Vitex glabrata suspension .... hydroxyecdysone production in V. glabrata cell suspension cultures. Determination of dry cell ... residue was dissolved in 3 ml methanol and vortexed with 2 ml hexane twice.

  6. Effect of NaCl on ionic content and distribution in suspension-cultured cells of the halophyte Sonneratia alba versus the glycophyte Oryza sativa.

    Science.gov (United States)

    Hayatsu, Manabu; Suzuki, Suechika; Hasegawa, Ai; Tsuchiya, Shinpei; Sasamoto, Hamako

    2014-09-15

    The effect of a high concentration of NaCl on the intra- (cytoplasmic matrix and vacuole) and extracellular (cell wall) distribution of Na, Cl, K, Mg, Ca, S, and P was investigated in suspension-cultured cells of the mangrove halophyte Sonneratia alba and compared to cultured cells of glycophytic rice (Oryza sativa). No significant differences were observed in ultrastructural features of cluster cells of both species cultured with and without 50mM NaCl. Quantitative X-ray microanalysis of cryosections of the cells cultured in the presence of 50mM NaCl showed that the Na concentration ([Na]) and Cl concentration ([Cl]) significantly increased in all three cell components measured. In S. alba, the [Na] was highest in the vacuole and lowest in the cytoplasmic matrix, while the [Cl] was highest in the cell wall and lowest in the cytoplasmic matrix. In O. sativa, however, the [Na] and [Cl] were highest in the cell wall, and the [Na] was lowest in the cytoplasmic matrix. Thus, the possible activities for Na and Cl transport from the cytoplasmic matrix into the vacuole were greater in S. alba than in O. sativa, suggesting that halophilic mangrove cells gain salt tolerance by transporting Na and Cl into their vacuoles. In O. sativa, the addition of NaCl to the culture medium caused no significant changes to the intracellular concentrations of various elements, such as K, P, S, Ca, and Mg, which suggests the absence of a direct relationship with the transport Na and Cl. In contrast, a marked decrease in the Ca concentration ([Ca]) in the cytoplasmic matrix and vacuole and an approximately two-fold increase in the P concentration ([P]) in the cytoplasmic matrix were found in S. alba, suggesting that the decrease in the [Ca] is related to the halophilic nature of S. alba (as indicated by the inward movement of Na(+) and Cl(-)). The possible roles of a Na(+)/Ca(2+) exchange mechanism in halophilism and the effect of the [P] on the metabolic activity under saline conditions are

  7. Volatiles identified from five stages of embryo development separated from a heterogeneous suspension culture of Daucus carota.

    Science.gov (United States)

    Kennedy, A H; Chamberlain, D; Wilson, G; Ryan, M F

    1991-11-01

    Five stages of embryo development were fractionated from a mature culture of Daucus carota (Gelbe Rheinsche), using a series of metal sieves. The composition of the population of embryos in each fraction was determined quantitatively from microscopic investigations. Volatiles from samples of tissue from six stages of development were trapped on activated charcoal cartridges. These volatiles, some of which may play a significant role in the interaction of the plant with the carrot root fly (Psila rosae), were analysed using gas chromatography/mass spectroscopy. The resulting chromatograms are arranged in order of embryo development. The progressive elaboration of the volatile profile reflects the increased biosynthetic capacity of the developing embryo.

  8. Cold Stress

    Science.gov (United States)

    ... Publications and Products Programs Contact NIOSH NIOSH COLD STRESS Recommend on Facebook Tweet Share Compartir Workers who ... cold environments may be at risk of cold stress. Extreme cold weather is a dangerous situation that ...

  9. Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts.

    Science.gov (United States)

    Emmerling, M; Seitz, H U

    1990-09-01

    A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc4Xyl3GalFuc). This nonasaccharide showed excellent "anti-auxin" properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 μM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.

  10. Evaluating and managing Cold War era historic properties : the cultural significance of U.S. Air Force defensive radar systems.

    Energy Technology Data Exchange (ETDEWEB)

    Whorton, M.

    1999-01-20

    Aircraft and later missile radar early warning stations played an important role in the Cold War. They are associated with important technological, social, political, and military themes of the Cold War and are worthy of preservation. The scope and scale of these systems make physical preservation impractical, but the U.S. Air Force program of historical evaluation and documentation of these systems will provide valuable information to future generations studying this historic period.

  11. The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures.

    Science.gov (United States)

    Andi, Seyed Ali; Gholami, Mansour; Ford, Christopher M

    2017-08-29

    Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.

  12. Influence of growth regulators and sucrose concentrations on growth and rosmarinic acid production in calli and suspension cultures of Coleus blumei.

    Science.gov (United States)

    Qian, Ju; Guiping, Liu; Xiujun, Liu; Xincai, Han; Hongmei, Liu

    2009-01-01

    Rosmarinic acid, which is reported to have adstringent, antibacterial, antiviral and antioxidant activities, is one of the most prominent secondary compounds in Coleus blumei (Lamiaceae). Rosmarinic acid (RA) production in different hybrids of C. blumei was estimated by HPLC. Conditions for HPLC were as follows: column, 150 x 4.6 mm; solvent system, methanol -0.1% phosphate (45 : 55); flow rate, 0.9 mL/min; detection: 325 nm. Two out of four hybrids of C. blumei (hy1; hy2) contain better rosmarinic acid production (0.9 and 1.0% dry weight, respectively) and the leaves have the highest rosmarinic acid production, followed by stems and roots. The hydroxyphenylpyruvate reductase (HPPR) gene expression levels were analysed by semi-quantitative RT-PCR. Hy3 shows highest level of HPPR gene expression out of four hybrids on genotype-specific patterns, and stems represent the highest level of HPPR gene expression among leaves, roots and stems. This was probably a result of the fact that the RA biosynthetic pathway was regulated by interactions of several enzymes necessary for biosynthesis. The explants from the hy1 leaves were used in subsequent studies on the effect of different growth regulators (2.0 mg L(-1) 6-benzyl-aminopurine (6-BA), different 2,4-dichlorophenxyaretic acid (2,4-D) and alpha-naphthaleneacetic (NAA) concentrations) and sucrose contents (1, 2, 3, 4, 5 or 6%) on culture growth and rosmarinic acid accumulation. On the effect of different growth regulators, the best result is obtained when the B5-medium supplemented is with 2.0 mg L(-1) 6-BA, 0.5 mg L(-1) NAA, 0.8 mg L(-1) 2,4-D and 2% sugar, and solidified with 0.8% agar. In this case, both growth index and rosmarinic acid accumulation reach a maximum, which is 49.7 and 25.3% (dry weight), respectively. The optimal medium for suspension culture growth contains 2.0 mg L(-1) 6-BA, 0.5 mg L(-1) NAA, 0.8 mg L(-1) 2,4-D, 600 mg L(-1) inositel and 2% sugar, and the rosmarinic acid production is 1.7% (dry weight

  13. Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure.

    Science.gov (United States)

    Shin, Yun-Ji; Chong, Yun-Jo; Yang, Moon-Sik; Kwon, Tae-Ho

    2011-12-01

    The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  14. Determination of Spoilage Microbiota of Pacific White Shrimp During Ambient and Cold Storage Using Next-Generation Sequencing and Culture-Dependent Method.

    Science.gov (United States)

    Yang, Sheng-Ping; Xie, Jing; Qian, Yun-Fang

    2017-05-01

    This study was conducted to determine the initial and spoilage microbiota of Pacific white shrimp during ambient and cold storage using next-generation sequencing (NGS) and a culture-dependent method. The quality changes were also evaluated by sensory analysis and total volatile basic nitrogen (TVB-N) values. After 1 d of storage, the psychrotrophic bacteria were only 5.97 log CFU/g, accounting for 1.1% of the mesophilic bacteria counts (7.94 log CFU/g). The psychrotrophic bacteria counts exceeded the counts of mesophilic bacteria for shrimp stored at 4 °C after 6 d of storage, indicating that psychrotrophic bacteria became predominant. The NGS was used to identify the bacterial species in samples stored at 25 and 4 °C. The results showed that the dominant microorganisms were Vibrio at 25 °C, and Acinetobacter, Psychrobacter, and Shewanella at 4 °C. By the culture-dependent method based on 16S rRNA gene and VITEK®2 CompactA system, it showed that the dominant microorganisms were Proteus spp. at 25 °C, and Shewanella putrefaciens, Acinetobacter johnsonii, and Aeromonas sobria at 4 °C. In conclusion, differences in results of microbiota analyzed by culture dependent and independent approaches were observed. The combination of both methodologies may provide more comprehensive information about the dominant spoilage microbiota in Pacific white shrimp during ambient and cold storage. © 2017 Institute of Food Technologists®.

  15. Bacterial Diversity and Bioprospecting for Cold-Active Hydrolytic Enzymes from Culturable Bacteria Associated with Sediment from Nella Fjord, Eastern Antarctica

    OpenAIRE

    Bo Chen; Yin-Xin Zeng; Hui-Rong Li; Yong Yu

    2011-01-01

    The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica (69°22′6″ S, 76°21′45″ E) was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychro...

  16. Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2014-01-01

    -amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected...... by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities.Conclusions: Many cold- and...

  17. Common Cold

    Science.gov (United States)

    ... nose, coughing - everyone knows the symptoms of the common cold. It is probably the most common illness. In ... avoid colds. There is no cure for the common cold. For relief, try Getting plenty of rest Drinking ...

  18. Citric acid production from partly deproteinized whey under non-sterile culture conditions using immobilized cells of lactose-positive and cold-adapted Yarrowia lipolytica B9.

    Science.gov (United States)

    Arslan, Nazli Pinar; Aydogan, Mehmet Nuri; Taskin, Mesut

    2016-08-10

    The present study was performed to produce citric acid (CA) from partly deproteinized cheese whey (DPCW) under non-sterile culture conditions using immobilized cells of the cold-adapted and lactose-positive yeast Yarrowia lipolytica B9. DPCW was prepared using the temperature treatment of 90°C for 15min. Sodium alginate was used as entrapping agent for cell immobilization. Optimum conditions for the maximum CA production (33.3g/L) in non-sterile DPCW medium were the temperature of 20°C, pH 5.5, additional lactose concentration of 20g/L, sodium alginate concentration of 2%, number of 150 beads/100mL and incubation time of 120h. Similarly, maximum citric acid/isocitric acid (CA/ICA) ratio (6.79) could be reached under these optimal conditions. Additional nitrogen and phosphorus sources decreased CA concentration and CA/ICA ratio. Immobilized cells were reused in three continuous reaction cycles without any loss in the maximum CA concentration. The unique combination of low pH and temperature values as well as cell immobilization procedure could prevent undesired microbial contaminants during CA production. This is the first work on CA production by cold-adapted microorganisms under non-sterile culture conditions. Besides, CA production using a lactose-positive strain of the yeast Y. lipolytica was investigated for the first time in the present study. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Phase II, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers.

    Science.gov (United States)

    Hayden, Frederick G; Turner, Ronald B; Gwaltney, Jack M; Chi-Burris, Kathy; Gersten, Merril; Hsyu, Poe; Patick, Amy K; Smith, George J; Zalman, Leora S

    2003-12-01

    Human rhinovirus (HRV) infections are usually self-limited but may be associated with serious consequences, particularly in those with asthma and chronic respiratory disease. Effective antiviral agents are needed for preventing and treating HRV illnesses. Ruprintrivir (Agouron Pharmaceuticals, Inc., San Diego, Calif.) selectively inhibits HRV 3C protease and shows potent, broad-spectrum anti-HRV activity in vitro. We conducted three double-blind, placebo-controlled clinical trials in 202 healthy volunteers to assess the activity of ruprintrivir in experimental HRV infection. Subjects were randomized to receive intranasal ruprintrivir (8 mg) or placebo sprays as prophylaxis (two or five times daily [2x/day or 5x/day] for 5 days) starting 6 h before infection or as treatment (5x/day for 4 days) starting 24 h after infection. Ruprintrivir prophylaxis reduced the proportion of subjects with positive viral cultures (for 5x/day dosing groups, 44% for ruprintrivir treatment group versus 70% for placebo treatment group [P=0.03]; for 2x/day dosing groups, 60% for ruprintrivir group versus 92% for placebo group [P=0.004]) and viral titers but did not decrease the frequency of colds. Ruprintrivir treatment reduced the mean total daily symptom score (2.2 for ruprintrivir treatment group and 3.3 for the placebo treatment group [P=0.014]) by 33%. Secondary endpoints, including viral titers, individual symptom scores, and nasal discharge weights, were also reduced by ruprintrivir treatment. Overall, ruprintrivir was well tolerated; blood-tinged mucus and nasal passage irritation were the most common adverse effects reported. Pharmacokinetic analysis of plasma and nasal ruprintrivir concentrations revealed intranasal drug residence with minimal systemic absorption. Results from these studies in experimental rhinoviral infection support continued investigation of intranasal ruprintrivir in the setting of natural HRV infection.

  20. Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    I. A. Siddiqui

    2013-12-01

    Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

  1. Mixed culture biofilms of Salmonella Typhimurium and cultivable indigenous microorganisms on lettuce show enhanced resistance of their sessile cells to cold oxygen plasma.

    Science.gov (United States)

    Jahid, Iqbal Kabir; Han, Noori; Zhang, Cheng-Yi; Ha, Sang-Do

    2015-04-01

    Control of foodborne pathogens in fresh produce is crucial for food safety, and numerous Salmonella Typhimurium (ST) outbreaks have been reported already. The present study was done to assess effectiveness of cold oxygen plasma (COP) against biofilms of ST mixed with cultivable indigenous microorganisms (CIM). ST and CIM were grown at 15 °C as monocultures and mixed cultures for planktonic state, biofilm on stainless steel, and lettuce leaves. Thereafter, the samples were treated with COP and surviving populations were counted using plate counting methods. Biofilms and stomatal colonization were examined using field emission scanning electron microscopy (FESEM) and food quality was assessed after treatment. Mixed cultures of ST and CIM showed an antagonistic interaction on lettuce but not on SS or in planktonic state. Mixed cultures showed significantly (p lettuce but not on SS or planktonic state. Shift from smooth to rugose colony type was found for planktonic and for biofilms on SS but not on lettuce for ST. Mixed culture biofilms colonized stomata on the inside as demonstrated by FESEM. Although, lettuce quality was not affected by COP, this technology has to be optimized for further development of the successful inactivation of complex multispecies biofilm structures presented by real food environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Rice callus suspension culture inhibits growth of cell lines of multiple cancer types and induces apoptosis in lung cancer cell line.

