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Sample records for rice suspension cells

  1. Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice.

    Science.gov (United States)

    Matsuno, Koya; Fujimura, Tatsuhito

    2014-03-01

    A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA accumulation levels increase with ABA concentration. On the other hand, higher concentrations of sucrose or inorganic phosphorus do not affect PA accumulation. Mutations in the genes RINO1, OsMIK, OsIPK1 and OsLPA1 have each been reported to confer low phytic acid phenotypes in seeds. Each of these genes is upregulated in cells cultured with ABA. OsITPK4 and OsITPK6 are upregulated in cells cultured with ABA and in developing seeds. These results suggest that the regulation of PA synthesis is similar between developing seeds and cells in this suspension culture system. This system will be a powerful tool for elucidating the regulation of PA synthesis.

  2. An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Yuan-Ling Chen; Hui-Lin Liang; Xing-Liang Ma; Su-Lin Lou; Yong-Yao Xie; Zhen-Lan Liu; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    Plant mutants are important bio-resources for crop breeding and gene functional studies.Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency.Here,we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells,with rice (Oryza sativa L.) as an example.We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22 h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations,including a number of important agronomic traits such as grain size,panicle size,grain or panicle shape,tiller number and angle,heading date,male sterility,and disease sensitivity.No mosaic mutant was observed in the mutant lines tested.In this mutant library,we obtained a mutant with an abnormally elongated uppermost internode.Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene,representing a successful example of this mutagenesis system.

  3. Screening anti-southern rice black-streaked dwarf virus drugs based on S7-1 gene expression in rice suspension cells.

    Science.gov (United States)

    Yu, Dandan; Wang, Zhenchao; Liu, Jing; Lv, Mingming; Liu, Jiaju; Li, Xiangyang; Chen, Zhuo; Jin, Linghong; Hu, Deyu; Yang, Song; Song, Baoan

    2013-08-28

    Southern rice black-streaked dwarf virus (SRBSDV) is a rice pathogen that had an outbreak in southern China in 2010 and caused significant crop losses. Therefore, screening for effective antiviral drugs against SRBSDV is very important. This study used rice suspension cells infected with SRBSDV by polyethylene glycol-mediated uptake for screening antiviral drugs. SRBSDV P7-1, which is coded by the S7-1 gene, has an intrinsic ability to self-interact to form tubules that play an important role in viral infection. Therefore, relative expression level of the SRBSDV S7-1 gene in infected rice suspension cells was assayed by real-time quantitative polymerase chain reaction to evaluate the antiviral activities of various drugs. Dufulin displayed the highest inhibitory activity against SRBSDV S7-1 expression. In addition, changes in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) activities were determined in inoculated and noninoculated cells. The results showed that both POD and PPO activities increased upon dufulin treatment. Furthermore, the validity of this approach was confirmed in an in vivo experiment in which dufulin was found to effectively inhibit SRBSDV.

  4. Evidence for 'silicon' within the cell walls of suspension-cultured rice cells.

    Science.gov (United States)

    He, Congwu; Wang, Lijun; Liu, Jian; Liu, Xin; Li, Xiuli; Ma, Jie; Lin, Yongjun; Xu, Fangsen

    2013-11-01

    Despite the ubiquity and beneficial role of silicon (Si) in plant biology, structural and chemical mechanisms operating at the single-cell level have not been extensively studied. To obtain insights regarding the effect of Si on individual cells, we cultivated suspended rice (Oryza sativa) cells in the absence and presence of Si and analyzed single cells using a combination of physical techniques including atomic force microscopy (AFM). Si is naturally present as a constituent of the cell walls, where it is firmly bound to the cell wall matrix rather than occurring within intra- or extracellular silica deposition, as determined by using inductively coupled plasma mass spectrometry (ICP-MS) and X-ray photoelectron spectroscopy (XPS). This species of Si, linked with the cell wall matrix, improves the structural stability of cell walls during their expansion and subsequent cell division. Maintaining cell shape is thereby enhanced, which may be crucial for the function and survival of cells. This study provides further evidence that organosilicon is present in plant cell walls, which broadens our understanding of the chemical nature of 'anomalous Si' in plant biology.

  5. 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.

  6. 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.

  7. Alpha-picolinic Acid Activates Diverse Defense Responses of Salicylic Acid-, Jasmonic Acid/Ethylene- and Ca2 -dependent Pathways in Arabidopsis and Rice Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGHai-Kuo; ZHANGXin; LIQun; HEZu-Hua

    2004-01-01

    Alpha-picolinic acid (PA) is an apoptosis inducer in animal cells, and could elicit hypersensitiv eresponse (HR) in rice, a monocotyledonous model plant. Here we report that PA is an HR inducer in plants. It induced HR in Arabidopsis, a dicotyledonous model plant, including the oxidative burst and cell death. We investigated defense signal transduction activated by PA through marker genes of particular defense pathways in Arabidopsis. The result indicated that both the salicylic acid-dependent and jasmonic acid/ethylene-dependent pathways were activated by PA, in which the marker defense genes PR-1, PR-2 and PDF 1.2 were all induced in dose-dependent and time-course manners. We also observed that the PAinduced reactive oxygen species (ROS) production in rice suspension cells was Ca2+-dependent. Together with our previous studies of PA-induced defense activation in rice, we conclude that PA acts as a nonspecific elicitor in plant defense and has a potential utilization in cellular model establishment of systemicac quired resistance (SAR) activation.

  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...

  9. Systematic secretome analyses of rice leaf and seed callus suspension-cultured cells: workflow development and establishment of high-density two-dimensional gel reference maps.

    Science.gov (United States)

    Jung, Young-Ho; Jeong, Seung-Hee; Kim, So Hee; Singh, Raksha; Lee, Jae-Eun; Cho, Yoon-Seong; Agrawal, Ganesh Kumar; Rakwal, Randeep; Jwa, Nam-Soo

    2008-12-01

    Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant

  10. O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role.

    Science.gov (United States)

    Kim, Jihye; Park, Heajin; Park, Byung Tae; Hwang, Hye Seong; Kim, Jae Il; Kim, Dae Kyong; Kim, Ha Hyung

    2016-10-14

    Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.

  11. Characterization of cell suspensions from solid tumors

    Energy Technology Data Exchange (ETDEWEB)

    Pallavicini, M.

    1985-07-10

    The desirable features of cells in suspension will necessarily be dependent upon the use for which the cells were prepared. Adequate cell yield or recovery is defined by the measurement to be performed. Retention of cellular morphology is important for microscopic identification of cell types in a heterogenous cell suspension, and may be used to determine whether the cells in suspension are representative of those in the tumor in situ. Different dispersal protocols may yield cells with different degrees of clonogenicity, as well as altered biochemical features, such as loss of cellular proteins, surface antigens, nucleotide pools, etc. The quality of the cell suspension can be judged by the degree of cell clumping and level of cellular debris, both of which impact on flow cytometric measurements and studies in which the number of cells be known accurately. Finally, if the data measured on the cells in suspension are to be extrapolated to phenomena occurring in the tumor in situ, it is desirable that the cells in suspension are representative of those in the solid tumor in vivo. This report compares characteristics of tumor cell suspensions obtained by different types of selected disaggregation methods. 33 refs., 2 figs., 4 tabs.

  12. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  13. Plasmodesmata in Arabidopsis thaliana suspension cells.

    Science.gov (United States)

    Bayer, E; Thomas, C L; Maule, A J

    2004-06-01

    A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.

  14. 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.

  15. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun'ai; WANG Jingang; FAN Jinping; GONG Shufang; CHE Daidi

    2008-01-01

    Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg-L-1.When transfered onto subculture media,fi-iable callus developed into embryogenic callus,which was used to establish cell suspension lines.Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles.The best subculturing cycle for the stable cell suspensions was 8-10 days.The best inoculum quantity was 1 mL PCV(Packed Cell Volume) per 40 mL culture fluid.

  16. Assessment of drug salt release from solutions, suspensions and in situ suspensions using a rotating dialysis cell

    DEFF Research Database (Denmark)

    Parshad, Henrik; Frydenvang, Karla; Liljefors, Tommy

    2003-01-01

    A rotating dialysis cell consisting of a small (10 ml) and a large compartment (1000 ml) was used to study the release of drug salt (bupivacaine 9-anthracene carboxylate) from (i). solutions, (ii). suspensions and (iii). in situ formed suspensions. Initial release experiments from suspensions...

  17. H_2O_2对水稻白叶枯病菌过氧化氢酶相关基因crg表达的诱导作用%Induction of bacterial catalase-related gene expression by H_2O_2 produced during interaction of rice suspension-cultured cells with Xanthomonas oryzae pv. oryzae or applied exogenously

    Institute of Scientific and Technical Information of China (English)

    周建波; 吴茂森; 胡俊; 何晨阳

    2009-01-01

    为了阐明H_2O_2对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)过氧化氢酶(CAT)相关基因(crg)表达的诱导作用,本研究定量分析了在水稻细胞-Xoo互作体系及其加入H_2O_2清除剂CAT后H_2O_2产量和crg表达;外源添加H_2O_2后的病菌生长和crg表达.结果表明:在互作条件下,H_2O_2含量稳定增加,10 h可达到峰值;在互作6 h时crg显著地被诱导表达;加入 CAT显著地降低了H_2O_2含量和crg表达;在外源H_2O_2胁迫条件下,H_2O_2以浓度效应的方式影响病菌增殖,显著地诱导了catB和srpA表达.因此,Xoo-水稻互作导致了H_2O_2的发生.无论是互作产生的还是外源的H_2O_2均显著地诱导了Xoo crg表达,从而活化了H_2O_2降解途径.%To elucidate the role of hydrogen peroxide (H_2O_2) produced during the interaction of rice suspension-cultured cells with Xanthomonas oryzae pv. oryzae (Xoo) or applied exogenously in inducing expression of bacterial catalase-related gene (crg), H_2O_2 production and crg expression during the rice-Xoo interaction, in which catalase (CAT) was exogenously added or not, were quantitatively analyzed. In vitro growth and crg expression of Xoo exposed to exogenously-applied H_2O_2 were quantitatively examined as well. Significant increase in H_2O_2 content and crg expression was observed during the interaction, while reduction in H_2O_2 concentration and crg expression was obviously found when CAT was exogenously added to the rice-Xoo interacting system. Growth in vitro was inhibited by exogenously-applied H_2O_2 in a dosage manner, which strongly induced the expression of catB and srpA. Therefore, H_2O_2 production was resulted from the rice-Xoo interaction, and crg expression was significantly induced by H_2O_2 either produced during the interaction or added exogenously.