    Science.gov (United States)

    Rahman, Nafeesa; Dhadi, Surendar Reddy; Deshpande, Aparna; Ramakrishna, Wusirika

    2016-11-02

    Cancer is one of the leading cause of mortality. Even though efficient drugs are being produced to treat cancer, conventional medicines are costly and have adverse effects. As a result, alternative treatments are being tried due to their low cost and little or no adverse effects. Our previous study identified one such alternative in rice callus suspension culture (RCSC) which was more efficient than Taxol® and Etoposide, in reducing the viability of human colon and renal cancer cells in culture with minimal or no effect on a normal cell line. In this study, we tested the effect of RCSC by studying the dynamics of lactate dehydrogenase (LDH) in lung cancer cell lines (NCI-H460 and A549), breast cancer cell lines (MDA-MB-231 and MCF-7) and colorectal cancer cell lines (SW620 and Caco-2) as well as their normal-prototypes. Complementary analysis for evaluating membrane integrity was performed by estimating LDH release in non-lysed cells and cell viability with WST-1 assay. Fluorescence microscopy with stains targeting nucleus and cell membrane as well as caspase 3/7 and Annexin V assays were performed. Real-time quantitative RT-PCR was performed to evaluate expression of 92 genes associated with molecular mechanisms of cancer in RCSC treated ling cancer cell line, NCI-H460 and its normal prototype, MRC-5. High performance liquid chromatography (HPLC) was used to collect RCSC fractions, which were evaluated on NCI-H460 for their anti-cancer activity. Lower dilutions of RCSC showed maximum reduction in total LDH indicating reduced viability in majority of the cancer cell lines tested with minimal or no effect on normal cell lines compared to the control. Complementary analysis based on LDH release in non-lysed cells and WST-1 assay mostly supported total LDH results. RCSC showed the best effect on the lung non-small carcinoma cell line, NCI-H460. Fluorescence microscopy analyses suggested apoptosis as the most likely event in NCI-H460 treated with RCSC. Gene expression

  3. Bacteria in the nose of young adults during wellness and rhinovirus colds - detection by culture and microarray methods in 100 nasal lavage specimens

    Science.gov (United States)

    Allen, E. Kaitlynn; Pitkäranta, Anne; Mäki, Minna; Hendley, J. Owen; Laakso, Sanna; Sale, Michèle M.; Winther, Birgit

    2013-01-01

    Background Patients with viral respiratory infections/viral rhinitis/common colds are often treated with antibiotic although there is little information on whether or how bacterial microbiota in the nose and nasopharynx might influence the course of viral illnesses. Methods To initiate investigation of possible interaction between viral respiratory illness and microbiota of the nose/nasopharynx, we utilized microarray technology to examine 100 nasal lavage fluid samples (NLF) for bacterial species and recorded the bacterial titer of culturable bacteria. Rhinovirus illnesses were induced by self-inoculation using the “finger to nose or eye natural transmission route” in ten otherwise healthy young adults. NLF samples were collected during wellness and at specific time points following experimental rhinovirus inoculation. Results The rhinovirus infection rate was 70%. There were no consistent changes in the prevalence of different bacterial species determined by microarray and bacterial titer by culture methods during rhinovirus infection. The bacterial profile in NLF samples showed high variability between volunteers but low variability in multiple NLF’s obtained before and following infection from the same volunteer. S. epidermidis/coagulase negative staphylococcus (CNS) were identified in all ten subjects. One or more bacterial sinus/otitis pathogens were identified by microarray in six of the ten volunteers. The microarray identified a few bacteria not included in traditional bacterial cultures. Conclusion Our pilot study showed that each of ten volunteers had a unique bacterial profile in the nose by microarray analysis and that bacterial load did not change during experimental rhinovirus colds. Larger scale studies are warranted. PMID:23801660

  4. Bacteria in the nose of young adults during wellness and rhinovirus colds: detection by culture and microarray methods in 100 nasal lavage specimens.

    Science.gov (United States)

    Allen, E Kaitlynn; Pitkäranta, Anne; Mäki, Minna; Hendley, J Owen; Laakso, Sanna; Sale, Michèle M; Winther, Birgit

    2013-09-01

    Patients  with  viral respiratory infections/viral rhinitis/common colds are often treated with antibiotic; however, there is little information on whether or how bacterial microbiota in the nose and nasopharynx might influence the course of viral illnesses. To initiate investigation of possible interaction between viral respiratory illness and microbiota of the nose/nasopharynx, we used microarray technology to examine 100 nasal lavage fluid (NLF) samples for bacterial species and recorded the bacterial titer of culturable bacteria. Rhinovirus illnesses were induced by self-inoculation using the "finger to nose or eye natural transmission route" in 10 otherwise healthy young adults. NLF samples were collected during wellness and at specific time points following experimental rhinovirus inoculation. The rhinovirus infection rate was 70%. There were no consistent changes in the prevalence of different bacterial species determined by microarray and bacterial titer by culture methods during rhinovirus infection. The bacterial profile in NLF samples showed high variability between volunteers but low variability in multiple NLFs obtained before and following infection from the same volunteer. Streptococcus epidermidis/coagulase-negative staphylococcus (CNS) were identified in all 10 subjects. One or more bacterial sinus/otitis pathogens were identified by microarray in 6 of the 10 volunteers. The microarray identified a few bacteria not included in traditional bacterial cultures. Our pilot study showed that each of the 10 volunteers had a unique bacterial profile in the nose by microarray analysis and that bacterial load did not change during experimental rhinovirus colds. Larger scale studies are warranted. © 2013 The Authors. International Forum of Allergy & Rhinology published by Wiley Periodicals, Inc., on behalf of ARS-AAOA, LLC.

  5. Phenolic acids identified in sorghum distillery residue demonstrated antioxidative and anti-cold-stress properties in cultured tilapia, Oreochromis mossambicus.

    Science.gov (United States)

    Lee, Shin-Mei; Lin, Jing-Jen; Liao, Chih-Yuan; Cheng, Hui-Ling; Pan, Bonnie Sun

    2014-05-21

    This study aimed to identify the bioactive compounds and evaluate the anti-cold-stress function of the sorghum distillery residue (SDR) using tilapia as an alternative animal model. The highest contents of water-soluble bioactive compounds in SDR were polyphenols, followed by tannins, anthocyanins, and flavonoids. SDR was extracted with double-distilled water, 95% ethanol, and ethyl acetate, separately. The ethanol extract (SDR-E) yielded the highest polyphenol content [15.03 mg/g of SDR dry weight (dw)], of which the EC50 value of R,R-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging efficiency was 0.56 ± 0.04 mg/mL. The SDR-E suppressed the oxidation of low-density lipoproteins (LDLs) more efficiently than that of other extracts. Tilapia fed a diet containing 3.6% SDR-E decreased accumulative mortality during cold stress, of 46.2%. The accumulative morality of the control was 92.9%. The phenolic acids identified in SDR included gallic acid (0.36 ± 0.08 mg/g of SDR dw), 3,4-dihydroxybenzoic acid (0.16 ± 0.12 mg/g of SDR dw), and 4-hydroxybenzoic acid (0.49 ± 0.23 mg/g of SDR dw). Diets supplemented with 0.5% 4-hydroxybenzoic acid fed to tilapia showed a lower mortality rate than that fed 1.0% 4-hydroxybenzoic acid, comparable to that of the tilapia fed 20% SDR. The latter showed lower mortality than that of the control. These results suggested that 4-hydroxybenzoic acid is one of the major anti-cold-stress compounds in SDR.

  6. Making a homefront without a battlefront: The manufacturing of domestic enemies in the early Cold War culture 

    Directory of Open Access Journals (Sweden)

    Jean-Paul Gabilliet

    2012-03-01

    Full Text Available Although the Cold War was an undeclared conflict without actual battlefront one of its earliest charcteristics was the emergence in the United States of a homefront-based “war culture” targetting domestic enemies. 1947 witnessed the rise in news media of anxieties over alleged threats to domestic stability: in the first few months of the year, a Crime Scare reactivating pre-war concerns about the Mob and, in the summer, the first reported UFO sightings. In both cases the media and public resp...

  7. Improved production of chlorogenic acid from cell suspension ...

    African Journals Online (AJOL)

    Purpose: To evaluate the potential of Lonicera macranthoides Hand. -Mazz. Yulei1 suspension culture system for enhanced production of the main secondary metabolite, chlorogenic acid. Methods: The callus of L. macranthoides Hand.-Mazz. “Yulei1” was suspension cultured in B5 liquid medium supplemented with ...

  8. Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

    Science.gov (United States)

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon; Choi, Young-Im; Lee, Byung-Hyun; Choi, Dong-Woog

    2012-09-01

    The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  9. Evidence that generation of inositol 1,4,5-trisphosphate and hydrolysis of phosphatidylinositol 4,5-bisphosphate are rapid responses following addition of fungal elicitor which induces phytoalexin synthesis in lucerne (Medicago sativa) suspension culture cells.

    Science.gov (United States)

    Walton, T J; Cooke, C J; Newton, R P; Smith, C J

    1993-05-01

    Treatment of lucerne suspension culture cells with glycoprotein elicitor from the phytopathogenic fungus Verticillium albo-atrum R & B triggers Ca(2+)-mediated induction of antimicrobial secondary metabolites termed phytoalexins. The present study investigated the possible role of polyphosphoinositide signal transduction in phytoalexin elicitation. Within 1 min of addition of elicitor to lucerne suspension culture cells we found a 100-160% (15-25 pmol/g fresh wt) increase in the level of compound with chromatographic and electrophoretic properties expected for an inositol trisphosphate (InsP3) and which was strongly bound by an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-specific binding protein; after 3 min the level of this compound had fallen below that observed prior to elicitor challenge. In 32P-prelabelled cells, the relative proportion of radioactivity which cochromatographed with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to have decreased by 48% 1 min after elicitor addition and that rapid depletion of membrane lipid radioactivity was specific to this lipid fraction. The rapid, transient increase in level of Ins(1,4,5)P3 and concomitant fall in PtdIns(4,5)P2 suggests that Ins(1,4,5)P3 generated by hydrolysis of PtdIns(4,5)P2 may provide a Ca(2+)-mobilizing signal in phytoalexin elicitation in lucerne.

  10. 2,3,5,4′- Tetrahydroxystilbene-2-O-β-D-glycoside Biosynthesis by Suspension Cells Cultures of Polygonum multiflorum Thunb and Production Enhancement by Methyl Jasmonate and Salicylic Acid

    Directory of Open Access Journals (Sweden)

    Li Shao

    2012-02-01

    Full Text Available Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA and kinetin (KT. Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glycoside (THSG, 56.39 mg/L of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA and salicylic acid (SA could increase THSG production. The most appropriate concentration of MeJA was 100 μmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L. The most appropriate concentration of SA was 125 μmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L.

  11. 2,3,5,4'- tetrahydroxystilbene-2-O-β-D-glycoside biosynthesis by suspension cells cultures of Polygonum multiflorum Thunb and production enhancement by methyl jasmonate and salicylic acid.

    Science.gov (United States)

    Shao, Li; Zhao, Shu-Jin; Cui, Tang-Bing; Liu, Zhong-Yu; Zhao, Wei

    2012-02-22

    Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA) and kinetin (KT). Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L) and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glycoside (THSG, 56.39 mg/L) of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA) and salicylic acid (SA) could increase THSG production. The most appropriate concentration of MeJA was 100 μmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L). The most appropriate concentration of SA was 125 μmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L).

  12. EDITORIAL: Colloidal suspensions Colloidal suspensions

    Science.gov (United States)

    Petukhov, Andrei; Kegel, Willem; van Duijneveldt, Jeroen

    2011-05-01

    Special issue in honour of Henk Lekkerkerker's 65th birthday Professor Henk N W Lekkerkerker is a world-leading authority in the field of experimental and theoretical soft condensed matter. On the occasion of his 65th birthday in the summer of 2011, this special issue celebrates his many contributions to science. Henk Lekkerkerker obtained his undergraduate degree in chemistry at the University of Utrecht (1968) and moved to Calgary where he received his PhD in 1971. He moved to Brussels as a NATO fellow at the Université Libre de Bruxelles and was appointed to an assistant professorship (1974), an associate professorship (1977) and a full professorship (1980) in physical chemistry at the Vrije Universiteit Brussel. In 1985 he returned to The Netherlands to take up a professorship at the Van 't Hoff Laboratory, where he has been ever since. He has received a series of awards during his career, including the Onsager Medal (1999) of the University of Trondheim, the Bakhuys Roozeboom Gold Medal (2003) of the Royal Dutch Academy of Arts and Sciences (KNAW), the ECIS-Rhodia European Colloid and Interface Prize (2003), and the Liquid Matter Prize of the European Physical Society (2008). He was elected a member of KNAW in 1996, was awarded an Academy Chair position in 2005, and has held several visiting lectureships. Henk's work focuses on phase transitions in soft condensed matter, and he has made seminal contributions to both the theoretical and experimental aspects of this field. Here we highlight three major themes running through his work, and a few selected publications. So-called depletion interactions may lead to phase separation in colloid-polymer mixtures, and Henk realised that the partitioning of polymer needs to be taken into account to describe the phase behaviour correctly [1]. Colloidal suspensions can be used as model fluids, with the time- and length-scales involved leading to novel opportunities, notably the direct observation of capillary waves at a

  13. Suspension as an Emergency Power

    National Research Council Canada - National Science Library

    Amanda L. Tyler

    2009-01-01

    ... Legislation B. Suspension During Reconstruction: Putting Down the Klan in South Carolina IV. UNDERSTANDING SUSPENSION AS AN EMERGENCY POWER A. Reading the Suspension Clause in Context B. Giving Meaning to the Suspension Power C. Mapping the Suspension Clause Within the Constitution V. SUSPENSION AND THE SEPARATION OF POWERS CONCLUSION [A] suspensio...

  14. Bacterial diversity and bioprospecting for cold-active hydrolytic enzymes from culturable bacteria associated with sediment from Nella Fjord, Eastern Antarctica.

    Science.gov (United States)

    Yu, Yong; Li, Hui-Rong; Zeng, Yin-Xin; Chen, Bo

    2011-01-31

    The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica (69°22'6″ S, 76°21'45″ E) was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychrophilic, 15 isolates were moderately psychrophilic, and 11 isolates were psychrotolerant. More than 72% of the isolates required sodium chloride to grow. Esterase, β-glucosidase and proteases activities at 4 °C were detected in more than 45% of the strains while approximately 21%, 15% and 12% of the strains possessed lipase, amylase and chitinase, respectively. These results indicate that a relatively high culturable bacterial diversity is present within marine sediment of Nella Fjord and it could serve as an ideal candidate region for bioprospecting.

  15. Bacterial Diversity and Bioprospecting for Cold-Active Hydrolytic Enzymes from Culturable Bacteria Associated with Sediment from Nella Fjord, Eastern Antarctica

    Science.gov (United States)

    Yu, Yong; Li, Hui-Rong; Zeng, Yin-Xin; Chen, Bo

    2011-01-01

    The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica (69°22′6″ S, 76°21′45″ E) was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychrophilic, 15 isolates were moderately psychrophilic, and 11 isolates were psychrotolerant. More than 72% of the isolates required sodium chloride to grow. Esterase, β-glucosidase and proteases activities at 4 °C were detected in more than 45% of the strains while approximately 21%, 15% and 12% of the strains possessed lipase, amylase and chitinase, respectively. These results indicate that a relatively high culturable bacterial diversity is present within marine sediment of Nella Fjord and it could serve as an ideal candidate region for bioprospecting. PMID:21566794

  16. Bacterial Diversity and Bioprospecting for Cold-Active Hydrolytic Enzymes from Culturable Bacteria Associated with Sediment from Nella Fjord, Eastern Antarctica

    Directory of Open Access Journals (Sweden)

    Bo Chen

    2011-01-01

    Full Text Available The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica (69°22′6″ S, 76°21′45″ E was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychrophilic, 15 isolates were moderately psychrophilic, and 11 isolates were psychrotolerant. More than 72% of the isolates required sodium chloride to grow. Esterase, b-glucosidase and proteases activities at 4 °C were detected in more than 45% of the strains while approximately 21%, 15% and 12% of the strains possessed lipase, amylase and chitinase, respectively. These results indicate that a relatively high culturable bacterial diversity is present within marine sediment of Nella Fjord and it could serve as an ideal candidate region for bioprospecting.

  17. Cold plate

    Energy Technology Data Exchange (ETDEWEB)

    Marroquin, Christopher M.; O' Connell, Kevin M.; Schultz, Mark D.; Tian, Shurong

    2018-02-13

    A cold plate, an electronic assembly including a cold plate, and a method for forming a cold plate are provided. The cold plate includes an interface plate and an opposing plate that form a plenum. The cold plate includes a plurality of active areas arranged for alignment over respective heat generating portions of an electronic assembly, and non-active areas between the active areas. A cooling fluid flows through the plenum. The plenum, at the non-active areas, has a reduced width and/or reduced height relative to the plenum at the active areas. The reduced width and/or height of the plenum, and exterior dimensions of cold plate, at the non-active areas allow the non-active areas to flex to accommodate surface variations of the electronics assembly. The reduced width and/or height non-active areas can be specifically shaped to fit between physical features of the electronics assembly.

  18. [Phylogenetic diversity and cold-adaptive hydrolytic enzymes of culturable psychrophilic bacteria associated with sea ice from high latitude ocean, Artic].

    Science.gov (United States)

    Yu, Yong; Li, Hui-Rong; Chen, Bo; Zeng, Yin-Xin; He, Jian-Feng

    2006-04-01

    The phylogenetic diversity of culturable psychrophilic bacteria associated with sea ice from high latitude sea (77 degrees 30'N - 81 degrees 12'N), Canadian Basin and Greenland sea Arctic, was investigated. A total of 37 psychrophilic strains were isolated using three different methods of ( i ) spread plate method: 100 microL of each dilution ice-melt sample was spreaded onto the surface of Marine 2216 agar (DIFCO laboratories, Detroit, MI) and incubated for 2 to 6 weeks at 4 degrees C; ( ii ) bath culture and spread plate method: 1 mL of sample was added to 9mL of NSW (unamended natural seawater, 0.2 microm prefiltered and autoclaved) and incubated for 1 months at - 1 degrees C, then spread plate method was used to isolate bacterial strains from the pre-cultured samples; ( iii ) cold shock, bath culture and spread plate method: samples were exposed to - 20 degrees C for 24h, then bacterial strains isolated by bath culture and spread plate method under aerobic conditions. Nearly half of psychrophilic strains are isolated by using method iii . 16S rDNA nearly full-length sequence analysis reveal that psychrophilic strains fall in two phylogenetic divisions, gamma-proteobacteria (in the genera Colwellia, Marinobacter, Shewanella, Thalassomonas, Glaciecola, Marinomonas and Pseudoalteromonas) and Cytophaga-Flexibacter-Bacteroides (in the genera Flavobacterium and Psychroflexus). Nine of bacterial isolates (BSi20007, BSi20497, BSi20517, BSi20537, BSi20170, BSi20001, BSi20002, BSi20675 and BSi20101) quite likely represent novel species (16S rDNA sequence similarity below 97%). One of strains (BSi20002) from Canadian Basin shows 100% sequence similarity to the Antarctic Weddell sea ice isolate Marinobacter sp. ANT8277, suggesting bacteria may have a bipolar distribution at the species level. AF283859 sequences were submitted to the BLAST search program of the National Center for Biotechnology Information website (NCBI, http://www. ncbi. nlm.nih. gov). Twenty sequences

  19. Laser effects on yeast cell suspensions

    Science.gov (United States)

    Grigorovici, A.; Despa, Sanda I.; Paunescu, Teodor G.

    1995-03-01

    The aim of this paper is to determine the effects produced by coherent electromagnetic radiation in the ultraviolet and visible range on the growth of a Saccharomyces cerevisiae cell suspension. There were made several experiments in which we used different irradiation parameters (power, irradiation time, wavelength) for pointing out those that produce the stimulation or inhibition of the cellular culture growth. Beyond the modifications that appeared in the culture evolution we investigated other physical and chemical changes induced by the laser light on yeast cell suspensions.

  20. Electrorheology of nanofiber suspensions

    National Research Council Canada - National Science Library

    Yin, Jianbo; Zhao, Xiaopeng

    2011-01-01

    .... In this review, we especially focus on the recent researches on electrorheology of various nanofiber-based suspensions, including inorganic, organic, and inorganic/organic composite nanofibers...

  1. Efficient amplification of chimeric adenovirus 5/40S vectors carrying the short fiber protein of Ad40 in suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Marta Miralles

    Full Text Available The human adenovirus 40 (Ad40 is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.

  2. Autologous epidermal cell suspension: A promising treatment for chronic wounds.

    Science.gov (United States)

    Zhao, Hongliang; Chen, Yan; Zhang, Cuiping; Fu, Xiaobing

    2016-02-01

    Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Skin grafting is an important treatment method for chronic wounds, which are refractory to conservative therapy. The technique involving epidermal cell suspensions was invented to enable the possibility of treating larger wounds with only a small piece of donor skin. Both uncultured and cultured autologous epidermal cell suspensions can be prepared and survive permanently on the wound bed. A systematic search was conducted of EMBASE, Cochrane Library, PubMed and web of science by using Boolean search terms, from the establishment of the database until May 31, 2014. The bibliographies of all retrieved articles in English were searched. The search terms were: (epithelial cell suspension OR keratinocyte suspension) and chronic and wound. From the included, 6 studies are descriptive interventions and discussed the use of autologous keratinocyte suspension to treat 61 patients' chronic wound. The various methods of preparation of epidermal cell suspension are described. The advantages and shortcomings of different carriers for epidermal cell suspensions are also summarised. Both uncultured and cultured autologous epidermal cell suspensions have been used to treat chronic wounds. Although the limitations of these studies include the small number of patient populations with chronic wounds and many important problems that remain to be solved, autologous epidermal cell suspension is a promising treatment for chronic wounds. Copyright © 2015 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  3. Cold Sore

    Science.gov (United States)

    ... pain Headache Cold sore Symptoms & causes Diagnosis & treatment Advertisement Mayo Clinic does not endorse companies or products. ... a Job Site Map About This Site Twitter Facebook Google YouTube Pinterest Mayo Clinic is a not- ...

  4. Cold Urticaria

    Science.gov (United States)

    ... management of physical urticaria. Annals of Allergy, Asthma & Immunology. 2013;111:235. Nov. 21, 2014 Original article: http://www.mayoclinic.org/diseases-conditions/cold-urticaria/basics/definition/CON-20034524 . Mayo Clinic Footer Legal Conditions ...

  5. Review of Cold War Freud, Psychiatry in Communist Europe, and Psiquiatría, Psicoánalisis y Cultura Comunista: Batallas Ideológicas en la Guerra Fria [Psychiatry, Psychoanalysis and Communist Culture: Ideological Battles in the Cold War].

    Science.gov (United States)

    Innamorati, Marco

    2017-08-01

    Reviews the books, Cold War Freud by D. Herzog (2016), Psychiatry in Communist Europe edited by M. Savelli and S. Marks (2015), and Psiquiatría, Psicoánalisis y Cultura Comunista: Batallas Ideológicas en la Guerra Fria [Psychiatry, psychoanalysis and communist culture: Ideological battles in the Cold War] by H. Vezzetti. On the whole, the three books show how the Cold War influenced, in various ways, psychiatric and psychotherapeutic cultures. Beyond the Iron Curtain, as one can perceive from the book edited by Savelli and Marks (2015), politics explicitly set the agenda for the psychological sciences, using them even to invent ad hoc nosologies, useful for purposes related to power. In the United States, on the other hand, as Herzog (2016) pinpoints, the political situation affected the same field, even if indirectly, as in the Christianization of a discipline-psychoanalysis-the creator of which proudly declared himself an atheist Jew. In other Western countries, the relationship between psychiatry and power could bring about paradoxical results. From Vezzetti's (2016) book, one can ascertain that psychiatric culture might assume an overtly opposing stance toward political power. Vezzetti scans the case of Argentina, and partly of France, but they were not isolated cases. In Italy, for example, a movement of radical psychiatrists understood their role as a necessary opposition to political power, having as an aim the "liberation" of patients locked up in the psychiatric hospitals (Foot, 2015). (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  6. Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris Enzimas do metabolismo da sacarose em cultura celular de Bauhinia forficata, Curcuma zedoaria e Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Marcia Ometto de Mello

    2001-09-01

    Full Text Available The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.O objetivo deste trabalho foi estudar as enzimas do metabolismo da sacarose em culturas de célula em suspensão de Bauhinia forficata Link, Curcuma zedoaria Roscoe e Phaseolus vulgaris L. A via da invertase foi identificada nas três espécies estudadas. A via da sacarose sintase também foi responsável pelo metabolismo da sacarose em células de Curcuma zedoaria e Phaseolus vulgaris. Foram encontradas atividades maiores que 300 nmol min-1 mg-1 de proteína das enzimas invertase ácida e alcalina, UDPglicose pirofosforilase e fosfoglicomutase no extrato celular das três espécies de plantas. A sacarose sintase mostrou atividade baixa nas células de Bauhinia forficata. À medida que a concentração de sacarose no meio de cultura diminuiu, a atividade da sacarose sintase aumentou em células de Curcuma zedoaria e Phaseolus vulgaris. Ao final do período de cultura, quando os carboidratos se tornaram limitantes, as atividades das enzimas glicolíticas reduziram-se gradualmente.

  7. Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures.

    Science.gov (United States)

    Petzinger, E; Frimmer, M

    1988-01-13

    Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.

  8. Induced accumulation of 20-hydroxyecdysone in cell suspension ...

    African Journals Online (AJOL)

    This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata, an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly ...

  9. Observation of antioxidant activity of leaves, callus and suspension ...

    African Journals Online (AJOL)

    ... of leaves, callus culture and cell suspension culture of J. gendarussa. Callus was induced from green and matured leaves of J. gendarussa cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) and ...

  10. In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae Morfogênese in vitro e estabelecimento de culturas de suspensão celular em Piper solmsianum C. DC. (Piperaceae

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena

    2009-03-01

    Full Text Available Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA. Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l-1 were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l-1 2,4-D and 2 mg.l-1 BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.Piper solmsianum é uma espécie herbácea do sudeste brasileiro onde vários compostos biologicamente ativos já foram identificados. O objetivo deste trabalho foi estabelecer suspensões celulares nesta espécie. Para tanto, foram utilizados explantes de pecíolos e folhas, retirados de plântulas cultivadas in vitro, os quais foram submetidos a diferentes combinações de reguladores de crescimento (AIA, ANA, 2,4-D e BAP. Foi obtida a neo-formação de raízes e brotos, estes últimos através do processo de organogênese indireta evidenciada por estudos histológicos. Para a indução e crescimento dos calos, foram avaliados, além das diferentes combinações de reguladores de crescimento, a suplementação ao meio de cultura de carvão ativado (1,5 mg.l-1 e o regime de luz. Culturas mantidas na luz, em meio de cultura suplementado com 0,2 mg.l-1 2,4-D e 2 mg

  11. Suspension Trauma / Orthostatic Intolerance

    Science.gov (United States)

    ... Emphasis Programs Directives Severe Violators TOPICS By Sector Construction Health Care Agriculture Maritime Oil and Gas Federal ... such fatalities often are referred to as "harnessinduced pathology" or "suspension trauma." Signs & symptoms that may be ...

  12. Urinary incontinence - retropubic suspension

    Science.gov (United States)

    ... your doctor will have you try bladder retraining, Kegel exercises, medicines, or other options. If you tried ... retropubic colposuspension; Needle suspension; Burch colposuspension Patient Instructions Kegel exercises - self-care Self catheterization - female Suprapubic catheter ...

  13. Rheology of organoclay suspension

    CSIR Research Space (South Africa)

    Hato, MJ

    2011-05-01

    Full Text Available The authors have studied the rheological properties of clay suspensions in silicone oil, where clay surfaces were modified with three different types of surfactants. Dynamic oscillation measurements showed a plateau-like behavior for all...

  14. Articulated suspension system

    Science.gov (United States)

    Bickler, Donald B. (Inventor)

    1989-01-01

    The invention provides a rough terrain vehicle which maintains a substantially constant weight, and therefore traction, on all wheels, despite one wheel moving considerably higher or lower than the others, while avoiding a very soft spring suspension. The vehicle includes a chassis or body to be supported and a pair of side suspensions at either side of the body. In a six wheel vehicle, each side suspension includes a middle wheel, and front and rear linkages respectively coupling the front and rear wheels to the middle wheel. A body link pivotally connects the front and rear linkages together, with the middle of the body link rising or falling by only a fraction of the rise or fall of any of the three wheels. The body link pivotally supports the middle of the length of the body. A transverse suspension for suspending the end of the body on the side suspensions includes a middle part pivotally connected to the body about a longitudinal axis and opposite ends each pivotally connected to one of the side suspensions along at least a longitudinal axis.

  15. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Directory of Open Access Journals (Sweden)

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  16. Reaction of detoxification mechanisms in suspension cultured spruce cells (Picea abies L. Karst.) to heavy metals in pure mixture and in soil eluates.