  18. CMOS based sensor for dielectric spectroscopy of biological cell suspension

    Science.gov (United States)

    Guha, S.; Schmalz, K.; Meliani, C.; Wenger, Ch

    2013-04-01

    In this work we investigate the use of microwave frequency range to measure the concentration of cells in a biological cell suspension. A theoretical model is discussed and the advantage of high frequency, which is to avoid dispersion mechanisms due to the cell parameters at lower frequencies (for example membrane capacitance), has been described. Interdigitated capacitor (IDC) has been proposed as the sensor for analysing the concentration of a cell species in the suspension. The read-out circuit is a VCO using the IDC and a pair of inductors as resonator. The capacitance of the IDC which is the function of the permittivity of the biological cell suspension determines the resonant frequency of the LC tank oscillator. Thus the concentration of cells in a solution, affecting its permittivity, is read out as the frequency of the oscillator.

  19. Transferring Gus gene into intact rice cells by low energy ion beam

    Science.gov (United States)

    Zengliang, Yu; Jianbo, Yang; Yuejin, Wu; Beijiu, Cheng; Jianjun, He; Yuping, Huo

    1993-06-01

    A new technique of transferring genes by low energy ion beam has been reported in this paper. The Gus and CAT (chloramphenicol acetyltransferase) genes, as "foreign" genetic materials, were introduced into the suspension cells and ripe embryos or rice by implantation of 20-30 keV Ar + at doses ranging from 1 × 10 15 to 4 × 10 15 ions/cm 2. The activities of CAT and Gus were detected in the cells and embryos after several weeks. The results indicate that the transfer was a success.

  20. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  1. Effect of annealing temperature on gelatinization of rice starch suspension as studied by rheological and thermal measurements.

    Science.gov (United States)

    Tsutsui, Kazumi; Katsuta, Keiko; Matoba, Teruyoshi; Takemasa, Makoto; Nishinari, Katsuyoshi

    2005-11-16

    The effect of annealing temperature (Ta) on the rheological behavior of 10 wt % rice starch suspension was investigated by the dynamic viscoelasticity, the differential scanning calorimetry (DSC), and the amount of leached out amylose and the swelling ratio of starch suspension. The rheological behaviors of the annealed samples are classified into three types in terms of Ta: Ta1, 48 and 55 degrees C, which are much lower than the gelatinization temperature, Tgel (=62 degrees C); Ta2, 58, 60, and 62 degrees C, which are almost the same as Tgel; and Ta3, 65, 68, 70, and 73 degrees C, which are much higher than Tgel. For the samples annealed at Ta2, the onset temperature of the storage and the loss moduli, G' and G'', increased with increasing T(a), and G' and G" in the temperature range from 65 to 90 degrees C gradually increased though smaller than those for the nonannealed sample, the control. This can be understood by the partial gelatinization; i.e., the leached out amylose prevents further amylose from leaching out. The rheological property of the samples annealed at Ta1 is not so different from that of the control, and the samples annealed at Ta3 are almost gelatinized. The rheological behavior of starch suspension can be controlled by Ta.

  2. 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...... units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10−6 M 2,4-D + 1 g/1 casein hydrolysate....

  3. Heparin promotes suspension adaptation process of CHO-TS28 cells by eliminating cell aggregation.

    Science.gov (United States)

    Li, Ling; Qin, Jun; Feng, Qiang; Tang, Hao; Liu, Rong; Xu, Liqing; Chen, Zhinan

    2011-01-01

    While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO-TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P cell aggregation elimination role at all concentrations (P cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10(4) cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO-TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.

  4. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  5. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  6. Spectral Representation Theory for Dielectric Behavior of Nonspherical Cell Suspensions

    Institute of Scientific and Technical Information of China (English)

    HUANG Ji-Ping; YU Kin-Wah; LEI Jun; SUN Hong

    2002-01-01

    Recent experiments revealed that the dielectric dispersion spectrum of fission yeast cells in a suspension was mainly composed of two sub-dispersions. The low-frequency sub-dispersion depended on the cell length, while the high-frequency one was independent of it. The cell shape effect was simulated by an ellipsoidal cell model but the comparison between theory and experiment was far from being satisfactory. Prompted by the discrepancy, we proposed the use of spectral representation to analyze more realistic cell models. We adopted a shell-spheroidal model to analyze the effects of the cell membrane. It is found that the dielectric property of the cell membrane has only a minor effect on the dispersion magnitude ratio and the characteristic frequency ratio. We further included the effect of rotation of dipole induced by an external electric field, and solved the dipole-rotation spheroidal model in the spectral representation.Good agreement between theory and experiment has been obtained.

  7. Shear induced diffusion in a red blood cell suspension

    Science.gov (United States)

    Podgorski, Thomas; Grandchamp, Xavier; Srivastav, Aparna; Coupier, Gwennou

    2012-11-01

    In the microcirculation, blood exhibits an inhomogeneous structure which results in the well know Fahraeus-Lindqvist effect : the apparent viscosity decreases when the diameter of the capillary decreases due to the formation of a marginal cell depletion layer (known as plasma skimming). This structure is a consequence of several phenomena, which include i) the migration of cells aways from walls due to lift forces and gradients of shear and ii) shear induced diffusion due to collisions and interactions among cells. We investigated these phenomena through experiments in simple shear and microchannel flows, with dilute suspensions of vesicles and blood cells. Pairwise interactions between suspended objects result in non-linear and flow-dependent diffusion, whose properties have been measured in different experiments for vesicles and blood cells. The injection of a sheet of concentrated blood cell suspension in a microchannel with a rectangular cross-section allows, through the measurement of its widening along the channel, to measure the diffusivity of blood cells, both in the local plane of shear and in the vorticity direction.

  8. Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

    Science.gov (United States)

    Liu, Yu-Kuo; Huang, Li-Fen; Ho, Shin-Lon; Liao, Chun-Yu; Liu, Hsin-Yi; Lai, Ying-Hui; Yu, Su-May; Lu, Chung-An

    2012-05-01

    To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

  9. Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines.

    Science.gov (United States)

    Shi, Y; Ryu, D D; Ballica, R

    1993-03-25

    Data on viscous (eta') and elastic (eta'') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 microm as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 microm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field.

  10. Auxin requirements of sycamore cells in suspension culture.

    Science.gov (United States)

    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C

    1983-04-01

    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  11. Axial dispersion in flowing red blood cell suspensions

    Science.gov (United States)

    Podgorski, Thomas; Losserand, Sylvain; Coupier, Gwennou

    2016-11-01

    A key parameter in blood microcirculation is the transit time of red blood cells (RBCs) through an organ, which can influence the efficiency of gas exchange and oxygen availability. A large dispersion of this transit time is observed in vivo and is partly due to the axial dispersion in the flowing suspension. In the classic Taylor-Aris example of a solute flowing in a tube, the combination of molecular diffusion and parabolic velocity profile leads to enhanced axial dispersion. In suspensions of non-Brownian deformable bodies such as RBCs, axial dispersion is governed by a combination of shear induced migration and shear-induced diffusion arising from hydrodynamic interactions. We revisit this problem in the case of RBC pulses flowing in a microchannel and show that the axial dispersion of the pulse eventually saturates with a final extension that depends directly on RBC mechanical properties. The result is especially interesting in the dilute limit since the final pulse length depends only on the channel width, exponent of the migration law and dimensionless migration velocity. In continuous flow, the dispersion of transit times is the result of complex cell-cell and cell-wall interactions and is strongy influenced by the polydispersity of the blood sample. The authors acknowledge support from LabEx TEC21 and CNES.

  12. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    Science.gov (United States)

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  13. Signal transduction events in aluminum-induced cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Woltering, E.J.

    2007-01-01

    In this study, some of the signal transduction events involved in AlCl3-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 ¿M AlCl3 showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation.

  14. Signal transduction events in aluminum-induced cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Woltering, E.J.

    2007-01-01

    In this study, some of the signal transduction events involved in AlCl3-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 ¿M AlCl3 showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation.

  15. Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P.tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L-1 2,4-D and 0.05 mg·L-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10 mg·L-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10 mg·L-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

  16. Characterization of aggregate size in Taxus suspension cell culture.

    Science.gov (United States)

    Kolewe, Martin E; Henson, Michael A; Roberts, Susan C

    2010-05-01

    Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 microm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R(2) > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.

  17. Enhancement of heat transfer in red cell suspensions in vitro experiments.

    Science.gov (United States)

    Carr, R T; Tiruvaloor, N R

    1989-05-01

    New data on laminar heat convection with red cell suspensions have been gathered for both heating and cooling. When compared to data for the suspending medium alone, it is apparent that the red cells enhance laminar heat transfer when Pe greater than 4. This is probably due to particle movements. These new data disagree with earlier studies which indicated no enhancement of heat transfer for blood cell suspensions. The data do agree with previous correlations for enhanced thermal transport in sheared suspensions.

  18. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    Science.gov (United States)

    Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

    2015-01-01

    Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-01

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size.

  2. 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.

  3. Growth and cell wall changes in rice roots during spaceflight.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Tanimoto, Eiichi

    2003-08-01

    We analyzed the changes in growth and cell wall properties of roots of rice (Oryza sativa L. cv. Koshihikari) grown for 68.5, 91.5, and 136 h during the Space Shuttle STS-95 mission. In space, most of rice roots elongated in a direction forming a constant mean angle of about 55 degrees with the perpendicular base line away from the caryopsis in the early phase of growth, but later the roots grew in various directions, including away from the agar medium. In space, elongation growth of roots was stimulated. On the other hand, some of elasticity moduli and viscosity coefficients were higher in roots grown in space than on the ground, suggesting that the cell wall of space-grown roots has a lower capacity to expand than the controls. The levels of both cellulose and the matrix polysaccharides per unit length of roots decreased greatly, whereas the ratio of the high molecular mass polysaccharides in the hemicellulose fraction increased in space-grown roots. The prominent thinning of the cell wall could overwhelm the disadvantageous changes in the cell wall mechanical properties, leading to the stimulation of elongation growth in rice roots in space. Thus, growth and the cell wall properties of rice roots were strongly modified under microgravity conditions during spaceflight.