    Science.gov (United States)

    Schröder, Peter; Fischer, Claudia; Debus, Reinhard; Wenzel, Andrea

    2003-01-01

    INTENTION, GOAL, BACKGROUND: The widespread and unconcerned use of chemicals in the past has led to an accumulation of pollutants in our environment. Numerous sites are polluted with a mixture of organic chemicals and heavy metals. The future use of these sites and the safe consumption of groundwater from these areas depends on our ability to assess risk by determining the bioavailability of trace levels of pollutants in the respective soil solutions. Soil eluates containing heavy metals in mixture as well as pure heavy metals in aqueous solution were added to a spruce cell culture to set up such a test system. The present study aims at evaluating the response of cultured spruce cells to heavy metals in aqueous solution, and at characterizing these basic cellular responses as potential biomarkers. In order to characterize cell reactions toward heavy metals, spruce cell cultures were incubated with CdSO4 (50 to 500 microM), Na2HAsO4 (1.5 to 80 microM) or PbCl2 (10 to 150 microM). Alternatively, the cells were incubated with a standard heavy metal mixture containing 80 microM Na2HAsO4, 150 microM CdSO4 and 150 microM PbCl2 in medium and with aqueous original soil eluates. Measurement of oxidative stress, antioxidants and basic detoxification enzymes involved in plant defence reactions were performed with the treated cells. After 5 hrs of incubation, the onset of a strong oxidative burst was observed. H2O2 concentrations exceeded 40 microM in the culture media after 20 hrs. Concomitantly, glutathione levels showed drastic changes indicating the influence of the metals and/or the H2O2 on antioxidative systems. Following cadmium treatment, GSH and GSSG were elevated by 50 and 200% above controls. Whereas arsenic doubled GSSG levels, treatment with lead did not cause significant changes. However, a mixture of the metals decreased both metabolites by 50%. The effect of the metals was concentration-dependent and disappeared at high concentrations. Furthermore, strong

  17. Cold fusion

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Suk Yong; You, Jae Jun [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-01-01

    Nearly every technical information is chased in the world. All of them are reviewed and analyzed. Some of them are chosen to study further more to review every related documents. And a probable suggestion about the excitonic process in deuteron absorbed condensed matter is proposed a way to cold fusion. 8 refs. (Author).

  18. Common cold

    Science.gov (United States)

    ... many health problems, including colds. DO NOT use antibiotics if they are not needed. Breastfeed infants if possible. Breast milk is known to protect against respiratory tract infections in children, even years after you stop breastfeeding. Drink plenty of fluids to help your immune ...

  19. Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis.

    Science.gov (United States)

    Zhou, Xin; Zhong, Jian-Jiang

    2011-05-01

    It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H(2)O(2) production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

  20. Coping with Cold Sores

    Science.gov (United States)

    ... Skating Living With Stepparents Be a Green Kid Cold Sores KidsHealth > For Kids > Cold Sores Print A ... sore." What's that? Adam wondered. What Is a Cold Sore? Cold sores are small blisters that is ...

  1. Cold fusion

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Suk Yong; Sung, Ki Woong; Kang, Joo Sang; Lee, Jong Jik [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1995-02-01

    So called `cold fusion phenomena` are not confirmed yet. Excess heat generation is very delicate one. Neutron generation is most reliable results, however, the records are erratic and the same results could not be repeated. So there is no reason to exclude the malfunction of testing instruments. The same arguments arise in recording {sup 4}He, {sup 3}He, {sup 3}H, which are not rich in quantity basically. An experiment where plenty of {sup 4}He were recorded is attached in appendix. The problem is that we are trying to search cold fusion which is permitted by nature or not. The famous tunneling effect in quantum mechanics will answer it, however, the most fusion rate is known to be negligible. The focus of this project is on the theme that how to increase that negligible fusion rate. 6 figs, 4 tabs, 1512 refs. (Author).

  2. A novel approach to regulate cell membrane permeability for ATP and NADH formation in Saccharomyces cerevisiae induced by air cold plasma

    Science.gov (United States)

    Dong, Xiaoyu; Liu, Tingting; Xiong, Yuqin

    2017-02-01

    Air cold plasma has been used as a novel method for enhancing microbial fermentation. The aim of this work was to explore the effect of plasma on membrane permeability and the formation of ATP and NADH in Saccharomyces cerevisiae, so as to provide valuable information for large-scale application of plasma in the fermentation industry. Suspensions of S. cerevisiae cells were exposed to air cold plasma for 0, 1, 2, 3, 4 and 5 min, and then subjected to various analyses prior to fermentation (0 h) and at the 9 and 21 h stages of fermentation. Compared with non-exposed cells, cells exposed to plasma for 1 min exhibited a marked increase in cytoplasmic free Ca2+ concentration as a result of the significant increase in membrane potential prior to fermentation. At the same time, the ATP level in the cell suspension decreased by about 40%, resulting in a reduction of about 60% in NADH prior to culturing. However, the levels of ATP and NADH in the culture at the 9 and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indicated that exposure of S. cerevisiae to air cold plasma could increase its cytoplasmic free Ca2+ concentration by improving the cell membrane potential, consequently leading to changes in ATP and NADH levels. Supported by National Natural Science Foundation of China (Nos. 21246012, 21306015 and 21476032).

  3. A contribution to the improvement of Listeria monocytogenes enumeration in cold-smoked salmon.

    Science.gov (United States)

    Besse, Nathalie Gnanou; Audinet, Nelly; Beaufort, Annie; Colin, Pierre; Cornu, Marie; Lombard, Bertrand

    2004-03-01

    For the enumeration of Listeria monocytogenes in food, a sensitive enumeration method based on membrane filtration followed by transfer of the filter to a selective medium has been developed. This study was carried out with cold-smoked salmon, a product likely to be contaminated with L. monocytogenes. The operating protocol utilizes three filtration runs in parallel (5, 15 and 30 ml) of a 1 in 10 dilution of the salmon suspension through 0.45-microm pore-size cellulose ester membranes, and then culture of the filters on Aloa agar (AES Laboratoires, Combourg, France). The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L. monocytogenes from both artificially and naturally contaminated cold-smoked salmon. Moreover, for several samples contaminated at low levels, L. monocytogenes could be recovered only by the filtration method. The examination of increasing volumes of salmon suspension enabled readable results to be obtained for all levels of L. monocytogenes and competitive microflora investigated. In most cases, the optimised operating protocol enabled 5.1 g of salmon to be examined, instead of 0.01-0.1 g with the reference EN ISO 11290-2 method, thus improving the sensitivity of the method.

  4. Estabelecimento de cultura de células em suspensão e identificação de flavonóides em Cordia verbenacea DC. Establishment of cell suspension cultures and flavonoid identification in Cordia verbenacea DC.

    Directory of Open Access Journals (Sweden)

    O.A. Lameira

    2009-01-01

    Full Text Available O trabalho teve como objetivos o estabelecimento de culturas de células em suspensão, extração, separação e identificação de flavonóides em extratos de folhas e de células em suspensão de Cordia verbenacea. Células dessa espécie, após terem sido subcultivadas três vezes no meio MS suplementado com 2,32 µM de cinetina + 10,74 µM de ANA a intervalos de 28 dias, apresentaram cinco estágios de crescimento: as fases lag, exponencial, linear, desaceleração e estacionária. O maior percentual de crescimento (37% ocorreu no período exponencial entre o quarto e o décimo segundo dia e o menor (3% na fase lag até o quarto dia. Para identificação de flavonóides, foram usados extratos submetidos à separação e purificação por CCD e CCL e os componentes obtidos submetidos à Espectroscopia de Ultravioleta, Espectrometria de Infravermelho e Massa e Ressonância Magnética Nuclear de Hidrogênio. Após as frações das amostras de folhas e células terem sido separadas pelo eluente ácido acético, foram identificados os componentes 7,4'-diidróxi-5'-carboximetóxi isoflavona e 7,4'-diidróxi-5'-metil isoflavona. Foi detectado maior concentração dessas substâncias nas células cultivadas in vitro.The aims of this study were to establish cell suspension cultures, as well as to extract, separate and identify flavonoids in Cordia verbenacea leaf extracts and cell suspensions. Cells of this species were subcultivated three times in MS culture medium supplemented with 2.32 µM kinetin + 10.74 µM NAA at 28-day intervals, showing five growth stages: lag, exponential, linear, deceleration and stationary phases. The highest growth rate (37% occurred in the exponential phase between the fourth and the twelfth day and the lowest growth rate (3%, in the lag phase until the fourth day. For flavonoid identification, extracts were separated and purified by TLC and LC and the obtained compounds were subjected to Ultraviolet Spectroscopy

  5. Culture

    OpenAIRE

    Smith, Timothy B.; Melanie M. Domenech Rodríguez; Bernal, Guillermo

    2011-01-01

    This article summarizes the definitions, means, and research of adapting psychotherapy to clients' cultural backgrounds. We begin by reviewing the prevailing definitions of cultural adaptation and providing a clinical example. We present an original meta-analysis of 65 experimental and quasi-experimental studies involving 8,620 participants. The omnibus effect size of d = .46 indicates that treatments specifically adapted for clients of color were moderately more effective with that clientele...

  6. Optimisation des conditions d'etablissement de suspensions ...

    African Journals Online (AJOL)

    En effet, l'établissement de suspensions cellulaires embryogènes a été obtenu au bout de 8 semaines, lorsque la première filtration a été faite après 2 semaines et que les amas de cals ont été broyés à chaque filtration. ... The growth of embryogenic cell suspension is similar on both culture media used (MS and N6).

  7. Mevastatin-induced inhibition of cell growth in avocado suspension ...

    African Journals Online (AJOL)

    Cell suspension cultures were established using soft, friable callus derived from nucellar tissue of 'Hass' avocado (Persea americana Mill.) seed from fruit harvested 190 days after full bloom. Cell cultures were maintained in liquid medium supplemented with naphthalene acetic acid (NAA), isopentenyl adenine (iP) and ...

  8. Enhancement of 20-hydroxyecdysone production in cell suspension ...

    African Journals Online (AJOL)

    ... most effective. The maximum 20-hydroxyecdysone productivity of about 1.31 mg/L/day was observed in culture with 10 mg/L 7-dehydrocholesterol. This data is the first indication that 7-dehydrocholesterol and ergosterol feeding are effective precursors for 20-hydroxyecdysone formation in plant cell suspension culture.

  9. Viscosity of colloidal suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, E.G.D. [Rockefeller Univ., New York, NY (United States); Schepper, I.M. de [Delft Univ. of Technology (Netherlands)

    1995-12-31

    Simple expressions are given for the effective Newtonian viscosity as a function of concentration as well as for the effective visco-elastic response as a function of concentration and imposed frequency, of monodisperse neutral colloidal suspensions over the entire fluid range. The basic physical mechanisms underlying these formulae are discussed. The agreement with existing experiments is very good.

  10. Flywheel Magnetic Suspension Developments

    Science.gov (United States)

    Palazzolo, Alan; Kenny, Andrew; Sifford, Curtiss; Thomas, Erwin; Bhuiyan, Mohammad; Provenza, Andrew; Kascak, Albert; Montague, Gerald; Lei, Shuliang; Kim, Yeonkyu; hide

    2002-01-01

    The paper provides an overview of many areas of the flywheel magnetic suspension (MS) R&D being performed at the Texas A&M Vibration Control and Electromechanics Lab (TAMU-VCEL). This includes system response prediction, actuator optimization and redundancy, controller realizations and stages, sensor enhancements and backup bearing reliability.

  11. Cryonic Suspension and the Law.

    Science.gov (United States)

    Smith, George P.; Hall, Clare

    1987-01-01

    Analyzes three central problems which adversely affect use, development, and perfection of cryonic suspension of individuals: the extent to which a physician may be guilty of malpractice in assisting with a suspension; the need for a recognition of suspension; and the present effect of the law's anachronistic treatment of estate devolution upon a…

  12. Culture

    OpenAIRE

    Coulangeon, Philippe

    2013-01-01

    Il n’est sans doute pas de notion aussi vaste et aussi polysémique en sciences sociales que la notion de culture, qui renvoie alternativement à l’ensemble des symboles, des significations, des valeurs et des manières de faire propres à un groupe et au domaine spécialisé des activités expressives, savantes et populaires. La notion de culture est ainsi tout autant mobilisée dans l’exploration des grandes thématiques de la sociologie (stratification, inégalités, institutions, mouvements sociaux)...

  13. Cultural

    Science.gov (United States)

    Wilbur F. LaPage

    1971-01-01

    A critical look at outdoor recreation research and some underlying premises. The author focuses on the concept of culture as communication and how it influences our perception of problems and our search for solutions. Both outdoor recreation and science are viewed as subcultures that have their own bodies of mythology, making recreation problems more difficult to...

  14. Cold-War Echoes in American Children.

    Science.gov (United States)

    Winn, Ira Jay

    1984-01-01

    The author believes a cold war ideology permeates our culture and poisons the minds of youth. The challenge to education is to awaken people to a historical and global perspective and raise public consciousness of the necessity for peace. (MD)

  15. cultural

    Directory of Open Access Journals (Sweden)

    Irene Kreutz

    2006-01-01

    Full Text Available Es un estudio cualitativo que adoptó como referencial teorico-motodológico la antropología y la etnografía. Presenta las experiencias vivenciadas por mujeres de una comunidad en el proceso salud-enfermedad, con el objetivo de comprender los determinantes sócio-culturales e históricos de las prácticas de prevención y tratamiento adoptados por el grupo cultural por medio de la entrevista semi-estructurada. Los temas que emergieron fueron: la relación entre la alimentación y lo proceso salud-enfermedad, las relaciones con el sistema de salud oficial y el proceso salud-enfermedad y lo sobrenatural. Los dados revelaron que los moradores de la comunidad investigada tienen un modo particular de explicar sus procedimientos terapéuticos. Consideramos que es papel de los profesionales de la salud en sus prácticas, la adopción de abordajes o enfoques que consideren al individuo en su dimensión sócio-cultural e histórica, considerando la enorme diversidad cultural en nuestro país.

  16. Heteropolar Magnetic Suspension

    Science.gov (United States)

    Misovec, Kathleen; Johnson, Bruce; Downer, James; Eisenhaure, David; Hockney, Richard

    1990-01-01

    Compact permanent-magnet/electromagnet actuator has six degrees of freedom. Heteropolar magnetic actuator conceived for use as actively controlled vibration-isolating suspension device. Exerts forces along, and torques about, all three principal coordinate axes to resist all three components of translational vibration and all three components of rotational vibration. Inner cylinder suspended magnetically within outer cylinder. Electro-magnet coils interact with fields of permanent magnets to provide active control of suspending force and torque.

  17. Cold symptoms (image)

    Science.gov (United States)

    Colds are caused by a virus and can occur year-round. The common cold generally involves a runny nose, nasal congestion, and ... symptoms include sore throat, cough, and headache. A cold usually lasts about 7 days, with perhaps a ...

  18. Cough & Cold Medicine Abuse

    Science.gov (United States)

    ... to Your Parents - or Other Adults Cough & Cold Medicine Abuse KidsHealth > For Teens > Cough & Cold Medicine Abuse ... resfriado Why Do People Use Cough and Cold Medicines to Get High? There's an ingredient in many ...

  19. Cold antihydrogen and CPT

    CERN Document Server

    Gabrielse, G; Bowden, N S; Oxley, P; Storry, C H; Wessels, M; Speck, A K; Estrada, J; Yesley, P S; Grzonka, D; Oelert, Walter; Schepers, G; Sefzick, T; Walz, J

    2002-01-01

    Progress in the quest for cold antihydrogen includes the first substantial accumulation of cold positrons and the first demonstration of positron cooling. Stacking of cold antiprotons is key to using the new antiproton decelerator facility at CERN. (22 refs).

  20. Culture

    OpenAIRE

    Dubois, Vincent

    2007-01-01

    Le découpage des spécialités sociologiques hésite habituellement entre une répartition thématique par domaines empiriquement distingués et un partage conceptuel reposant sur des orientations de recherche. La sociologie de la culture n'échappe pas à cette oscillation. De prime abord, elle couvre un secteur plus ou moins clairement délimité, qui englobe la sociologie de l'art et ce qui est socialement désigné comme relevant de la « vie culturelle ». Elle regroupe alors un ensemble de subdivisio...

  1. Culture

    OpenAIRE

    Boas, Franz

    2003-01-01

    L’un des objets de l’enquête anthropologique, pour laquelle des éléments peuvent être obtenus par l’étude des sociétés existantes, est l’inter-dépendance des phénomènes culturels. Alors que dans l’étude des processus de diffusion et de développement parallèle les caractères et la distribution de traits singuliers sont communément les objets de l’analyse, nous sommes conduits, ici, à considérer la culture, dans toutes ses manifestations, comme un tout. Les inventions, la vie économique, la str...

  2. Culture-independent characterization of bacterial communities associated with the cold-water coral Lophelia pertusa in the northeastern Gulf of Mexico

    Science.gov (United States)

    Kellogg, C.A.; Lisle, J.T.; Galkiewicz, J.P.

    2009-01-01

    Bacteria are recognized as an important part of the total biology of shallow-water corals. Studies of shallow-water corals suggest that associated bacteria may benefit the corals by cycling carbon, fixing nitrogen, chelating iron, and producing antibiotics that protect the coral from other microbes. Cold-water or deep-sea corals have a fundamentally different ecology due to their adaptation to cold, dark, high-pressure environments and as such have novel microbiota. The goal of this study was to characterize the microbial associates of Lophelia pertusa in the northeastern Gulf of Mexico. This is the first study to collect the coral samples in individual insulated containers and to preserve coral samples at depth in an effort to minimize thermal shock and evaluate the effects of environmental gradients on the microbial diversity of samples. Molecular analysis of bacterial diversity showed a marked difference between the two study sites, Viosca Knoll 906/862 (VK906/862) and Viosca Knoll 826 (VK826). The bacterial communities from VK826 were dominated by a variety of unknown mycoplasmal members of the Tenericutes and Bacteroidetes, whereas the libraries from VK906/862 were dominated by members of the Proteobacteria. In addition to novel sequences, the 16S rRNA gene clone libraries revealed many bacterial sequences in common between Gulf of Mexico Lophelia corals and Norwegian fjord Lophelia corals, as well as shallow-water corals. Two Lophelia-specific bacterial groups were identified: a cluster of gammaproteobacteria related to sulfide-oxidizing gill symbionts of seep clams and a group of Mycoplasma spp. The presence of these groups in both Gulf and Norwegian Lophelia corals indicates that in spite of the geographic heterogeneity observed in Lophelia-associated bacterial communities, there are Lophelia-specific microbes. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

  3. 31 CFR 10.82 - Expedited suspension.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Expedited suspension. 10.82 Section... INTERNAL REVENUE SERVICE Rules Applicable to Disciplinary Proceedings § 10.82 Expedited suspension. (a... suspension. A suspension under this section will commence on the date that written notice of the suspension...

  4. Cold energy

    Science.gov (United States)

    Wallace, John P.

    2015-12-01

    Deviations in Q for resonant superconducting radio frequency niobium accelerator cavities are generally correlated with resistivity loss mechanisms. Field dependent Qs are not well modeled by these classical loss mechanisms, but rather can represent a form of precision cavity surface thermometry. When the field dependent Q variation shows improvement with increasing B field level the classical treatment of this problem is inadequate. To justify this behavior hydrogen as a ubiquitous impurity in niobium, which creates measurable property changes, even at very low concentrations is typically considered the cause of such anomalous behavior. This maybe the case in some instances, but more importantly any system operating with a highly coherent field with a significant time dependent magnetic component at near 2° K will have the ability to organize the remaining free spins within the London penetration depth to form a coupled energy reservoir in the form of low mass spin waves. The niobium resonant cavities are composed of a single isotope with a large nuclear spin. When the other loss mechanisms are stripped away this may be the gain medium activated by the low level residual magnetic fields. It was found that one resonant cavity heat treatment produced optimum surface properties and then functioned as a MASER extracting energy from the 2° K thermal bath while cooling the cavity walls. The cavity operating in this mode is a simulator of what can take place in the wider but not colder universe using the cosmic microwave background (CMB) as a thermal source. The low mass, long lifetimes, and the scale of the magnetic spin waves on the weakly magnetized interstellar medium allows energy to be stored that is many orders of magnitude colder than the cosmic microwave background. A linear accelerator cavity becomes a tool to explore the properties of the long wave length magnetic spin waves that populate this cold low energy regime.

  5. [The "culture of survival" and international public health in Latin America: the Cold War and the eradication of diseases in the mid-twentieth century].

    Science.gov (United States)

    Cueto, Marcos

    2015-01-01

    This article analyzes the main campaigns run by international agencies and national health bodies to eradicate infectious diseases in rural Latin America in the 1940s and 1950s. The political dimensions of the period have been studied but there has been little attention as yet to the health dimensions. This article proposes the concept of a "culture of survival" to explain the official public health problems of states with limited social policies that did not allow the exercise of citizenship. Public health, as part of this culture of survival, sought a temporary solution without confronting the social problems that led to infections and left a public health legacy in the region.

  6. Growing Bacteriophage M13 in Liquid Culture.

    Science.gov (United States)

    Green, Michael R; Sambrook, Joseph

    2017-11-01

    Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼10(12) pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque. © 2017 Cold Spring Harbor Laboratory Press.

  7. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Directory of Open Access Journals (Sweden)

    Priscila Cardoso Cristovam

    2008-10-01

    Full Text Available OBJETIVO: Avaliar a importância da presença de células 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo obtido de rimas córneo-esclerais. MÉTODOS: Rimas de diferentes doadores tiveram seus estroma posterior e endotélio removidos (n=6. Cada rima foi dividida em três segmentos iguais, que foram colocados em cultura em três diferentes condições: um segmento foi colocado na placa de cultura com o lado epitelial para cima (Grupo A. Os dois segmentos restantes foram tripsinizados e a suspensão de células obtida foi cultivada com (Grupo B ou sem (Grupo C células 3T3 irradiadas. As células foram mantidas em meio de cultura "supplemental hormonal epithelial médium" (SHEM, a migração epitelial e a formação de clones nos grupos A, B e C foram avaliadas pela microscopia de contraste de fase e por coloração pela rodamina B. Os resultados foram comparados estatisticamente. RESULTADOS: O crescimento de células epiteliais foi observado em 4/6 rimas (Grupo A. Todas as suspensões de células epiteliais que foram cultivadas com células 3T3 (Grupo B formaram clones. Nenhuma adesão ou formação de clones verdadeiros (holo ou meroclones foi observada na cultura de células que foi cultivada sem 3T3 (Grupo C (p=0,009. CONCLUSÕES: Suspensão de células epiteliais límbicas obtidas de rimas córneo-esclerais no modelo utilizado precisa ser cultivada com células 3T3 para formar clones e estabelecer colônias epiteliais com perspectivas para uso terapêutico na reconstrução da superfície ocular.PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR. METHODS: Corneo-scleral rims from different donors (n=6 had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the

  8. 48 CFR 209.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Suspension. 209.407... OF DEFENSE ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 209.407 Suspension. ...

  9. The need to be cold : cold warriors

    Energy Technology Data Exchange (ETDEWEB)

    Gregoire, L.

    2008-10-15

    This article discussed the changing climate of Ellesmere Island and the adaptation of the Inuit in response to the climate change, with particular reference to Canada's most northern community of Grise Fiord. Because of the changing climate, the vast northern landscape that the Inuit navigated for centuries by reading its subtle signs is becoming warmer, softer, and unpredictable. The geographic history and demographics of Grise Fiord were described. The community's main water supply comes from a glacier which is sinking. The negative impacts of ice shrinkage on this northern community and on the environment were presented. These included more international shipping through the Arctic, more resource exploration, a greater risk of environmental contamination, and reduced habitat for the polar bears and seals that eat, mate, and reproduce on the ice. Climate change impacts on the sea and sea ice were also discussed. Several photographs illustrating the changing climate were presented. The article noted that climate change could destroy the Inuit culture, making climate change an issue of human rights, notably the right to live connected to the land and the right to be cold. It was concluded that in one generation, Inuit were swept up by both a social and an economic upheaval. In one more generation, they will undergo an environmental shift. 13 figs.

  10. Mapping and characterisation of the sorghum cell suspension ...

    African Journals Online (AJOL)

    Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step ...

  11. Cold Signaling and Cold Response in Plants

    OpenAIRE

    Kenji Miura; Tsuyoshi Furumoto

    2013-01-01

    Plants are constantly exposed to a variety of environmental stresses. Freezing or extremely low temperature constitutes a key factor influencing plant growth, development and crop productivity. Plants have evolved a mechanism to enhance tolerance to freezing during exposure to periods of low, but non-freezing temperatures. This phenomenon is called cold acclimation. During cold acclimation, plants develop several mechanisms to minimize potential damages caused by low temperature. Cold respons...

  12. Cold Stress and the Cold Pressor Test

    Science.gov (United States)

    Silverthorn, Dee U.; Michael, Joel

    2013-01-01

    Temperature and other environmental stressors are known to affect blood pressure and heart rate. In this activity, students perform the cold pressor test, demonstrating increased blood pressure during a 1- to 2-min immersion of one hand in ice water. The cold pressor test is used clinically to evaluate autonomic and left ventricular function. This…

  13. Cold wave lotion poisoning

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002693.htm Cold wave lotion poisoning To use the sharing features on this page, please enable JavaScript. Cold wave lotion is a hair care product used ...

  14. Paroxysmal cold hemoglobinuria (PCH)

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/000557.htm Paroxysmal cold hemoglobinuria (PCH) To use the sharing features on this page, please enable JavaScript. Paroxysmal cold hemoglobinuria (PCH) is a rare blood disorder in ...

  15. Cold-induced metabolism

    NARCIS (Netherlands)

    van Marken Lichtenbelt, W.D.; Daanen, A.M.

    2003-01-01

    Cold-induced metabolism. van Marken Lichtenbelt WD, Daanen HA. Department of Human Biology, Maastricht University, Maastricht, The Netherlands. PURPOSE OF REVIEW: Cold response can be insulative (drop in peripheral temperature) or metabolic (increase in energy expenditure). Nonshivering

  16. Conceptualizing Cold Disasters

    DEFF Research Database (Denmark)

    Lauta, Kristian Cedervall; Dahlberg, Rasmus; Vendelø, Morten Thanning

    2017-01-01

    In the present article, we explore in more depth the particular circumstances and characteristics of governing what we call ‘cold disasters’, and thereby, the paper sets out to investigate how disasters in cold contexts distinguish themselves from other disasters, and what the implications hereof...... are for the conceptualization and governance of cold disasters. Hence, the paper can also be viewed as a response to Alexander’s (2012a) recent call for new theory in the field of disaster risk reduction. The article is structured in four overall parts. The first part, Cold Context, provides an overview of the specific...... conditions in a cold context, exemplified by the Arctic, and zooms in on Greenland to provide more specific background for the paper. The second part, Disasters in Cold Contexts, discusses “cold disasters” in relation to disaster theory, in order to, elucidate how cold disasters challenge existing...

  17. Cold knife cone biopsy

    Science.gov (United States)

    ... biopsy; Pap smear - cone biopsy; HPV - cone biopsy; Human papilloma virus - cone biopsy; Cervix - cone biopsy; Colposcopy - cone biopsy Images Female reproductive anatomy Cold cone biopsy Cold cone removal References American ...

  18. 49 CFR 238.427 - Suspension system.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Suspension system. 238.427 Section 238.427... Equipment § 238.427 Suspension system. (a) General requirements. (1) Suspension systems shall be designed to... equipment. (2) Passenger equipment shall meet the safety performance standards for suspension systems...

  19. 78 FR 57525 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-09-19

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  20. 48 CFR 2909.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Suspension. 2909.407... CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 2909.407 Suspension. (a) The Senior... authorized to make an exception, regarding suspension by another agency suspending official under the...

  1. 14 CFR 1267.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Suspension. 1267.670 Section 1267.670... WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 1267.670 Suspension. Suspension means an action taken by a..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  2. 22 CFR 1008.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Suspension. 1008.670 Section 1008.670 Foreign... ASSISTANCE) Definitions § 1008.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  3. 2 CFR 182.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 2 Grants and Agreements 1 2010-01-01 2010-01-01 false Suspension. 182.670 Section 182.670 Grants... Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from... guidance on nonprocurement debarment and suspension (2 CFR part 180, which implements Executive Orders...

  4. 40 CFR 36.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Suspension. 36.670 Section 36.670... REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 36.670 Suspension. Suspension means... contracts (48 CFR part 9, subpart 9.4) and the common rule, Government-wide Debarment and Suspension...

  5. 78 FR 5734 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-01-28

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  6. 77 FR 53775 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-09-04

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  7. 29 CFR 94.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Suspension. 94.670 Section 94.670 Labor Office of the... § 94.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a... Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive Order 12689...

  8. 45 CFR 1173.670 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Suspension. 1173.670 Section 1173.670 Public... (FINANCIAL ASSISTANCE) Definitions § 1173.670 Suspension. Suspension means an action taken by a Federal..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  9. 45 CFR 1641.11 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Suspension. 1641.11 Section 1641.11 Public Welfare Regulations Relating to Public Welfare (Continued) LEGAL SERVICES CORPORATION DEBARMENT, SUSPENSION AND REMOVAL OF RECIPIENT AUDITORS Suspension § 1641.11 Suspension. (a) IPAs suspended from providing audit...

  10. 77 FR 2646 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-01-19

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  11. 31 CFR 19.1015 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Suspension. 19.1015 Section 19.1015 Money and Finance: Treasury Office of the Secretary of the Treasury GOVERNMENTWIDE DEBARMENT AND SUSPENSION (NONPROCUREMENT) Definitions § 19.1015 Suspension. Suspension is an action taken by a suspending...

  12. 41 CFR 105-68.1015 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Suspension. 105-68.1015 Section 105-68.1015 Public Contracts and Property Management Federal Property Management Regulations...-GOVERNMENTWIDE DEBARMENT AND SUSPENSION (NONPROCUREMENT) Definitions § 105-68.1015 Suspension. Suspension is an...