  4. Feruloyl oligosaccharides from cell walls of suspension-cultured spinach cells and sugar beet pulp.

    Science.gov (United States)

    Ishii, T

    1994-06-01

    Cell walls of suspension-cultured spinach cells and sugar beet pulp were separately hydrolyzed with Driselase. A feruloyl arabinobiose was isolated from both spinach cells and sugar beet. Four feruloyl oligosaccharides were obtained from sugar beet. The four oligosaccharides were characterized by NMR spectroscopy, methylation analysis and FAB-MS.

  5. Plant regeneration from mesophyll protoplast of indica rice Qiugui'ai 11 (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    JIANYuyu; JintaanankulSuwan

    1998-01-01

    In the recent decade, plant regeneration from protoplast has been obtained through embryogenic cell suspension cultures of rice. However, not only the establishment of embryogenic call suspension cultures of rice was difficult, but also the protoplasts became less and less regenerable and the genetic change was gradu ally accumulated during the prolonged culture.Since 1976 (Deka.), extensive efforts have been made to induce sustained division and regenerate plants from rnesophyll protoplasts of rice, but not successful.

  6. 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

  7. Influence of Flow Behavior of Alginate-Cell Suspensions on Cell Viability and Proliferation.

    Science.gov (United States)

    Ning, Liqun; Guillemot, Arthur; Zhao, Jingxuan; Kipouros, Georges; Chen, Xiongbiao

    2016-07-01

    Tissue scaffolds with living cells fabricated by three-dimensional bioprinting/plotting techniques are becoming more prevalent in tissue repair and regeneration. In the bioprinting process, cells are subject to process-induced forces (such as shear force) that can result in cell damage and loss of cell function. The flow behavior of the biomaterial solutions that encapsulate living cells in this process plays an important role. This study used a rheometer to examine the flow behavior of alginate solution and alginate-Schwann cell (RSC96), alginate-fibroblast cell (NIH-3T3), and alginate-skeletal muscle cell (L8) suspensions during shearing with respect to effects on cell viability and proliferation. The flow behavior of all the alginate-cell suspensions varied with alginate concentration and cell density and had a significant influence on the viability and proliferation of the cells once sheared as well as on the recovery of the sheared cells. These findings provide a mean to preserve cell viability and/or retain cell proliferation function in the bioprinting process by regulating the flow behavior of cell-biomaterial suspensions and process parameters.

  8. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  9. Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2005-11-01

    Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

  10. Convective flows of colloidal suspension in an inclined closed cell

    Science.gov (United States)

    Smorodin, Boris; Cherepanov, Ivan; Ishutov, Sergey

    2016-12-01

    The nonlinear spatiotemporal evolution of convective flows is numerically investigated in the case of colloidal suspension filling an inclined closed cell heated from below. The bifurcation diagram (the dependency of the Nusselt number on the Rayleigh number) is obtained. The characteristics of the wave and steady patterns are investigated depending on heat intensity. The travelling wave changing travel direction and the non-regular oscillatory flow are found to be stable solutions within a certain interval of the Rayleigh number. Temporal Fourier decomposition is used together with other diagnostic tools to analyse the complex bifurcation and spatiotemporal properties caused by the interplay of the gravity-induced gradient of concentration and convective mixing of the fluid. It is shown that a more complex flow structure exists at a lower heating intensity (Rayleigh number).

  11. Comparison of the oxygen exchange between photosynthetic cell suspensions and detached leaves of Euphorbia characias L

    Energy Technology Data Exchange (ETDEWEB)

    Carrier, P.; Chagvardieff, P.; Tapie, P. (C.E.N. Cadarache, Saint-Paul lez Durance (France))

    1989-11-01

    Using a mass-spectrometric {sup 16}O{sub 2}/{sup 18}O{sub 2}-isotope technique, we compared the nature and the relative importance of oxygen exchange in photomixotrophic (PM) and photoautotrophic (PA) suspensions of Euphorbia characias L. with those in intact leaves of the same species. Young and mature leaves, dividing and nondividing cell suspensions were characterized in short-term experiments. On chlorophyll basis, the gross photosynthetic activities at CO{sub 2} saturating concentration of PA and PM suspensions varied little from those of leaves. On dry weight basis, gross photosynthesis of PA suspensions was equal to that of leaves because of their similar chlorophyll content. This was not the case in PM suspensions where gross photosynthesis was lower and largely varied during the growth cycle. The CO{sub 2} compensation point of PA cells was much higher than that of leaves. Oxygen uptakes were analyzed in terms of mitochondrial respiration, photorespiration and light stimulation of oxygen uptake (LSOU), often identified to Mehler-type reactions. In Pa and PM suspensions, mitochondrial respiration rates were higher than in leaves by a factor of 1.5 to 4.5. In PM suspensions, photorespiration and LSOU were observed only in nondividing cells. Photorespiration and LSOU rates were comparable in PA suspensions and leaves. Our results demonstrate that photorespiration of PA suspensions has not been affected by the 2% CO{sub 2} concentration imposed during 2 years of culture.

  12. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Zhaohua PEng [Mississippi State University; Ronald, Palmela [UC-Davis; Wang, Guo-Liang [The Ohio State University

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  13. Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

    OpenAIRE

    Bauwens, Celine L.; Toms, Derek; Ungrin, Mark

    2016-01-01

    Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization l...

  14. A numerical model of localized convection cells of Euglena suspensions

    Science.gov (United States)

    Iima, Makoto; Shoji, Erika; Yamaguchi, Takayuki

    2014-11-01

    Suspension of Euglena gracilis shows localized convection cells when it is illuminated form below with strong light intensity. Experiments in an annular container shows that there are two elementary localized structures. One consists of a pair of convection cells and a single region where number density of Euglena is high. The other consists a localized traveling wave. Based on the measurements of the flux of number density, we propose a model of bioconvection incorporating lateral phototaxis effect proportional to the light intensity gradient. Using pseudo spectral method, we performed numerical simulation of this model. We succeed in reproducing one of the localized structures, a convection pair with single region of high number density. Also, when the aspect ratio is large, there are a parameter region where the localized structure and conductive state are both stable, which is suggested by experiments. Spatial distribution of the number density implies that the accumulation of microorganism due to the convective flow causes such bistability. CREST(PJ74100011) and KAKENHI(26400396).

  15. Evaluating Kinetic Composing of Cell Wall for Low-Fiber Mutation Rice

    Institute of Scientific and Technical Information of China (English)

    SHEN Heng-sheng; CHEN Jun-chen; ZENG Da-li; TU Jie-feng; TANG Bao-sha; TENG Sheng

    2004-01-01

    The work compared the differences of low fiber mutation rice (LF, Nendao) that selectedthrough gamma-ray (γ) with parental variety Shuangkezao (CK) on their biologicaldevelopment and cell wall composing after rice heading stage. Comparing with parentalrice, LF rice revealed an advantage on its vegetative growing by increasing the yieldsof leave blade, leave sheath and stem for 27.77, 30.19 and 37.96% respectively. And thecellulose content of LF rice straw was decreased remarkably for 23.9%, the hemicellulose,lignin and biogenic silicon contents were increased contrarily for 11.94, 8.79 and 5.60%respectively. Moreover, the crude protein content was increased by 20.71% for LF rice andwith an improvement on its solubility for 63.49% concomitantly. The results indicatedthat the Iow-fiber mutation rice exhibited its potential as a fodder-rice variety or asdual-purpose rice to improve fiber degradability of straw.

  16. Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures.

    Science.gov (United States)

    Menges, Margit; Murray, James A H

    2004-02-01

    We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

  17. Microchannel-free collection and single-cell isolation of yeast cells in a suspension using liquid standing wave

    Science.gov (United States)

    Matsutani, Akihiro; Takada, Ayako

    2016-11-01

    We demonstrate a microchannel-free collection method at nodes of liquid standing waves by the vertical vibration of a suspension including yeast cells. The pattern formation of the collection of cells using standing waves in a suspension was investigated by varying the frequency and waveform of vibrations. The single-cell isolation of yeast cells was achieved using a microenclosure array set at the nodes. In addition, we succeeded in the microchannel-free collection of yeast cells in a suspension, where patterns were formed by tapping vibration. The proposed technique is very simple and we believe that it will be useful for single-cell analysis and investigation.

  18. Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

    Institute of Scientific and Technical Information of China (English)

    MAO Shengji; GU Hongya; QU Lijia; CHEN Zhangliang

    2003-01-01

    The strategy of the two-component system, composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.

  19. Measuring NO Production by Plant Tissues and Suspension Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Jan Vitecek; Vilem Reinohl; Russell L.Jones

    2008-01-01

    We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants.The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap.The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors.We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources.NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected,as was the production of NO from nitrite by the incubation medium of barley aleurone.Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up.We could also measure NO production from elicitor-treated suspension cultured cells using this set-up.Further,we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues.In addition to the dramatic effects that pH can have on fluorescence from DAF-FM,the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results.Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

  20. 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

  1. Transplantation of autologous noncultured epidermal cell suspension in treatment of patients with stable vitiligo

    Institute of Scientific and Technical Information of China (English)

    XU Ai-e; WEI Xiao-dong; CHENG Dong-qing; ZHOU He-fen; QIAN Guo-pei

    2005-01-01

    @@ Treatment of vitiligo by transplantation of noncultured melanocytes containing keratino-cytes has been successful since 1992,1 We report the encouraging results of autologous epidermal cell suspension in the treatment of 24 patients with stable vitiligo since 1998.

  2. Feruloyl Oligosaccharides from Cell Walls of Suspension-Cultured Spinach Cells and Sugar Beet Pulp : STRUCTURE AND FUNCTION OF CELLS

    OpenAIRE

    Tadashi, Ishii; Forestry and Forest Products Research Institute

    1994-01-01

    Cell walls of suspension-cultured spinach cells and sugar beet pulp were separately hydrolyzed with Driselase. A feruloyl arabinobiose was isolated from both spinach cells and sugar beet. Four feruloyl oligosaccharides were obtained from sugar beet. The four oligosaccharides were characterized by NMR spectroscopy, methylation analysis and FAB-MS.