  13. 15 CFR 29.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Suspension. 29.670 Section 29.670... WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 29.670 Suspension. Suspension means an action taken by a..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  14. 13 CFR 147.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Suspension. 147.670 Section 147...-FREE WORKPLACE (NONPROCUREMENT) Definitions § 147.670 Suspension. Suspension means an action taken by a..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  15. 78 FR 2622 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-01-14

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  16. 10 CFR 607.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Suspension. 607.670 Section 607.670 Energy DEPARTMENT OF... ASSISTANCE) Definitions § 607.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  17. 76 FR 9666 - Suspension of Community Eligibility

    Science.gov (United States)

    2011-02-22

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  18. 29 CFR 1471.1015 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Suspension. 1471.1015 Section 1471.1015 Labor Regulations Relating to Labor (Continued) FEDERAL MEDIATION AND CONCILIATION SERVICE GOVERNMENTWIDE DEBARMENT AND SUSPENSION (NONPROCUREMENT) Definitions § 1471.1015 Suspension. Suspension is an action taken by a suspending...

  19. 22 CFR 133.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Suspension. 133.670 Section 133.670 Foreign... ASSISTANCE) Definitions § 133.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  20. 43 CFR 43.670 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Suspension. 43.670 Section 43.670 Public... WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 43.670 Suspension. Suspension means an action taken by a..., subpart 9.4) and 2 CFR part 180. Suspension of a recipient is a distinct and separate action from...

  1. 22 CFR 312.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Suspension. 312.670 Section 312.670 Foreign... § 312.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a... Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive Order 12689...

  2. 34 CFR 84.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Suspension. 84.670 Section 84.670 Education Office of... ASSISTANCE) Definitions § 84.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  3. 77 FR 7537 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-02-13

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  4. 19 CFR 146.82 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Suspension. 146.82 Section 146.82 Customs Duties U... (CONTINUED) FOREIGN TRADE ZONES Penalties; Suspension; Revocation § 146.82 Suspension. (a) For cause. The... for a period not to exceed 90 days. Upon order of the Board the suspension may be continued. If...

  5. 77 FR 24858 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-04-26

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  6. 75 FR 5890 - Suspension of Community Eligibility

    Science.gov (United States)

    2010-02-05

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  7. 76 FR 39782 - Suspension of Community Eligibility

    Science.gov (United States)

    2011-07-07

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  8. 22 CFR 1509.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Suspension. 1509.670 Section 1509.670 Foreign... ASSISTANCE) Definitions § 1509.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  9. 22 CFR 210.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Suspension. 210.670 Section 210.670 Foreign... (FINANCIAL ASSISTANCE) Definitions § 210.670 Suspension. Suspension means an action taken by a Federal agency..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  10. 45 CFR 1206.1-4 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Suspension. 1206.1-4 Section 1206.1-4 Public... GRANTS AND CONTRACTS-SUSPENSION AND TERMINATION AND DENIAL OF APPLICATION FOR REFUNDING Suspension and Termination of Assistance § 1206.1-4 Suspension. (a) General. The responsible Corporation official may suspend...

  11. 31 CFR 20.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Suspension. 20.670 Section 20.670...-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 20.670 Suspension. Suspension means an action taken..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  12. 78 FR 68999 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-11-18

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  13. 29 CFR 1472.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Suspension. 1472.670 Section 1472.670 Labor Regulations... DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 1472.670 Suspension. Suspension means an... CFR part 9, subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement...

  14. 24 CFR 21.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Suspension. 21.670 Section 21.670... GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (GRANTS) Definitions § 21.670 Suspension. Suspension means an... CFR part 9, subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement...

  15. 77 FR 9856 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-02-21

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  16. 49 CFR 32.670 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Suspension. 32.670 Section 32.670 Transportation... ASSISTANCE) Definitions § 32.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  17. 21 CFR 1405.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Suspension. 1405.670 Section 1405.670 Food and... (FINANCIAL ASSISTANCE) Definitions § 1405.670 Suspension. Suspension means an action taken by a Federal..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  18. 50 CFR 13.27 - Permit suspension.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 1 2010-10-01 2010-10-01 false Permit suspension. 13.27 Section 13.27... GENERAL PERMIT PROCEDURES Permit Administration § 13.27 Permit suspension. (a) Criteria for suspension... Government. Such suspension shall remain in effect until the issuing officer determines that the permittee...

  19. 45 CFR 1155.670 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Suspension. 1155.670 Section 1155.670 Public... ASSISTANCE) Definitions § 1155.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  20. 78 FR 57523 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-09-19

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  1. 5 CFR 919.1015 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 2 2010-01-01 2010-01-01 false Suspension. 919.1015 Section 919.1015 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT (CONTINUED) CIVIL SERVICE REGULATIONS (CONTINUED) GOVERNMENTWIDE DEBARMENT AND SUSPENSION (NONPROCUREMENT) Definitions § 919.1015 Suspension. Suspension is an...

  2. 78 FR 2624 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-01-14

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  3. 76 FR 2596 - Suspension of Community Eligibility

    Science.gov (United States)

    2011-01-14

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  4. 75 FR 52861 - Suspension of Community Eligibility

    Science.gov (United States)

    2010-08-30

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  5. 28 CFR 83.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Suspension. 83.670 Section 83.670... WORKPLACE (GRANTS) Definitions § 83.670 Suspension. Suspension means an action taken by a Federal agency..., subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement), that...

  6. 39 CFR 957.27 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Suspension. 957.27 Section 957.27 Postal Service... SUSPENSION FROM CONTRACTING § 957.27 Suspension. (a) Any firm or individual suspended under chapter 3, section 7 of the Postal Service Purchasing Manual who believes that the suspension has not been in...

  7. 7 CFR 3021.670 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Suspension. 3021.670 Section 3021.670 Agriculture... Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from... Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive Order 12689...

  8. 76 FR 5284 - Suspension of Community Eligibility

    Science.gov (United States)

    2011-01-31

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  9. 78 FR 69001 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-11-18

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  10. 45 CFR 630.670 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Suspension. 630.670 Section 630.670 Public Welfare... DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 630.670 Suspension. Suspension means an action... CFR part 9, subpart 9.4) and the common rule, Government-wide Debarment and Suspension (Nonprocurement...

  11. 20 CFR 439.670 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Suspension. 439.670 Section 439.670 Employees... ASSISTANCE) Definitions § 439.670 Suspension. Suspension means an action taken by a Federal agency that...-wide Debarment and Suspension (Nonprocurement), that implements Executive Order 12549 and Executive...

  12. 75 FR 9111 - Suspension of Community Eligibility

    Science.gov (United States)

    2010-03-01

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...), that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  13. 77 FR 63753 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-10-17

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  14. 77 FR 2650 - Suspension of Community Eligibility

    Science.gov (United States)

    2012-01-19

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  15. 78 FR 5736 - Suspension of Community Eligibility

    Science.gov (United States)

    2013-01-28

    ... SECURITY Federal Emergency Management Agency 44 CFR Part 64 Suspension of Community Eligibility AGENCY...) that are scheduled for suspension on the effective dates listed within this rule because of... measures prior to the effective suspension date given in this rule, the suspension will not occur and a...

  16. A Method for Collecting Single Cell Suspensions Using an Ultrasonic Pump.

    Science.gov (United States)

    Nakao, Misa; Kurashina, Yuta; Imashiro, Chikahiro; Takemura, Kenjiro

    2018-01-01

    The presence of cell aggregates in cell suspensions may reduce cell culture efficiency because they can induce apoptosis and inhibit proliferation. To avoid this problem, this study proposes a novel method for collecting single cell suspensions from culture chambers for subculture using an ultrasonic pump driven by the squeeze film effect. First, we developed a cell culture device consisting of a cell culture substrate with a piezoelectric ceramic disk glued to the back, so that we can elicit resonance vibration of the substrate. A glass pipe is then placed vertically against the cell culture substrate with a slight gap (corresponding to cell diameter) between the pipe and the substrate. By exciting an out-of-plane resonance vibration of the cell culture substrate, we can collect a cell suspension from the cell culture chamber. Since the gap distance between the glass pipe and the cell culture substrate corresponds to cell diameter, the collected cell suspension only contains single cells. We evaluated the capability of the developed cell suspension pumping system and the proliferation of the collected cells with C2C12 myoblast cells. The ratio of single cells in the cell suspension was improved by up to 9.6% compared with that of suspensions collected by the control method (traditional pipetting). Moreover, after cultivating the collected cells for 72 hr, the cells collected by our method proliferated 13.6% more than those collected by the control method. These results suggest that the proposed method has great potential for improving the cultivation efficiency of adhesive cell culture.

  17. Bacterial diversity and bioprospecting for cold-active enzymes from culturable bacteria associated with sediment from a melt water stream of Midtre Lovenbreen glacier, an Arctic glacier.

    Science.gov (United States)

    Vardhan Reddy, Puram Vishnu; Shiva Nageswara Rao, Singireesu Soma; Pratibha, Mambatta Shankaranarayanan; Sailaja, Buddhi; Kavya, Bakka; Manorama, Ravoori Ruth; Singh, Shiv Mohan; Radha Srinivas, Tanuku Naga; Shivaji, Sisinthy

    2009-10-01

    Culturable bacterial diversity of Midtre Lovenbreen glacier, an Arctic glacier, was studied using 12 sediment samples collected from different points, along a transect, from the snout of Midtre Lovenbreen glacier up to the convergence point of the melt water stream with the sea. Bacterial abundance appeared to be closer to the convergence point of the glacial melt water stream with the sea than at the snout of the glacier. A total of 117 bacterial strains were isolated from the sediment samples. Based on 16S rRNA gene sequence analyses, the isolates (n=117) could be categorised in to 32 groups, with each group representing a different taxa belonging to 4 phyla (Actinobacteria, Bacilli, Flavobacteria and Proteobacteria). Representatives of the 32 groups varied in their growth temperature range (4-37 degrees C), in their tolerance to NaCl (0.1-1M NaCl) and in the growth pH range (2-13). Only 14 of 32 representative strains exhibited amylase, lipase and (or) protease activity and only one isolate (AsdM4-6) showed all three enzyme activities at 5 and 20 degrees C respectively. More than half of the isolates were pigmented. Fatty acid profile studies indicated that short-chain fatty acids, unsaturated fatty acids, branched fatty acids, cyclic and cis fatty acids are predominant in the psychrophilic bacteria.

  18. "Point de suspension"

    CERN Multimedia

    2004-01-01

    CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This spectacle in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local population. There will be three performances for...

  19. "Point de suspension"

    CERN Multimedia

    2004-01-01

    CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This wondrous show in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local population. There will be three perfo...

  20. "Point de suspension"

    CERN Document Server

    2004-01-01

    http://www.cern.ch/cern50/ CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This wondrous show in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local pop...

  1. Preparation, characterisation and antibacterial activity of a florfenicol-loaded solid lipid nanoparticle suspension.

    Science.gov (United States)

    Wang, Ting; Chen, Xiaojin; Lu, Mengmeng; Li, Xihe; Zhou, WenZhong

    2015-12-01

    A florfenicol-loaded solid lipid nanoparticle (FFC-SLN) suspension was prepared by hot homogenisation and ultrasonic technique. The suspension was characterised for its release profile, stability, toxicity, and the physicochemical properties of the nanoparticles. Antibacterial activity of the suspension was evaluated in vitro and in vivo. The results showed that the mean diameter, polydispersity index and zeta potential of the nanoparticles were 253 ± 3 nm, 0.409 ± 0.022 and 47.5 ± 0.21 mV, respectively. In vitro release profile showed the FFC-SLN suspension had sustained release effect. The minimum inhibition concentration values of the FFC-SLN suspension were 6 and 3 µg/mL against Staphylococcus aureus and Escherichia coli respectively, compared with 3.5 and 2 µg/mL of native florfenicol. The suspension was relatively stable at 4°C and less stable at room temperature during 9 months storage. Although the nanoparticle carriers exhibited cytotoxicity in cell cultures, the LD50 of the lyophilised dry power of the suspension was higher than 5 g/kg body weight. Mortality protection against E. coli lethal infection in mice showed that the nanoparticle suspension had much better efficacy (6/10) than native drug (1/10). These results indicate that FFC-SLN suspension could be a promising formulation in veterinary medicine.

  2. Cold storage of rat hepatocyte spheroids.

    Science.gov (United States)

    Liu, Hongling; Yu, Yue; Glorioso, Jaime; Mao, Shennen; Rodysil, Brian; Amiot, Bruce P; Rinaldo, Piero; Nyberg, Scott L

    2014-01-01

    Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

  3. Suspension biomechanics of swimming microbes.

    Science.gov (United States)

    Ishikawa, Takuji

    2009-10-06

    Micro-organisms play a vital role in many biological, medical and engineering phenomena. Some recent research efforts have demonstrated the importance of biomechanics in understanding certain aspects of micro-organism behaviours such as locomotion and collective motions of cells. In particular, spatio-temporal coherent structures found in a bacterial suspension have been the focus of many research studies over the last few years. Recent studies have shown that macroscopic properties of a suspension, such as rheology and diffusion, are strongly affected by meso-scale flow structures generated by swimming microbes. Since the meso-scale flow structures are strongly affected by the interactions between microbes, a bottom-up strategy, i.e. from a cellular level to a continuum suspension level, represents the natural approach to the study of a suspension of swimming microbes. In this paper, we first provide a summary of existing biomechanical research on interactions between a pair of swimming micro-organisms, as a two-body interaction is the simplest many-body interaction. We show that interactions between two nearby swimming micro-organisms are described well by existing mathematical models. Then, collective motions formed by a group of swimming micro-organisms are discussed. We show that some collective motions of micro-organisms, such as coherent structures of bacterial suspensions, are satisfactorily explained by fluid dynamics. Lastly, we discuss how macroscopic suspension properties are changed by the microscopic characteristics of the cell suspension. The fundamental knowledge we present will be useful in obtaining a better understanding of the behaviour of micro-organisms.

  4. Engine Cold Start

    Science.gov (United States)

    2015-09-01

    UNCLASSIFIED UNCLASSIFIED ENGINE COLD START INTERIM REPORT TFLRF No. 469 by Douglas M. Yost Gregory A. T. Hansen U.S...not return it to the originator. UNCLASSIFIED UNCLASSIFIED ENGINE COLD START INTERIM REPORT TFLRF No. 469 by Douglas M. Yost...TITLE AND SUBTITLE Engine Cold Start 5a. CONTRACT NUMBER W56HZV-09-C-0100 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Douglas

  5. Vaccine production: upstream processing with adherent or suspension cell lines.

    Science.gov (United States)

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered.

  6. Active Control of Suspension Bridges

    DEFF Research Database (Denmark)

    Thoft-Christensen, Palle

    In this paper some recent research on active control of very long suspension bridges, is presented. The presentation is based on research work at Aalborg University, Denmark. The active control system is based on movable flaps attached to the bridge girder. Wind load on bridges with or without...... flaps attached to the girder is briefly presented. A simple active control system is discussed. Results from wind tunnel experiments with a bridge section show that flaps can be used effectively to control bridge girder vibrations. Flutter conditions for suspension bridges with and without flaps...

  7. Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor.

    Science.gov (United States)

    Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Samadian, Azam; Baharvand, Hossein

    2012-11-01

    Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (∼2×10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells" to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess

  8. Characterizing Fullerene Nanoparticles in Aqueous Suspensions

    Science.gov (United States)

    Studies have indicated that fullerenes can form stable colloidal suspensions in water when introduced to the aqueous phase through solvent exchange, sonication, or extended mixing. The colloidal suspensions created using these techniques have effective aqueous phase concentratio...

  9. Heavy vehicle pitch dynamics and suspension tuning

    OpenAIRE

    Cao, Dongpu; Rakheja, Subhash; Su, Chun-Yi

    2008-01-01

    The influence of suspension tuning of passenger cars on bounce and pitch ride performance has been explored in a number of studies, while only minimal efforts have been made for establishing similar rules for heavy vehicles. This study aims to explore pitch dynamics and suspension tunings of a two-axle heavy vehicle with unconnected suspension, which could also provide valuable information for heavy vehicles with coupled suspensions. Based on a generalised pitch-plane model of a two-axle heav...

  10. Suspension-Firing of Biomass

    DEFF Research Database (Denmark)

    Shafique Bashir, Muhammad; Jensen, Peter Arendt; Frandsen, Flemming

    2012-01-01

    This paper is the second of two papers, describing probe measurements of deposit buildup and removal (shedding), conducted in a 350 MWth suspension-fired boiler, firing straw and wood. Investigations of deposit buildup and shedding have been made by use of an advanced online deposit probe and a s...

  11. Rotational diffusion in dense suspensions

    NARCIS (Netherlands)

    Hagen, M. H. J.; Frenkel, D.; Lowe, C.P.

    1999-01-01

    We have computed the rotational diffusion coefficient for a suspension of hard spheres. We find excellent agreement with experimental results over a density range up to, and including, the colloidal crystal. However, we find that theories derived to second order in the volume fraction overestimate

  12. Transmission function of pneumatic suspension

    OpenAIRE

    Turenko, A.; Bogomolov, V.; Klimenko, V.; Shilov, A.

    2006-01-01

    The transmission function of pneumatic suspension at assumption, that walls of pneumatic elastic element is absolute not stretched; the rubber buffers of compression and of retreat are absent; description of shock absorber is linear and symmetric; the processes of compression and expansion of air are adiabatic; motion of the oscillating system carry out without separation of wheel from a road is received.

  13. 49 CFR 570.61 - Suspension system.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Suspension system. 570.61 Section 570.61 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY... 10,000 Pounds § 570.61 Suspension system. (a) Suspension condition. Ball joint seals shall not be cut...

  14. 49 CFR 570.8 - Suspension systems.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Suspension systems. 570.8 Section 570.8 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY... Pounds or Less § 570.8 Suspension systems. (a) Suspension condition. Ball joint seals shall not be cut or...

  15. 49 CFR 393.207 - Suspension systems.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Suspension systems. 393.207 Section 393.207... NECESSARY FOR SAFE OPERATION Frames, Cab and Body Components, Wheels, Steering, and Suspension Systems § 393.207 Suspension systems. (a) Axles. No axle positioning part shall be cracked, broken, loose or missing...

  16. 45 CFR 1210.3-3 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Suspension. 1210.3-3 Section 1210.3-3 Public... § 1210.3-3 Suspension. (a) The ACTION State Director may suspend a Volunteer for up to 30 days in order... Volunteer. Suspension is not warranted if the State Director determines that sufficient grounds already...

  17. 48 CFR 2509.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Suspension. 2509.407 Section 2509.407 Federal Acquisition Regulations System NATIONAL SCIENCE FOUNDATION ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 2509.407 Suspension. ...

  18. 32 CFR 26.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Suspension. 26.670 Section 26.670 National... GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 26.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from...

  19. 41 CFR 105-74.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Suspension. 105-74.670...-GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 105-74.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from...

  20. 48 CFR 609.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Suspension. 609.407 Section 609.407 Federal Acquisition Regulations System DEPARTMENT OF STATE COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 609.407 Suspension. ...

  1. 25 CFR 558.5 - License suspension.

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 2 2010-04-01 2010-04-01 false License suspension. 558.5 Section 558.5 Indians NATIONAL... MANAGEMENT OFFICIALS § 558.5 License suspension. (a) If, after the issuance of a gaming license, the... tribe shall suspend such license and shall notify in writing the licensee of the suspension and the...

  2. 48 CFR 1309.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Suspension. 1309.407 Section 1309.407 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 1309.407 Suspension. ...

  3. 36 CFR 1212.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Suspension. 1212.670 Section... GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 1212.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from...

  4. 25 CFR 23.52 - Grant suspension.

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Grant suspension. 23.52 Section 23.52 Indians BUREAU OF... Grant Administration Provisions and Requirements § 23.52 Grant suspension. (a) When a grantee has... assistance, suspend the grant. The notice preceding the suspension shall include the effective date of the...

  5. 22 CFR 34.20 - Suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Suspension. 34.20 Section 34.20 Foreign Relations DEPARTMENT OF STATE CLAIMS AND STOLEN PROPERTY DEBT COLLECTION Collection Adjustments § 34.20 Suspension. The suspension of collection action shall be made in accordance with the standards set forth in...

  6. 32 CFR 552.80 - Suspension period.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 3 2010-07-01 2010-07-01 true Suspension period. 552.80 Section 552.80 National... Suspension period. All solicitation privileges suspended by installation commanders will be for a specific time. Normally, it will not exceed 2 years. When the suspension period expires, the agent may reapply...

  7. 48 CFR 509.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Suspension. 509.407 Section 509.407 Federal Acquisition Regulations System GENERAL SERVICES ADMINISTRATION COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 509.407 Suspension. ...

  8. 7 CFR 3015.123 - Suspension.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Suspension. 3015.123 Section 3015.123 Agriculture... AGRICULTURE UNIFORM FEDERAL ASSISTANCE REGULATIONS Grant and Subgrant Closeout, Suspension and Termination § 3015.123 Suspension. (a) When a recipient has materially failed to comply with the provisions...

  9. 21 CFR 520.1806 - Piperazine suspension.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Piperazine suspension. 520.1806 Section 520.1806... DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.1806 Piperazine suspension. (a) Specifications. Each milliliter of suspension contains piperazine monohydrochloride equivalent to...

  10. 48 CFR 9.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Suspension. 9.407 Section 9.407 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 9.407 Suspension. ...

  11. 48 CFR 909.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Suspension. 909.407 Section 909.407 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 909.407 Suspension. ...

  12. 48 CFR 809.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Suspension. 809.407 Section 809.407 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 809.407 Suspension. ...

  13. 48 CFR 1509.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Suspension. 1509.407 Section 1509.407 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension and Ineligibility 1509.407 Suspension. ...

  14. 21 CFR 522.1289 - Lufenuron suspension.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Lufenuron suspension. 522.1289 Section 522.1289 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Lufenuron suspension. (a) Specifications. Each milliliter of sterile aqueous suspension contains 10...

  15. 32 CFR 776.82 - Interim suspension.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 5 2010-07-01 2010-07-01 false Interim suspension. 776.82 Section 776.82... Complaint Processing Procedures § 776.82 Interim suspension. (a) Where the Rules Counsel determines there is... interim suspension, pending completion of a professional responsibility investigation. The covered...

  16. 48 CFR 409.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Suspension. 409.407 Section 409.407 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension and Ineligibility 409.407 Suspension. ...

  17. 48 CFR 2009.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Suspension. 2009.407 Section 2009.407 Federal Acquisition Regulations System NUCLEAR REGULATORY COMMISSION COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 2009.407 Suspension. ...

  18. 75 FR 24494 - Nonprocurement Debarment and Suspension

    Science.gov (United States)

    2010-05-05

    ...; ] ELECTION ASSISTANCE COMMISSION 2 CFR Chapter 58 Nonprocurement Debarment and Suspension AGENCY: U.S... proposed debarment and suspension regulations. These proposed regulations will apply to nonprocurement... Management and Budget (OMB) in a document on nonprocurement debarment and suspension published in the Federal...

  19. 21 CFR 520.1630 - Oxfendazole suspension.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Oxfendazole suspension. 520.1630 Section 520.1630... DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.1630 Oxfendazole suspension. (a) Specifications. Each milliliter of suspension contains: (1) 90.6 milligrams (mg) oxfendazole (9...

  20. 32 CFR 552.79 - Suspension action.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 3 2010-07-01 2010-07-01 true Suspension action. 552.79 Section 552.79 National... Suspension action. (a) When suspended for cause, immediately notify the company and the agent, in writing, of the reason. When the installation commander determines that suspension should be extended throughout...

  1. 36 CFR 223.141 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Suspension. 223.141 Section... DISPOSAL OF NATIONAL FOREST SYSTEM TIMBER Suspension and Debarment of Timber Purchasers § 223.141 Suspension. (a) The suspending official may, in the public interest, suspend a purchaser on the basis of...

  2. 22 CFR 127.8 - Interim suspension.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Interim suspension. 127.8 Section 127.8 Foreign... Interim suspension. (a) The Managing Director of the Directorate of Defense Trade Controls or the Director of the Office of Defense Trade Controls Compliance is authorized to order the interim suspension of...

  3. 38 CFR 48.670 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Suspension. 48.670...) GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 48.670 Suspension. Suspension means an action taken by a Federal agency that immediately prohibits a recipient from...

  4. 48 CFR 309.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Suspension. 309.407 Section 309.407 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 309.407 Suspension. ...

  5. 48 CFR 1409.407 - Suspension.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Suspension. 1409.407 Section 1409.407 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR COMPETITION AND ACQUISITION PLANNING CONTRACTOR QUALIFICATIONS Debarment, Suspension, and Ineligibility 1409.407 Suspension. ...

  6. Working in the Cold

    Centers for Disease Control (CDC) Podcasts

    2016-02-08

    During the winter, many workers are outdoors, working in cold, wet, icy, or snowy conditions. Learn how to identify symptoms that tell you there may be a problem and protect yourself from cold stress.  Created: 2/8/2016 by National Institute for Occupational Safety and Health (NIOSH).   Date Released: 2/8/2016.

  7. COLD-WORKED HARDWARE

    Directory of Open Access Journals (Sweden)

    N. M. Strizhak

    2007-01-01

    Full Text Available The different types of cold-worked accessory are examined in the article. The necessity of development of such type of accessory in the Republic of Belarus due to requirements of market is shown. High emphasis is placed on the methods of increase of plasticity of cold-worked accessory from usual mill of RUP and CIS countries.

  8. Cold-induced retrotransposition of fish LINEs.

    Science.gov (United States)

    Chen, Shue; Yu, Mengchao; Chu, Xu; Li, Wenhao; Yin, Xiujuan; Chen, Liangbiao

    2017-08-20

    Classes of retrotransposons constitute a large portion of metazoan genome. There have been cases reported that genomic abundance of retrotransposons is correlated with the severity of low environmental temperatures. However, the molecular mechanisms underlying such correlation are unknown. We show here by cell transfection assays that retrotransposition (RTP) of a long interspersed nuclear element (LINE) from an Antarctic notothenioid fish Dissostichus mawsoni (dmL1) could be activated by low temperature exposure, causing increased dmL1 copies in the host cell genome. The cold-induced dmL1 propagation was demonstrated to be mediated by the mitogen-activated protein kinases (MAPK)/p38 signaling pathway, which is activated by accumulation of reactive oxygen species (ROS) in cold-stressed conditions. Surprisingly, dmL1 transfected cells showed an increase in the number of viable cells after prolonged cold exposures than non-transfected cells. Features of cold inducibility of dmL1 were recapitulated in LINEs of zebrafish origin both in cultured cell lines and tissues, suggesting existence of a common cold-induced LINE amplification in fishes. The findings reveal an important function of LINEs in temperature adaptation and provid insights into the MAPK/p38 stress responsive pathway that shapes LINE composition in fishes facing cold stresses. Copyright © 2017. Published by Elsevier Ltd.

  9. Cold Osmotic Shock in Saccharomyces cerevisiae

    Science.gov (United States)

    Patching, J. W.; Rose, A. H.

    1971-01-01

    Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Exponentially growing cells from batch cultures grown in defined medium at 30 C, after being suspended in 0.8 m mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mm MgCl2, accumulated the nonmetabolizable solutes d-glucosamine-hydrochloride and 2-aminoisobutyrate at slower rates than unshocked cells; shocked cells retained their viability. Storage of unshocked batch-grown cells in buffer at 10 C led to an increase in ability to accumulate glucosamine, and further experiments were confined to cells grown in a chemostat under conditions of glucose limitation, thereby obviating the need for storing cells before use. A study was made of the effect of the different stages in the cold osmotic shock procedure, including the osmotic stress, the chelating agent, and the cold Mg2+-containing diluent, on viability and solute-accumulating ability. Growth of shocked cells in defined medium resembled that of unshocked cells; however, in malt extract-yeast extract-glucose-peptone medium, the shocked cells had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low-molecular-weight compounds and about 6 to 8% of the cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase and l-leucine-β-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase, were released when yeast cells were subjected to cold osmotic shock. PMID:5001201

  10. Effects of aluminum on organic acid metabolism and secretion by red spruce cell suspension cultures and the reversal of Al effects on growth and polyamine metabolism by exogenous organic acids

    Science.gov (United States)

    Rakesh Minocha; Stephanie Long

    2004-01-01

    In the absence of added Al, the concentration of succinate in cultured red spruce (Picea rubens Sarg.) cells was 15-20 times higher (> 600 nmol g-1FW) than that of citrate or oxalate and 4-6 times higher than that of malate. Addition of AICIJ (effective monomeric Al concentrations of 0.23 and 0.48...

  11. Human whole body cold adaptation.

    NARCIS (Netherlands)

    Daanen, Hein A.M.; Van Marken Lichtenbelt, Wouter D.

    2016-01-01

    Reviews on whole body human cold adaptation generally do not distinguish between population studies and dedicated acclimation studies, leading to confusing results. Population studies show that indigenous black Africans have reduced shivering thermogenesis in the cold and poor cold induced

  12. Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C.

    Science.gov (United States)

    Seo, Jin Seok; Kim, Yeon Jung; Cho, Jong Moon; Baek, Eric; Lee, Gyun Min

    2013-06-01

    The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

  13. Effects of photoperiodically induced reproductive diapause and cold hardening on the cold tolerance of Drosophila montana.