  3. Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension.

    Science.gov (United States)

    Monteil, Dominique T; Shen, Xiao; Tontodonati, Giulia; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-07-15

    Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.

  4. Agronomic traits and RAPD analysis of two mutants derived from rice somatic cell culturing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Genetic variation, including agronomic trait variation, often occurs in somatic cell culturing. In this study, we compared the main agronomic traits of two rice mutants, M3 and M14, which were derived from Shenxiangjing 5 somatic cell culturing. Significant differences were found between the two mutants and the wild rice Shenxiangjing 5 (Table 1). Results were as follows:

  5. Signal transduction events in aluminum-induced cell death in tomato suspension cells.

    Science.gov (United States)

    Yakimova, Elena T; Kapchina-Toteva, Veneta M; Woltering, Ernst J

    2007-06-01

    In this study, some of the signal transduction events involved in AlCl(3)-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 microM AlCl(3) showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation. Cell death was effectively inhibited by protease and human caspase inhibitors indicating a cell death execution mechanism with similarities to animal apoptosis. Cell death was suppressed by application of antoxidants and by inhibitors of phospholipase C (PLC), phospholipase D (PLD) and ethylene signalling pathways. The results suggest that low concentrations of heavy metal ions stimulate both PLC and PLD signalling pathways leading to the production of reactive oxygen species (ROS) and subsequent cell death executed by caspase-like proteases.

  6. Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain.

    Science.gov (United States)

    Palmer, Richard; Cornuault, Valérie; Marcus, Susan E; Knox, J Paul; Shewry, Peter R; Tosi, Paola

    2015-03-01

    Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

  7. 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 p27(Kip1) 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.

  8. Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis.

    Science.gov (United States)

    Hetherington, P. R.; Fry, S. C.

    1993-11-01

    Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.

  9. Elimination of acute muelogenous leukemic cells from marrow and tumor suspensions in the rat with 4-hydroperoxycyclophosphamide

    Energy Technology Data Exchange (ETDEWEB)

    Sharkis, S.J.; Santos, G.W.; Colvin, M.

    1980-03-01

    Cell suspensions of normal rat marrow mixed with rat acute myelogenous leukemic cells were prepared and incubated in vitro with graded doses of 4-hydroperoxycyclophosphamide (4HC). The cell suspensions were injected into rats prepared with a lethal dose of total body irradiation. Animals injected with these cells survived fatal irradiation induced aplasia. In a dose related manner 4HC was able to purge tumor cells from the cell mixtures. Thus, animals given cell suspensions incubated with the lower doses of 4HC showed prolonged survived before death from leukemia and animals given cell suspensions incubated with higher doses of 4HC survival lethal irradiation without the subsequent appearance of leukemia. These studies clearly establish that tumor cells may be eliminated from normal marrow suspensions without completely destroying the pluripotent stem cells.

  10. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    Science.gov (United States)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  11. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.; Harren, F.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2¿3 days which indicates the existence

  12. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Yakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.J.; Harren, F.J.M.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 23 days which indicates the existence

  13. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.; Harren, F.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2¿3 days which indicates the existence

  14. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Yakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.J.; Harren, F.J.M.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 23 days which indicates the existence

  15. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  16. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  17. Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases

    NARCIS (Netherlands)

    Lens, P.N.L.; Gastesi, R.; Lettinga, G.

    2003-01-01

    This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell

  18. Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases

    NARCIS (Netherlands)

    Lens, P.N.L.; Gastesi, R.; Lettinga, G.

    2003-01-01

    This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell s

  19. Zinc-dependent protection of tobacco and rice cells from Aluminum-induced superoxide-mediated cytotoxicity

    Directory of Open Access Journals (Sweden)

    Cun eLin

    2015-12-01

    Full Text Available Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2 and rice (Oryza sativa L., cv. Nipponbare, was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco, suggesting that this phenomenon (Al3+/Zn2+ interaction can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e. change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored.

  20. Computer Simulation Study of Collective Phenomena in Dense Suspensions of Red Blood Cells under Shear

    CERN Document Server

    Krüger, Timm

    2012-01-01

    The rheology of dense red blood cell suspensions is investigated via computer simulations based on the lattice Boltzmann, the immersed boundary, and the finite element methods. The red blood cells are treated as extended and deformable particles immersed in the ambient fluid. In the first part of the work, the numerical model and strategies for stress evaluation are discussed. In the second part, the behavior of the suspensions in simple shear flow is studied for different volume fractions, particle deformabilities, and shear rates. Shear thinning behavior is recovered. The existence of a shear-induced transition from a tumbling to a tank-treading motion is demonstrated. The transition can be parameterized by a single quantity, namely the effective capillary number. It is the ratio of the suspension stress and the characteristic particle membrane stress. At the transition point, a strong increase in the orientational order of the red blood cells and a significant decrease of the particle diffusivity are obser...

  1. Glucosidase II β-subunit, a novel substrate for caspase-3-like activity in rice, plays as a molecular switch between autophagy and programmed cell death

    Science.gov (United States)

    Cui, Jing; Chen, Bing; Wang, Hongjuan; Han, Yue; Chen, Xi; Zhang, Wei

    2016-01-01

    Endoplasmic reticulum (ER) stress activates unfolded protein response (UPR) and autophagy. However, prolonged, severe stresses activate programmed cell death (PCD) in both animal and plant cells. Compared to the well-studied UPR pathway, the molecular mechanisms of ER-stress-induced PCD are less understood. Here, we report the identification of Gas2, the glucosidase II β subunit in the ER, as a potential switch between PCD and autophagy in rice. MS analysis identified Gas2, GRP94, and HSP40 protein in a purified caspase-3-like activity from heat stressed rice cell suspensions. The three corresponding genes were down-regulated under DTT-induced ER stress. Gas2 and GRP94 were localized to the ER, while HSP40 localized to the cytoplasm. Compared to wild-type, a Gas2 RNAi cell line was much sensitive to DTT treatment and had high levels of autophagy. Both caspase-3 and heat-stressed cell suspension lysate could cleave Gas2, producing a 14 kDa N-terminal fragment. Conditional expression of corresponding C-terminal fragment resulted in enhanced caspase-3-like activity in the protoplasts under heat stress. We proposed that mild ER stress causes down-regulation of Gas2 and induces autophagy, while severe stress results in Gas2 cleavage by caspase-3-like activity and the cleavage product amplifies this activity, possibly participating in the initiation of PCD. PMID:27538481

  2. Search for cell-wall-degrading enzymes of world-wide rice grains by PCR and their effects on the palatability of rice.

    Science.gov (United States)

    Nakamura, Sumiko; Machida, Keisuke; Ohtsubo, Ken'ichi

    2012-01-01

    Such rice cultivars as Japonica, Japonica-Indica hybrid, Javanica and Indica, were evaluated for their main chemical components (amylose content and protein content), pasting property of rice flour (consistency), physical property of the cooked rice grains (adhesion, L3), and enzyme activities (cellulase and xylanase). The amylose content, cellulase activity and xylanase activity showed significant positive or negative correlation with the pasting property (consistency) of rice flour (r = 0.89, r = 0.58, r = 0.70, respectively) and with the physical property of the cooked rice grains (adhesion, L3: r = -0.51, r = -0.61, r = -0.71, respectively) at the level of 1%. Endogenous xylanase and cellulase played important roles to determine the texture of the cooked rice grains similarly to the amylose content. Part of the DNA sequences of the α-glucosidase gene differed among the Japonica, Japonica-Indica hybrid and Indica subspecies. We found discriminative DNA bands appearing by PCR, corresponding to 1,4-β-xylanase and endo-1,4-β-glucanase 13 in the case of Indica rice, Indica-Japonica hybrid rice, and Javanica rice (non-Japonica subspecies). The equation for estimating the physical property (adhesion) of cooked rice grains by PCR was improved by adding novel primers related to the cell-wall-degrading enzymes.

  3. Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors.

    Science.gov (United States)

    Mukherjee, S; Ghosh, B; Jha, S

    2000-01-07

    Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.

  4. 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.

  5. 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...... sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions...

  6. Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Begum, Parvin, E-mail: parvinchy@ees.hokudai.ac.jp; Fugetsu, Bunshi

    2013-09-15

    Highlights: • This study was set up to explore potential influence of graphene on T87 cells. • Fragmented nuclei, membrane damage, mitochondrial dysfunction were observed. • ROS increased, ROS are key mediators in the cell death signaling pathway. • Translocation of graphene into cells and an endocytosis-like structure was observed. • Graphene entering into the cells by endocytosis. -- Abstract: The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0–80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2′,7′-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS.

  7. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Jong, de A.J.; Yakimova, E.T.; Kapchina, V.M.; Woltering, E.J.

    2002-01-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and

  8. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Jong, de A.J.; Yakimova, E.T.; Kapchina, V.M.; Woltering, E.J.

    2002-01-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and

  9. 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

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

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract 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

  11. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  12. Studies on the Programmed Cell Death in Rice During Starchy Endosperm Development

    Institute of Scientific and Technical Information of China (English)

    LI Rui; LAN Sheng-yin; XU Zhen-xiu

    2004-01-01

    Morphological variations of the nucleus in starchy endosperm cell were observed by the electron-transmisson microscope during endosperm development in rice. Along with the development of the starchy endosperm,the nuclei of the cells showed chromatin condensation,the typical feature of programmed cell death(PCD). The nuclei also showed nucleus deformation,disruption of nuclear envelope,nucleoplasm leaking into the cytoplasm and nucleus disintegration resulting in nuclear residue formation. From the nucleus deformation to the nucleus disintegration,the morphological changes of the nucleus were orderly progressive. This indicated that the cell death of starchy endosperm in rice was programmed cell death. Evans Blue staining observation showed that the cell death was initially detected in the central part of starchy endosperm in rice,then expanded outward. The activities of superoxide dismutase(SOD)and catalase(CAT)in rice starchy endosperm both descended continuously as development progressed. The analysis of DNA of rice starchy endosperm did not show the presence of DNA laddering. The above results showed that the cell death of starchy endosperm in rice was a special form of PCD.