    Science.gov (United States)

    Vesala, Laura; Hoikkala, Anneli

    2011-01-01

    Coping with seasonal and daily variation in environmental conditions requires that organisms are able to adjust their reproduction and stress tolerance according to environmental conditions. Females of Drosophila montana populations have adapted to survive over the dark and cold winters at high latitudes and altitudes by spending this season in photoperiodically controlled reproductive diapause and reproducing only in spring/summer. The present study showed that flies of a northern population of this species are quite tolerant of low temperatures and show high seasonal and short-term plasticity in this trait. Culturing the flies in short day length (nearly all females in reproductive diapause), as well as allowing the flies to get cold hardened before the cold treatment, increased the cold tolerance of both sexes both in chill coma recovery time test and in mortality assay. Chill coma recovery time test performed for the females of two additional D. montana populations cultured in a day length where about half of the females enter diapause, also showed that diapause can increase female cold tolerance even without a change in day length. Direct linkage between diapause and cold tolerance was found in only two strains representing a high-altitude population of the species, but the phenomenon will certainly be worth of studying in northern and southern populations of the species with larger data sets. 2010 Elsevier Ltd. All rights reserved.

  14. Suspensions with reduced violin string modes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, B H; Ju, L; Blair, D G [School of Physics, University of Western Australia, Crawley 6009, WA (Australia)

    2006-03-02

    We discuss the possibility of significantly reducing the number and Q-factor of violin string modes in the mirror suspension. Simulations of a bar-flexure suspension and an orthogonal ribbon have shown a reduction in the number of violin string modes when compared to a normal ribbon suspension. By calculating the expected suspension thermal noise, we find that the orthogonal ribbon provides a promising suspension alternative. A lower number of violin modes oscillating in the direction of the laser and a reduction in violin mode peak values of at least 23dB can be achieved with a slight increase in thermal noise above 40Hz.

  15. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

    DEFF Research Database (Denmark)

    de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F

    2014-01-01

    polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model...

  16. Effective viscosity of actively swimming algae suspensions

    Science.gov (United States)

    Ewoldt, Randy; Caretta, Lucas; Chengala, Anwar; Sheng, Jian

    2010-11-01

    Suspensions of actively swimming microorganisms exhibit an effective viscosity which may depend on volume fraction, cell shape, and the nature of locomotion (e.g. "pushers" vs. "pullers"). Here we report experimental measurements of shear viscosity for suspensions of unicellular green algae (Dunaliella primolecta, a biflagellated "puller"). We use a cone-and-plate rheometer to measure the dynamic shear viscosity for both motile and non-motile suspensions of D. primolecta. Viscosity increases with concentration for both cases, but the active suspensions of "pullers" have a comparatively lower effective viscosity than passive suspensions. This observation contrasts recently proposed theories which predict that "pullers" should instead have a higher viscosity than non-motile suspensions. Additionally, we observe shear-induced migration of active suspensions and consider its impact on the resulting effective shear viscosity.

  17. Stacking of cold antiprotons

    CERN Document Server

    Gabrielse, G; Oxley, P; Speck, A K; Storry, C H; Tan, J N; Wessels, M; Grzonka, D; Oelert, W; Schepers, G; Sefzick, T; Walz, J; Pittner, H; Hänsch, T W; Hessels, E A

    2002-01-01

    The stacking of cold antiprotons is currently the only way to accumulate the large numbers of the cold antiprotons that are needed for low energy experiments. Both the largest possible number and the lowest possible temperature are desired, especially for the production and study of cold antihydrogen. The antiprotons accumulated in our particle trap have an energy 10/sup 10/ times lower than the energy of those delivered by CERN's Antiprotons Decelerator (AD). The number accumulated (more than 0.4 million in this demonstration) is linear in the number of accepted high energy antiproton pulses (32 in this demonstration). Accumulation efficiencies and losses are measured and discussed. (12 refs).

  18. Robust Tensioned Kevlar Suspension Design

    Science.gov (United States)

    Young, Joseph B.; Naylor, Bret J.; Holmes, Warren A.

    2012-01-01

    One common but challenging problem in cryogenic engineering is to produce a mount that has excellent thermal isolation but is also rigid. Such mounts can be achieved by suspending the load from a network of fibers or strings held in tension. Kevlar fibers are often used for this purpose owing to their high strength and low thermal conductivity. A suite of compact design elements has been developed to improve the reliability of suspension systems made of Kevlar.

  19. Particle Suspension Mechanisms - Supplemental Material

    Energy Technology Data Exchange (ETDEWEB)

    Dillon, M B

    2011-03-03

    This supplemental material provides a brief introduction to particle suspension mechanisms that cause exfoliated skin cells to become and remain airborne. The material presented here provides additional context to the primary manuscript and serves as background for designing possible future studies to assess the impact of skin cells as a source of infectious aerosols. This introduction is not intended to be comprehensive and interested readers are encouraged to consult the references cited.

  20. Dynamics of magnetic nanoparticle suspensions

    OpenAIRE

    Singh, Vanchna; Banerjee, Varsha; Sharma, Manish

    2012-01-01

    We study the dynamics of a suspension of magnetic nanoparticles. Their relaxation times are strongly size-dependent. The dominant mode of relaxation is also governed by the size of the particles. As a result the dynamics is greatly altered due to polydispersity in the sample. We study the effect of polydispersity on the response functions. These exhibit significant changes as the parameters characterizing polydispersity are varied. We also provide a procedure to extract the particle size dist...

  1. [Use of UV rays for the disinfection of water. III. UV sensitivity of Legionella pneumophila of different ages in cold and warm drinking water].

    Science.gov (United States)

    Martiny, H; Seidel, K; Rüden, H

    1989-05-01

    In drinking water the sensitivity of L. pneumophila serogroup 1 type Philadelphia and L. pneumophila serogroup 5 type Dallas were studied with a flowthrough u.v. light treatment apparatus. By washing purified cells from broth cultures were used as inoculum directly ( = young cultures) or kept 2-3 weeks in drinking water in the dark ( = old cultures). A decrease of 99,9999% was found after u.v. treatment by 19 mWs/cm2 for young cultures and by 15 mWs/cm2 for old cultures. Reductions of 99,9999% were obtained by 16 mWs/cm2 in cold drinking water (13-16 degrees C) and by 13 mWs/cm2 in warm drinking water (45-47 degrees C). L. pneumophila serogroup 1 and L. pneumophila serogroup 5 show a very similar susceptibility to u.v.-irradiation. Reductions of 99,9999% were obtained by 14 mWs/cm2 and 15 mWs/cm2, respectively. Thus L. pneumophila-suspensions proved to be more sensitive to u.v.-irradiation than E. coli oder E. faecium in earlier experiments.

  2. Polymorphism in Bacterial Flagella Suspensions

    Science.gov (United States)

    Schwenger, Walter J.

    Bacterial flagella are a type of biological polymer studied for its role in bacterial motility and the polymorphic transitions undertaken to facilitate the run and tumble behavior. The naturally rigid, helical shape of flagella gives rise to novel colloidal dynamics and material properties. This thesis studies methods in which the shape of bacterial flagella can be controlled using in vitro methods and the changes the shape of the flagella have on both single particle dynamics and bulk material properties. We observe individual flagellum in both the dilute and semidilute regimes to observe the effects of solvent condition on the shape of the filament as well as the effect the filament morphology has on reptation through a network of flagella. In addition, we present rheological measurements showing how the shape of filaments effects the bulk material properties of flagellar suspensions. We find that the individual particle dynamics in suspensions of flagella can vary with geometry from needing to reptate linearly via rotation for helical filaments to the prevention of long range diffusion for block copolymer filaments. Similarly, for bulk material properties of flagella suspensions, helical geometries show a dramatic enhancement in elasticity over straight filaments while block copolymers form an elastic gel without the aid of crosslinking agents.

  3. Heating up cold agglutinins

    National Research Council Canada - National Science Library

    Stone, Marvin J

    2010-01-01

    In this issue of Blood, Berentsen and coworkers describe a high response rate which is durable in some patients who receive combination fludarabine and rituximab for chronic cold agglutinin disease (CAD...

  4. Chilling Out With Colds

    Science.gov (United States)

    ... a little earlier for a few nights. De-stress. Kids who are stressed out feel worse when they have colds. Relax and use the time to read, listen to music, or watch a movie. In other words, chill ...

  5. Cold Vacuum Drying Facility

    Data.gov (United States)

    Federal Laboratory Consortium — Located near the K-Basins (see K-Basins link) in Hanford's 100 Area is a facility called the Cold Vacuum Drying Facility (CVDF).Between 2000 and 2004, workers at the...

  6. Cold-induced metabolism

    NARCIS (Netherlands)

    Van Marken Lichtenbelt, Wouter D.; Daanen, Hein A M

    Purpose of review: Cold response can be insulative (drop in peripheral temperature) or metabolic (increase in energy expenditure). Nonshivering thermogenesis by sympathetic, norepinephrine-induced mitochondrial heat production in brown adipose tissue is a well known component of this metabolic

  7. Cold-induced metabolism

    NARCIS (Netherlands)

    Lichtenbelt, W. van Marken; Daanen, H.A.M.

    2003-01-01

    Purpose of review Cold response can be insulative (drop in peripheral temperature) or metabolic (increase in energy expenditure). Nonshivering thermogenesis by sympathetic, norepinephrine-induced mitochondrial heat production in brown adipose tissue is a well known component of this metabolic

  8. Dence Cold Matter

    Directory of Open Access Journals (Sweden)

    Stavinskiy Alexey

    2014-04-01

    Full Text Available Possible way to create dense cold baryonic matter in the laboratory is discussed. The density of this matter is comparable or even larger than the density of neutron star core. The properties of this matter can be controlled by trigger conditions. Experimental program for the study of properties of dense cold matter for light and heavy ion collisions at initial energy range √sNN~2-3GeV is proposed..

  9. Drag Reduction of Bacterial Cellulose Suspensions

    Directory of Open Access Journals (Sweden)

    Satoshi Ogata

    2011-01-01

    Full Text Available Drag reduction due to bacterial cellulose suspensions with small environmental loading was investigated. Experiments were carried out by measuring the pressure drop in pipe flow. It was found that bacterial cellulose suspensions give rise to drag reduction in the turbulent flow range. We observed a maximum drag reduction ratio of 11% and found that it increased with the concentration of the bacterial cellulose suspension. However, the drag reduction effect decreased in the presence of mechanical shear.

  10. Introducing Dual Suspension System in Road Vehicles

    Directory of Open Access Journals (Sweden)

    Imtiaz Hussain

    2013-04-01

    Full Text Available The main objective of suspension system is to reduce the motions of the vehicle body with respect to road disturbances. The conventional suspension systems in road vehicles use passive elements such as springs and dampers to suppress the vibrations induced by the irregularities in the road. But these conventional suspension systems can suppress vibrations to a certain limit. This paper presents a novel idea to improve the ride quality of roads vehicles without compromising vehicle?s stability. The paper proposes the use of primary and secondary suspension to suppress the vibrations more effectively.

  11. Time Varying Behavior of the Loudspeaker Suspension

    DEFF Research Database (Denmark)

    Pedersen, Bo Rohde; Agerkvist, Finn T.

    2007-01-01

    The suspension part of the electrodynamic loudspeaker is often modelled as a simple linear spring with viscous damping, however the dynamic behaviour of the suspension is much more complicated than predicted by such a simple model. At higher levels the compliance becomes non-linear and often...... changes during excitation at high levels. This paper investigates how the compliance of the suspension depends on the excitation, i.e. level and frequency content. The measurements are compared with other known measurement methods of the suspension....

  12. Cold regions isotope applications

    Energy Technology Data Exchange (ETDEWEB)

    Perrigo, L.D.; Divine, T.E.

    1976-04-01

    Pacific Northwest Laboratories (PNL) started the Cold Regions Isotope Applications Program in FY-1975 to identify special conditions in the Arctic and similar geographic areas (Cold Regions) where radioisotope power, heater, or sterilization systems would be desirable and economically viable. Significant progress was made in the first year of this program and all objectives for this initial 12-month period were achieved. The major conclusions and recommendations resulting for this effort are described below. The areas of interest covered include: radiosterilization of sewage; heating of septic tanks; and radioisotope thermoelectric generators as power sources for meteorological instruments and navigational aids. (TFD)

  13. Commemoration of a cold war

    DEFF Research Database (Denmark)

    Farbøl, Rosanna

    2015-01-01

    and heritage sites as case studies, this article sheds new light on the politics of history involved in Cold War commemoration. It suggests that the Cold War is commemorated as a war, yet this war memory is of a particular kind: it is a war memory without victims.......This article brings together the fields of Cold War studies and memory studies. In Denmark, a remarkable institutionalisation of Cold War memory has taken place in the midst of a heated ideological battle over the past and whether to remember the Cold War as a ‘war’. Using Danish Cold War museums...

  14. Introduction: the human sciences and Cold War America.

    Science.gov (United States)

    Isaac, Joel

    2011-01-01

    Studies of the history of the human sciences during the Cold War era have proliferated over the past decade--in JHBS and elsewhere. This special issue focuses on the connections between the behavioral sciences and the culture and politics of the Cold War in the United States. In the recent literature, there is a tendency to identify the Cold War human sciences with two main paradigms: that of psychocultural analysis, on the one hand, and of the systems sciences, on the other. The essays in the special issue both extend understanding of each of these interpretive frameworks and help us to grasp their interconnection. © 2011 Wiley Periodicals, Inc.

  15. Photoelectrochemical studies on aqueous suspensions of some ...

    Indian Academy of Sciences (India)

    pensions meet these requirements. The suspension media are equally as important as the suspension particles in generating an efficient photolysis process. Previous studies used semi- conductor particles as major producers of photonic outcome through heterogeneous charge transfer processes at the par- ticle/electrolyte ...

  16. 49 CFR 238.227 - Suspension system.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Suspension system. 238.227 Section 238.227 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Equipment § 238.227 Suspension system. On or after November 8, 1999— (a) All passenger equipment shall...

  17. Phase diagram of vertically vibrated dense suspensions

    NARCIS (Netherlands)

    von Kann, S.; Snoeijer, Jacobus Hendrikus; van der Meer, Roger M.

    2014-01-01

    When a hole is created in a layer of a dense, vertically vibrated suspension, phenomena are known to occur that defy the natural tendency of gravity to close the hole. Here, an overview is presented of the different patterns that we observed in a variety of dense particulate suspensions.

  18. Active suspension for a field sprayer boom

    DEFF Research Database (Denmark)

    Nielsen, Henrik Skovsgaard; Sørensen, Paul Haase

    1998-01-01

    The possibilities of implementing an active boom suspension is investigated. The performance improvement of an active suspension over a traditional passive one is studied in simulation, and shows a significant improvement. A closed-loop control system involving two ultrasonic distance transducers...

  19. Time Variance of the Suspension Nonlinearity

    DEFF Research Database (Denmark)

    Agerkvist, Finn T.; Pedersen, Bo Rohde

    2008-01-01

    . This paper investigates the changes in compliance the driving signal can cause, this includes low level short duration measurements of the resonance frequency as well as high power long duration measurements of the non-linearity of the suspension. It is found that at low levels the suspension softens...

  20. 32 CFR 1609.5 - Suspension.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Suspension. 1609.5 Section 1609.5 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM UNCOMPENSATED PERSONNEL § 1609.5 Suspension. The Director of Selective Service may suspend from duty any uncompensated person...