  13. Embedding Arabidopsis Plant Cell Suspensions in Low-Melting Agarose Facilitates Altered Gravity Studies

    Science.gov (United States)

    Kamal, Khaled Y.; van Loon, Jack J. W. A.; Medina, F. Javier; Herranz, Raúl

    2017-02-01

    Gravity plays a role in modulating plant growth and development and its alteration induces changes in these processes. Microgravity research has recently been extended to the use of in vitro plant cell cultures which are considered as an ideal model system to study cell proliferation and growth. In general, among the ground-based facilities available for microgravity simulation, the 2D pipette clinostat had been previously considered a suitable facility to be used for unicellular biological models although studies using single plant cell cultures raised some concerns. The incompatibility comes from the standard requirement of shaking a suspension culture for assuring its viability and active proliferation status in the control samples. Moreover, a related issue applies to the use of the random positioning machine (RPM) for cell suspension experiments. Here, we demonstrate an alternative culture method based on the immobilization of the culture before the altered gravity treatment occurs, such that it behaves as a solid object. Our immobilization procedure preserved plant cell culture viability without compromising basic cell properties as viability, morphology, cell cycle phases distribution, or chromatin organization, when compared with a standard cell suspension under shaking as a control. This approach should allow the space biology community to improve the quantity and quality of plant cell results in future simulated microgravity experiments or spaceflight opportunities.

  14. DIGLUCOSYLATION OF SALICYL ALCOHOL BY CELL SUSPENSION CULTURES OF SOLANUM LACINIATUM

    Institute of Scientific and Technical Information of China (English)

    ACHMAD SYAHRANI; FRANSISCA HARTUTI; GUNAWAN INDRAYANTO; ALISTAIR L.WILKINS

    2001-01-01

    A new biotransformation product, salicyl alcohol-7-O-β-D-(β-l,6-D-glucopyranosyl)-gluco pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

  15. Accumulation of podophyllotoxin and related lignans in cell suspension cultures of Linum album

    NARCIS (Netherlands)

    Smollny, T.; Wichers, H.; Kalenberg, S.; Shahsavari, A.; Petersen, M.; Alfermann, A.W.

    1998-01-01

    Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotox

  16. Comparative metabolite profiling of the insecticide thiamethoxam in plant and cell suspension culture of tomato.

    Science.gov (United States)

    Karmakar, Rajib; Bhattacharya, Ramcharan; Kulshrestha, Gita

    2009-07-22

    The metabolism of thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-yl-methyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene (nitro) amine] was investigated in whole plant, callus, and heterotrophic cell suspension culture of aseptically and field grown tomato (Lycopersicon esculentum Mill.) plants. The structure of the metabolites was elucidated by chromatographic (HPLC) and spectroscopic (IR, NMR, and MS) methods. Thiamethoxam metabolism proceeded by the formation of a urea derivative, a nitroso product, and nitro guanidine. Both urea and nitro guanidine metabolites further degraded in plants, and a mechanism has been proposed. In the plant, organ-specific differences in thiamethoxam metabolism were observed. Only one metabolite was formed in whole plant against four in callus and eight metabolites in cell suspension culture under aseptic conditions. Out of six metabolites of thiamethoxam in tomato fruits in field conditions, five were similar to those formed in the cell suspension culture. In the cell suspension culture, thiamethoxam degraded to maximum metabolites within 72 h, whereas in plants, such extensive conversion could only be observed after 10 days.

  17. Biotransformation of isonitrosoacetophenone (2-keto-2-phenyl-acetaldoxime) in tobacco cell suspensions

    CSIR Research Space (South Africa)

    Madala, NE

    2012-07-01

    Full Text Available Biotechnology Letters July 2012/ Vol. 34 No.7, pp 1351-1356 Biotransformation of isonitrosoacetophenone (2-keto-2- phenyl-acetaldoxime) in tobacco cell suspensions Ntakadzeni E. Madala 1 , P. A. Steenkamp 2 , L. A. Piater 1 and I. A. Dubery 1 1...

  18. Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions.

    Science.gov (United States)

    Broggi, Achille; Cigni, Clara; Zanoni, Ivan; Granucci, Francesca

    2016-04-20

    The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

  19. Analysis of impedance measurements of a suspension of microcapsules using a variable length impedance measurement cell

    Directory of Open Access Journals (Sweden)

    Dejan Krizaj

    2012-10-01

    Full Text Available Electrical impedance measurements of the suspensions have to take into account the double layer impedance that is due to a very thin charged layer formed at the electrode-electrolite interface. A dedicated measuring cell that enables variation of the distance between the electrodes was developed for investigation of electrical properties of suspensions using two electrode impedance measurements. By varying the distance between the electrodes it is possible to separate the double layer and the suspension impedance from the measured data. From measured and extracted impedances electrical lumped models have been developed. The error of non inclusion of the double layer impedance has been analyzed. The error depends on the frequency of the measurements as well as on the distance between the electrodes.

  20. Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco.

    Science.gov (United States)

    Han, Jung-Yeon; Wang, Hong-Yan; Choi, Yong-Eui

    2014-02-01

    Dammarenediol-II is biologically active tetracyclic triterpenoid, which is basic compound of ginsenoside saponin. Here, we established the dammarenediol-II production via a cell suspension culture of transgenic tobacco overexpressing PgDDS. Dammarenediol-II synthase catalyzes the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is the basic triterpene skeleton in dammarene-type saponin (ginsenosides) in Panax ginseng. Dammarenediol-II is a useful candidate both for pharmacologically active triterpenes and as a defense compound in plants. Dammarenediol-II is present in the roots of P. ginseng in trace amounts because it is an intermediate product in triterpene biosynthesis. In this work, we established the production of dammarenediol-II via cell suspension culture of transgenic tobacco. The dammarenediol-II synthase gene (PgDDS) isolated from P. ginseng was introduced into the Nicotiana tobacum genome under the control of 35S promoter by Agrobacterium-mediated transformation. Accumulation of dammarenediol-II in transgenic tobacco plants occurred in an organ-specific manner (roots > stems > leaves > flower buds), and transgenic line 14 (T14) exhibited a high amount (157.8 μg g⁻¹ DW) of dammarenediol-II in the roots. Dammarenediol-II production in transgenic tobacco plants resulted in reduced phytosterol (β-sitosterol, campesterol, and stigmasterol) contents. A cell suspension culture was established as a shake flask culture of a callus derived from root segments of transgenic (T14) plants. The amount of dammarenediol-II production in the cell suspension reached 573 μg g⁻¹ dry weight after 3 weeks of culture, which is equivalent to a culture volume of 5.2 mg dammarenediol-II per liter. Conclusively, the production of dammarenediol-II in a cell suspension culture of transgenic tobacco can be applied to the large-scale production of this compound and utilized as a source of pharmacologically active medicinal materials.

  1. Programmed cell death features in apple suspension cells under low oxygen culture.

    Science.gov (United States)

    Xu, Chang-jie; Chen, Kun-song; Ferguson, Ian B

    2004-02-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  2. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  3. 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.

  4. Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Herranz, Raúl; van Loon, Jack J. W. A.; Christianen, Peter C. M.; Medina, F. Javier

    2016-06-01

    Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.

  5. Optimization of Lycopene Extraction from Tomato Cell Suspension Culture by Response Surface Methodology

    OpenAIRE

    Lu, Chi-Hua; Engelmann, Nancy J.; Lila, Mary Ann; Erdman, John W

    2008-01-01

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combi...

  6. Morphological characterization of cells in concentrated suspensions using multispectral diffuse optical tomography.

    Science.gov (United States)

    Hajihashemi, Mohammad Reza; Li, Xiaoqi; Jiang, Huabei

    2012-10-01

    Based on a non-spherical model of particle scattering, we investigate the capabilities and limitations of a T-matrix based inverse algorithm to morphologically characterize cells in concentrated suspensions. Here the cells are modeled as randomly orientated spheroidal particles with homogenous dielectric properties and suspended in turbid media. The inverse algorithm retrieves the geometrical parameters and the concentration of cells simultaneously by inverting the reduced scattering coefficient spectra obtained from multispectral diffuse optical tomography (MS-DOT). Both round and spheroidal cells are tested and the role of multiple and higher order scattering of particles on the performance of the algorithm is evaluated using different concentrations of cells.

  7. Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Mori, Ryuji; Saiki, Mizue; Nakamura, Yukiko; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

    2002-09-01

    We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.

  8. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  9. Evaluation of the oxidative burst in suspension cell culture of Phaseolus vulgaris.

    Science.gov (United States)

    Janisch, Kerstin; Schempp, Harald

    2004-01-01

    Plants respond to the attack of pathogens with the oxidative burst, a production of reactive oxygen species (ROS). In this work a cell culture suspension of Phaseolus vulgaris was used to investigate the oxidative burst triggered by a conidia suspension of different races of Colletotrichum lindemuthianum. As a defence response of the cells a two-phase peak was observed with all used races of Colletotrichum lindemuthianum, varying only in the produced amounts of hydrogen peroxide. Findings with additives such as superoxide dismutase (SOD), diphenyleneiodonium (DPI) and catalase gave rise to the conclusion that more superoxide radicals were produced than be detectable with Amplex Red as hydrogen peroxide. It is assumed that the conversion of the superoxide radical is spontaneous and not driven via a cell-derived superoxide dismutase. The addition of low-molecular cell wall components (ergosterol, glucosamine, galactosamine) showed clearly that compounds like this act as elicitors and thus are involved in triggering the burst. Furthermore, an evaluation of the metabolizing capacities of hydrogen peroxide of the suspension culture cells revealed the enormous capacity of the cells to detoxify this ROS.

  10. Origination of turbulence in dense suspensions of sperm cells

    Science.gov (United States)

    Denissenko, Petr; Kirkman-Brown, Jackson; Smith, David; Kantsler, Vasily

    2014-11-01

    Motile micro-organisms with pushing flagella, such as sperm cells, can be directed by ``one way'' microchannels with ratchet teeth-like wall configuration. We use an array of such micro-channels to gradually concentrate human spermatozoa in a circular arena of 1 mm diameter and 200 micron depth. Velocities of individual cells are measured by particle tracking and velocity of cell-carrying fluid is measured using PIV. At high concentrations, fluid velocities and the velocity fluctuations of individual cells exceeding that of individual swimmers in the dilute regime by an order of magnitude have been measured. Velocity correlations are calculated to study evolution of characteristic length scales as the cell concentration increases. Results are discussed in the context of self-organisation phenomena in active fluids and cooperation of sperm cells.

  11. 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.

  12. The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

    Science.gov (United States)

    Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

    2016-01-15

    In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats.

  13. A simple and efficient method for the long-term preservation of plant cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Boisson Anne-Marie

    2012-01-01

    Full Text Available Abstract Background The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority. Results Sycamore (Acer pseudoplatanus and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed. Conclusion We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.

  14. On the dielectric relaxation of biological cell suspensions: the effect of the membrane electrical conductivity.

    Science.gov (United States)

    Di Biasio, A; Cametti, C

    2011-06-01

    Due to the mismatch of the electrical parameters (the permittivity ϵ' and the electrical conductivity σ) of the membrane of a biological cell with the ones of the cytosol and the extracellular medium, biological cell suspensions are the site, under the influence of an external electric field, of large dielectric relaxations in the radiowave frequency range. However, a point still remains controversial, i.e., whether or not the value of membrane conductivity σ(s) might be extracted from the de-convolution of the dielectric spectra or otherwise if it would be more reasonable to assign to the membrane conductivity a value equal to zero. This point is not to be considered with superficiality since it concerns an a priori choice which ultimately influences the values of the electrical parameters deduced from this technique. As far as this point is concerned, the opinion of the researchers in this field diverges. We believe that, at least within certain limits, the membrane conductivity can be deduced from the shape of the relaxation spectra. We substantiate this thesis with two different examples concerning the first a suspension of human normal erythrocyte cells and the second a suspension of human lymphocyte cells. In both cases, by means of an accurate fitting procedure based on the Levenberg-Marquardt method for complex functions, we can evaluate the membrane conductivity σ(s) with its associated uncertainty. The knowledge of the membrane electrical conductivity will favor the investigation of different ion transport mechanisms across the cell membrane.

  15. Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells

    Institute of Scientific and Technical Information of China (English)

    Jian-You Li; Ai-Liang Jiang; Wei Zhang

    2007-01-01

    Salt stressed rice root tips were used to investigate the changes of reactive oxygen species (ROS) and antioxidant enzymes at the early stages of programmed cell death (PCD). The results indicated that 500 mmol/L NaCl treatment could lead to specific features of PCD in root tips, such as DNA ladder, nuclear condense and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and progressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. In the very early phase of PCD, an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase. A short period of increase in the activity was followed by prolonged impairment. Thus,we conclude that salt can induce PCD in rice root tip cells, and propose that in the early phase of rice root tip cell PCD, salt stress-induced oxidative burst increased the antioxidant enzyme activity, which, In turn, scavenged the ROS and abrogated PCD. Also, when the stress is prolonged, the antioxidant system is damaged and accumulated ROS induces the PCD process, which leads to cytochrome c release and nuclear change.

  16. 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.

  17. Extracellular Hydrolysis of Starch in Sugarcane Cell Suspensions 12

    Science.gov (United States)

    Maretzki, A.; dela Cruz, A.; Nickell, L. G.

    1971-01-01

    Evidence is presented for the increased excretion of amylolytic enzymes into a sugarcane cell culture medium when starch was substituted for sucrose as an energy source. The excretion was further enhanced by the inclusion of 1 μm gibberellic acid in the nutrient medium. The growth rate of the cells increased after they became adapted to starch relative to cells grown on sucrose, but the rate of amylolytic enzyme excretion remained unaltered. Amylolytic enzymes in the medium included α-amylase but the identity of one or more other enzymes related to starch hydrolysis remains in doubt. PMID:16657831

  18. Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Feria-Romero, Iris; Lazo, Elizabeth; Ponce-Noyola, Teresa; Cerda-García-Rojas, Carlos M; Ramos-Valdivia, Ana C

    2005-06-01

    Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

  19. Survival of Suspension-cultured Sycamore Cells Cooled to the Temperature of Liquid Nitrogen.

    Science.gov (United States)

    Sugawara, Y; Sakai, A

    1974-11-01

    Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) which were immersed in liquid nitrogen after prefreezing to the temperatures from -30 to -50 C in the presence of dimethylsulfoxide and glucose as cryoprotective additive could proliferate vigorously when rewarmed rapidly in water at 40 C. For maintaining high viability of the cells after immersion in liquid nitrogen, it seems to be essential to use the cells at the later lag phase or the early cell division phase. This study provides a possibility for long term preservation in liquid nitrogen of plant-cultured lines.

  20. Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

    Science.gov (United States)

    Shakhbazau, Antos; Mirfeizi, Leila; Walsh, Tylor; Wobma, Holly M; Kumar, Ranjan; Singh, Bhagat; Kallos, Michael S; Midha, Rajiv

    2016-02-01

    Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

  1. Collision rates for rare cell capture in periodic obstacle arrays strongly depend on density of cell suspension.

    Science.gov (United States)

    Cimrák, I

    2016-11-01

    Recently, computational modelling has been successfully used for determination of collision rates for rare cell capture in periodic obstacle arrays. The models were based on particle advection simulations where the cells were advected according to velocity field computed from two dimensional Navier-Stokes equations. This approach may be used under the assumption of very dilute cell suspensions where no mutual cell collisions occur. We use the object-in-fluid framework to demonstrate that even with low cell-to-fluid ratio, the optimal geometry of the obstacle array significantly changes. We show computational simulations for ratios of 3.5, 6.9 and 10.4% determining the optimal geometry of the periodic obstacle arrays. It was already previously demonstrated that cells in periodic obstacle arrays follow trajectories in two modes: the colliding mode and the zig-zag mode. The colliding mode maximizes the cell-obstacle collision frequency. Our simulations reveal that for dilute suspensions and for suspensions with cell-to-fluid ratio 3.5%, there is a range of column shifts for which the cells follow colliding trajectories. However we showed, that for 6.9 and 10.4%, the cells never follow colliding trajectories.

  2. Secondary metabolite production in Hypericum perforatum L. cell suspensions upon elicitation with fungal mycelia from Aspergillus flavus

    Directory of Open Access Journals (Sweden)

    Gadzovska-Simic Sonja

    2012-01-01

    Full Text Available We investigated the production of phenylpropanoids (phenolic compounds, flavanols, flavonols and anthocyanins and naphtodianthrones (hypericins in elicited Hypericum perforatum L. cell suspensions. To determine whether secondary metabolite production could be enhanced, Hypericum cell suspensions were exposed to mycelia extract from the fungus Aspergillus flavus. Elicited Hypericum cell suspension cultures displayed reduced growth and viability and a modification of secondary metabolites production. Anthocyanins were only stimulated in fungal-elicited cell suspensions. Secondary metabolite production in elicited Hypericum cells revealed an antagonism between the flavonoid/naphtodianthrone and anthocyanin pathways. The data suggest a modification of the channeling of the phenylpropanoid compounds. Together, these results represent useful data for monitoring the channeling in different secondary metabolite pathways during the scaled-up production of naphtodianthrones for medicinal uses.

  3. A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

    Science.gov (United States)

    Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

    2016-02-01

    Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

  4. Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

    Institute of Scientific and Technical Information of China (English)

    Xing Yang; Zhaoying Zhou; Mingfei Xiao; Ying Wu; Shangfeng Liu

    2006-01-01

    A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed. The substrate and the electrodes of the chip were made of glass and gold, respectively. The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline, culture medium, living cell suspension etc.) by scanning from 10Hz to 45kHz. A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension. An actual circuit was also built and tested to verify the 6-element circuit model proposed. The micro-EIS chip has several advantages including the use of small sample volumes, high resolution and ease of operation. It shows good application prospects in the areas of cellular electrophysiology, drug screening and bio-sensors etc.

  5. 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.

  6. Low-frequency dielectric dispersion of bacterial cell suspensions.

    Science.gov (United States)

    Asami, Koji

    2014-07-01

    Dielectric spectra of Escherichia coli cells suspended in 0.1-10 mM NaCl were measured over a frequency range of 10 Hz to 10 MHz. Low-frequency dielectric dispersion, so-called the α-dispersion, was found below 10 kHz in addition to the β-dispersion, due to interfacial polarization, appearing above 100 kHz. When the cells were killed by heating at 60°C for 30 min, the β-dispersion disappeared completely, whereas the α-dispersion was little influenced. This suggests that the plasma (or inner) membranes of the dead cells are no longer the permeability barrier to small ions, and that the α-dispersion is not related to the membrane potential due to selective membrane permeability of ions. The intensity of the α-dispersion depended on both of the pH and ionic strength of the external medium, supporting the model that the α-dispersion results from the deformation of the ion clouds formed outside and inside the cell wall containing charged residues. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture ... Even though. Arabidopsis provides for an excellent model system for .... stained, destained and imaged using a Molecular Imager PharosFX ... system, are dynamic and heterogeneous, being com- posed of a ...

  8. Cell Biological Characterization of Male Meiosis and Pollen Development in Rice

    Institute of Scientific and Technical Information of China (English)

    Chang-Bin CHEN; Yun-Yuan XU; Hong MA; Kang CHONG

    2005-01-01

    Little systematic analysis has been undertaken in rice (Oryza sativa L.) on the stages of male meiosis from leptotene to telophase Ⅱ or of pollen development from microspores to mature pollen grains.The present study describes multiple stages in detail from analysis of rice chromosome spreading with staining of 4',6-diamidino-2-phenylindole. The description of normal wild-type male meiosis provides an important morphological reference for analyses of meiotic mutants. Meiosis in rice is largely similar to those of the well characterizing model plants Arabidopsis thaliana L. and Zea mays L. However, rice meiosis differs from that in Arabidopsis in that rice meiosis I is followed by the formation of a cell plate, instead of an organelle band that forms between the two nuclei and persist through meiosis Ⅱ. This suggests a difference in the control of organelle biogenesis and distribution and cytokinesis. Our results should facilitate studies of rice meiosis and pollen development using molecular genetic and cell biological approaches.

  9. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

    Science.gov (United States)

    Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

    2015-12-01

    Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

  10. C-27 AND C-3 GLUCOSYLATION OF DIOSGENIN BY CELL SUSPENSION CULTURES OF COSTUS SPECIOSUS

    Institute of Scientific and Technical Information of China (English)

    GUNAWAN INDRAYANTO; SITI ZUMAROH; ACHMAD SYAHRANI; ALISTAIR L. WILKINS

    2001-01-01

    3-O-[β-D-glucopyranosyl-(l″→ 2′)-β-D-glucopyranosyl], 27-O-β-D-glucopyranosyl-(25R)-spir ost-5-ene-3β,27-diol was isolated from cell suspension cultures of Costus speciosus, following incubation with diosgenin, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C NMR spectral data, and positive and negative ion ESMS spectral data.

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

    OpenAIRE

    Solís Ramos, Laura Yesenia; Miranda Carballo, Laura; Valdez Melara, Marta

    2013-01-01

    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 industria...

  12. Putrescine facilitated enhancement of capsaicin production in cell suspension cultures of Capsicum frutescens.

    Science.gov (United States)

    Sudha, Govindaswamy; Ravishankar, Gokare A

    2003-04-01

    Putrescine treatment (0.1 mmol/L) influenced enhancement of growth and capsaicin production in the cell suspension cultures of C. frutescens. The administration of polyamine inhibitor DFMA (alpha-DL-difluoromethylarginine) resulted in a reduction of the growth, capsaicin content and the endogenous titres of polyamines (PAs). The capsaicin synthase activity was also higher in the putrescine (Put) treated cultures. Ethylene levels were lower in the cultures treated with putrescine. This study suggested that Put facilitates growth and capsaicin production.

  13. Solid oxide fuel cell electrolytes produced via very low pressure suspension plasma spray and electrophoretic deposition

    OpenAIRE

    Fleetwood, James D

    2014-01-01

    Solid oxide fuel cells (SOFCs) are a promising element of comprehensive energy policies due to their direct mechanism for converting the oxidization of fuel, such as hydrogen, into electrical energy. Both very low pressure plasma spray and electrophoretic deposition allow working with high melting temperature SOFC suspension based feedstock on complex surfaces, such as in non-planar SOFC designs. Dense, thin electrolytes of ideal composition for SOFCs can be fabricated with each of these proc...

  14. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

    OpenAIRE

    Liangyun Zhou; Jian Yang; Guang Yang; Chuanzhi Kang; Wenjuan Xiao; Chaogeng Lv; Sheng Wang; Jinfu Tang; Lanping Guo

    2016-01-01

    Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphe...

  15. Isolation of fatty acids and aromatics from cell suspension cultures of Lavandula angustifolia.

    Science.gov (United States)

    Topçu, Gülaçti; Herrmann, Gabriele; Kolak, Ufuk; Gören, C; Porzel, Andrea; Kutchan, Toni M

    2007-02-01

    Cell suspension cultures of Lavandula angustifolia Mill. ssp. angustifolia (syn.: L. officinalis Chaix.) afforded a fatty acid composition, cis and trans p-coumaric acids (=p-hydroxy cinnamic acids), and beta-sitosterol. The fatty acid composition was analyzed by GC-MS, and the structures of the isolated three compounds were determined by 1H- and 13C-NMR, and MS spectroscopic techniques.

  16. Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2004-06-30

    A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism. Copyright 2004 Wiley Periodicals, Inc.

  17. Ce4+-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    葛志强; 元英进; 王艳东; 马振毅; 胡宗定

    2002-01-01

    The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged "DNA ladder" on agarose gel electrophoresis. TdT-mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′-OH termini. These results suggest that Ce4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product-Taxol.

  18. [Massive multiplication of coffee (Coffee arabica L. cv. Catimor) through embryogenic cell suspension culture].

    Science.gov (United States)

    Flermoso-Gallardo, L; Menóndez-Yuffá, A

    2000-01-01

    Cell suspensions offer several advantages as a system for massive propagation because of the high rates of multiplication, the higher homogeneity in the culture conditions and the possibility of automatization. In this study, different experimental conditions were analyzed to establish embryogenic cell suspension cultures of coffee. The best conditions to establish the embryogenic cell suspension cultures of coffee were as follows: coffee leaf sections were cultivated during 12 weeks (Stage I) in a solid medium with the Murashige and Skoog salts, 2 mg/l kinetin and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (medium 1). Under these conditions the explants formed a callus tissue that was transferred to a liquid medium containing 5 mg/l of 6-benzylamlno-purine (medium 2). After 12 days in a shaking liquid medium (Stage II), the cultures were sieved and were maintained In the same media, which was renewed every eight days (Stage III). This method yielded 1884 embryos in 50 ml; placing the embryos under conditions for germination yielded plantlets of normal appearance.

  19. Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata).

    Science.gov (United States)

    Cheema, G S

    1989-02-01

    Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.

  20. Nanometer-scale sizing accuracy of particle suspensions on an unmodified cell phone using elastic light scattering.

    Directory of Open Access Journals (Sweden)

    Zachary J Smith

    Full Text Available We report on the construction of a Fourier plane imaging system attached to a cell phone. By illuminating particle suspensions with a collimated beam from an inexpensive diode laser, angularly resolved scattering patterns are imaged by the phone's camera. Analyzing these patterns with Mie theory results in predictions of size distributions of the particles in suspension. Despite using consumer grade electronics, we extracted size distributions of sphere suspensions with better than 20 nm accuracy in determining the mean size. We also show results from milk, yeast, and blood cells. Performing these measurements on a portable device presents opportunities for field-testing of food quality, process monitoring, and medical diagnosis.

  1. [Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

    Science.gov (United States)

    Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

    2015-01-01

    The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

  2. The role of cell replacement in benthic–pelagic coupling by suspension feeders

    Science.gov (United States)

    2016-01-01

    Benthic–pelagic coupling through suspension feeders and their detrital pathways is integral to carbon transport in oceans. In food-poor ecosystems however, a novel mechanism of carbon recycling has been proposed that involves direct uptake of dissolved carbon by suspension feeders followed by shedding of cells as particulate carbon. We studied cell replacement rates in a range of cold-water sponge species to determine how universal this mechanism might be. We show that cell replacement rates of feeding epithelia in explants vary from 30 hours up to 7 days, and change during different seasons and life-history stages. We also found that feeding epithelia are not replaced through direct replication but instead arise from a population of stem cells that differentiate and integrate into epithelial tissues. Our results reveal a surprising amount of complexity in the control of cell processes in sponges, with cell turnover depending on environmental conditions and using stem cells as rate-limiting mechanisms. Our results also suggest that for species in cold water with high particulate organic matter, cell turnover is not the mechanism delivering carbon flux to surrounding communities. PMID:28018632

  3. Numerical simulation of dielectric spectra of aqueous suspensions of non-spheroidal differently shaped biological cells

    Science.gov (United States)

    di Biasio, Antonio; Ambrosone, Luigi; Cametti, Cesare

    2009-01-01

    The effect of shape on the dielectric and conductometric spectra of aqueous suspensions of non-spheroidal biological cells has been investigated by means of numerical simulation methods. This work extends our previous investigation directed to biological cell systems where a superficial electric charge distribution is present on the outer interface of the cell membrane. This generalization results in a more composite dielectric spectra, where a low-frequency and a high-frequency contribution are expected. We consider different geometries, from ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles to budded vesicles, which model a biological cell during different processes of biological relevance. The overview of the evolution of the dielectric spectra with the progressive change in the cell shape, maintaining constant the electrical properties of the different media involved and the fractional volume of the dispersed cells, offers a preliminary opportunity to separate contributions derived exclusively from the geometry to those due to the bulk and/or interface polarizations. These aspects are particularly relevant since dielectric spectroscopy of biological cell suspensions has proved its effectiveness in the characterization of the passive electrical properties of the cell membrane and also in controlled manipulations of biological systems.

  4. Translation of cell therapies to the clinic: characteristics of cell suspensions in large-diameter injection cannulae.

    Science.gov (United States)

    Torres, Eduardo M; Trigano, Matthieu; Dunnett, Stephen B

    2015-01-01

    With the use of cell replacement therapies as a realistic prospect for conditions such as Parkinson's and Huntington's diseases, the logistics of the delivery of cell suspensions to deep brain targets is a topic for consideration. Because of the large cannulae required for such procedures, we need to consider the behavior of cell suspensions within the cannulae if we are to ensure that the injected cells are distributed as intended within the target tissue. We have investigated the behavior of primary embryonic cell suspensions of neural tissue, in cannulae of different diameters, using a protocol designed to mimic the handling and injection of cells during clinical application. Internal cannula diameter had a large effect on the distribution of cells during their dispensation from the syringe. In vertical or near vertical cannulae, cells settled toward the tip of the needle, and were dispensed unevenly, with the majority of cells emerging in the first 10-20% of the injectate. In horizontal or near-horizontal cannulae, we observed the opposite effect, such that few cells were dispensed in the first 80% of the injectate, and the majority emerged in the final 10-20%. Use of a glass cannula showed that the results obtained using the horizontal cannula were caused by settling and adherence of the cells on the side of the cannulae, such that during dispensation, the overlying, cell-free solution was dispensed first, prior to the emergence of the cells. We show that the behavior of cells in such cannulae is affected by the cannula diameter, and by the material of the cannula itself. In horizontal cannulae, uneven expulsion of cells from the needle can be ameliorated by regular rotation of the cannula during the procedure. We discuss the potential impact of these observations on the translation of cell therapies to the clinic.

  5. Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production

    OpenAIRE

    FARJAMINEZHAD, Reza; Nasser ZARE; ASGHARI-ZAKARIA, Rasool; Farjaminezhad, Manoochehr

    2013-01-01

    Iranian poppy (Papaver bracteatum) is an important medicinal plant that is the main source of the opium alkaloids codeine, morphine, and thebaine. To establish an efficient protocol for cell suspension culture and growth, the effects of different plant growth regulators (2,4-D, NAA, BAP, and kinetin) on callus induction and cell suspension culture of Iranian poppy were evaluated. The maximum percentage of callus induction (86.67%) and fresh weight of callus were obtained in MS medium suppleme...

  6. Influence of particle size and concentration on the diffuse backscattering of polarized light from tissue phantoms and biological cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Hielscher, A.H.; Mourant, J.R.; Bigio, I.J. [Los Alamos National Laboratory, Bioscience and Biotechnology, CST-4, MS E535, Los Alamos, New Mexico 87545 (United States)

    1997-01-01

    We present experimental results that show the spatial variations of the diffuse-backscattered intensity when linearly polarized light is incident upon highly scattering media. Experiments on polystyrene-sphere and Intralipid suspensions demonstrate that the radial and azimuthal variations of the observed pattern depend on the concentration, and anisotropy factor {ital g} of the particles that constitute the scattering medium. Measurements performed on biological-cell suspensions show the potential of this method for cell characterization. {copyright} 1997 Optical Society of America

  7. Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

    Science.gov (United States)

    Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

    That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

  8. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    Science.gov (United States)

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A.; Ko, Minoru S.H.; Motohashi, Tsutomu; Kunisada, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. PMID:18624284

  9. 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.

  10. System for the exposure of cell suspensions to power-frequency electric fields.

    Science.gov (United States)

    Kaune, W T; Frazier, M E; King, A J; Samuel, J E; Hungate, F P; Causey, S C

    1984-01-01

    A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line. In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated. Electric fields in the range 0.02-3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5%. The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 microT . The temperatures of the experimental and control cell suspensions are held fixed within +/- 0.1 degrees C by a water bath. Special chambers were developed to hold cell cultures during exposure and sham exposure. Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16-17 h, approximately the same as for CHO cells grown under standard cell-culture conditions.

  11. Evaluation of limonoid production in suspension cell culture of Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Elisângela Fumagali Gerolino

    2015-10-01

    Full Text Available ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L. Osbeck, Rutaceae and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds, callus cultures (originating from hypocotyls, cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight. Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

  12. [Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor].

    Science.gov (United States)

    Li, Hui-hua; Yao, De-heng; Xu, Jian; Wang, Wei; Chang, Qiang; Su, Ming-hua

    2015-05-01

    Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.

  13. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors.

    Science.gov (United States)

    zur Nieden, Nicole I; Cormier, Jaymi T; Rancourt, Derrick E; Kallos, Michael S

    2007-05-01

    Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.

  14. Where the linearized Poisson-Boltzmann cell model fails: Spurious phase separation in charged colloidal suspensions

    Science.gov (United States)

    Tamashiro, M. N.; Schiessel, H.

    2003-07-01

    The Poisson-Boltzmann (PB) spherical Wigner-Seitz cell model—introduced to theoretically describe suspensions of spherical charged colloidal particles—is investigated at the nonlinear and linearized levels. The linearization of the mean-field PB functional yields linearized Debye-Hückel-type equations agreeing asymptotically with the nonlinear PB results in the weak-coupling (high-temperature) limit. Both the canonical (fixed number of microions) as well as the semigrand-canonical (in contact with an infinite salt reservoir) cases are considered and discussed in a unified linearized framework. In disagreement with the exact nonlinear PB solution inside a Wigner-Seitz cell, the linearized theory predicts the occurrence of a thermodynamical instability with an associated phase separation of the homogeneous suspension into dilute (gas) and dense (liquid) phases, being thus a spurious result of the linearization. We show that these artifacts, although thermodynamically consistent with quadratic expansions of the nonlinear functional and osmotic pressure, may be traced back to the nonfulfillment of the underlying assumptions of the linearization. This raises questions about the reliability of the prediction of gas/liquid-like phase separation in deionized aqueous suspensions of charged colloids mediated by monovalent counterions obtained by linearized theories.

  15. Ghost Cell Suspensions as Blood Analogue Fluid for Macroscopic Particle Image Velocimetry Measurements.

    Science.gov (United States)

    Jansen, Sebastian V; Müller, Indra; Nachtsheim, Max; Schmitz-Rode, Thomas; Steinseifer, Ulrich

    2016-02-01

    Spatially resolved measurement of blood flow is of great interest in the development of artificial blood-carrying devices such as blood pumps, heart valve prostheses, and oxygenators. Particle image velocimetry (PIV) is able to measure instantaneous velocity fields in a plane with high accuracy and is being used more frequently for the development of such devices. However, as this measurement technique is based on optical access, blood flow at physiological hematocrit values is difficult to measure due to its low transparency and multiscattering properties. So far, only very small dimensions (in the range of 400 μm) can be measured using PIV. A suspension of ghost cells (GCs) offers a higher optical transparency than blood while having a similar rheological behavior. In this study, a procedure for the production of GC suspensions containing a very low intracellular hemoglobin concentration is presented. With the help of multiple rounds of controlled cell lysis, the intracellular hemoglobin concentration could be decreased to a point where a standard macroscopic PIV measurement was possible. A velocity profile of a 44% GC suspension in a circular channel with a diameter of 9.5 mm was measured with high spatial resolution. Meanwhile, the rheological behavior was found to be comparable with blood.

  16. 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

  17. 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

  18. Anti-invasive activity against cancer cells of phytochemicals in red jasmine rice (Oryza sativa L.).

    Science.gov (United States)

    Pintha, Komsak; Yodkeeree, Supachai; Pitchakarn, Pornsirit; Limtrakul, Pornngarm

    2014-01-01

    Red rice contains pharmacological substances including phenolics, oryzanol, tocotrienol and tocopherol. Recently, red rice extract has been employed as a source of antioxidants for inhibition of tumor growth. This study was carried out to evaluate the anti-invasion effects of red rice extract fractions on cancer cells. It was found that at 100 μg/ml of crude ethanolic extract (CEE), hexane fraction (Hex) and dichloromethane fraction (DCM) could reduce HT1080 and MDA-MB-231 cancer cell invasion. Hex and DCM revealed higher potency levels than CEE, whereas an ethyl acetate fraction (EtOAc) had no effect. Gelatin zymography revealed that Hex decreased the secretion and activity of matrix metalloproteinase-2 and -9 (MMP-2 and-9). In contrast, the DCM fraction exhibited slightly effect on MMPs secretion and had no effect on MMPs activity. Collagenase activity was significantly inhibited by the Hex and DCM fractions. High amounts of γ-oryzanol and γ-tocotrienol were found in the Hex and DCM fractions and demonstrated an anti-invasion property. On the other hand, proanthocyanidin was detected only in the CEE fraction and reduced MDA-MB-231 cells invasion property. These observations suggest that proanthocyanidin, γ-oryzanol and γ-tocotrienol in the red rice fractions might be responsible for the anti invasion activity. The red rice extract may have a potential to serve as a food-derived chemotherapeutic agent for cancer patients.

  19. MIL2 (MICROSPORELESS2) regulates early cell differentiation in the rice anther.

    Science.gov (United States)

    Hong, Lilan; Tang, Ding; Shen, Yi; Hu, Qing; Wang, Kejian; Li, Ming; Lu, Tiegang; Cheng, Zhukuan

    2012-10-01

    The formation of diverse, appropriately patterned cell types is critical in the development of all complex multicellular organisms. In flowering plants, anther patterning is a complex process essential for successful sexual reproduction. However, few genes regulating this process have been characterized to date. We report here that the gene MICROSPORELESS2 (MIL2) regulates early anther cell differentiation in rice (Oryza sativa). The anthers of mil2 mutants were characterized using molecular markers and cytological examination. The MIL2 gene was cloned and its expression pattern was analyzed through RNA in situ hybridization. The localization of the MIL2 protein was observed by immunostaining. MIL2 encodes the rice homolog of the Arabidopsis TAPETUM DETERMINANT1 (TPD1) protein. However, mil2 anthers display phenotypes different from those of tpd1 mutants, with only two layers of anther wall cells formed. MIL2 has an expression pattern distinct from that of TPD1. Its transcripts and proteins predominate in inner parietal cells, but show little accumulation in reproductive cells. Our results demonstrate that MIL2 is responsible for the differentiation of primary parietal cells into secondary parietal cells in rice anthers, and suggest that rice and Arabidopsis anthers might share similar regulators in anther patterning, but divergent mechanisms are employed in these processes. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  20. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  1. A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy.

    Science.gov (United States)

    Mihara, Keiko; Nakayama, Tomofumi; Saitoh, Hisato

    2015-09-01

    Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation. All-trans-retinoic-acid-exposed differentiated HL-60 cells, which have properties similar to neutrophils, can also adhere efficiently to coverslips in FBS-free medium. Because the procedure is not complex and special equipment is not required, the simplicity and cost effectiveness of this FBS-free cell adhesion protocol may be beneficial to researchers who are interested in assessing the structure and function of suspension cells using microscopy. Copyright © 2015 John Wiley & Sons, Inc.

  2. Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens.

    Science.gov (United States)

    Baldes, R; Moos, M; Geider, K

    1987-03-01

    Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.

  3. 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...

  4. 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...... 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...

  5. APOPTOSIS AND TAXOL PRODUCTION IN SUSPENSION CULTURES OF Taxus spp.CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    l.lntroductionSuspension cultures of Taxal chinensis var.mairei frequently accumulate Taxol (paclitaxel),which is clinically effective amineoplanic agent.TSXol is known to act by enhancing thePOlymeriZation of tubulin in the initiation andextension of microtubules, ac has been shown toinduce apoptosis in human and animal cellslll.Apoptosis, also known as programmed cell death, isthe active process of cell death which occurs duringdevelopment and in resPOnse tO enviboental cues ofa multicellular organism. In...

  6. Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

    Science.gov (United States)

    Cervera, Laura; Kamen, Amine A

    2018-01-01

    Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10(6) cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.

  7. Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L.; Vega-Sánchez, Miguel E.; Williams, Brian; Chiniquy, Dawn M.; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G.; Willats, William G. T.; Scheller, Henrik V.; Ronald, Pamela C.; Bartley, Laura E.

    2016-08-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

  8. 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

    improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...... 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...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after...

  9. 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.

  10. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert

    2011-01-01

    Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.

  11. A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems.

    Science.gov (United States)

    Valadez-Bustos, Ma Guadalupe; Aguado-Santacruz, Gerardo Armando; Tiessen-Favier, Axel; Robledo-Paz, Alejandrina; Muñoz-Orozco, Abel; Rascón-Cruz, Quintin; Santacruz-Varela, Amalio

    2016-04-01

    Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures.

  12. 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.

  13. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures.

    Science.gov (United States)

    Schillberg, Stefan; Raven, Nicole; Fischer, Rainer; Twyman, Richard M; Schiermeyer, Andreas

    2013-01-01

    Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

  14. 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

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon...... 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...

  15. Establishment of suspension cell culture of Gymnema sylvestre R.Br.- A threatened anti-diabetic plant

    Directory of Open Access Journals (Sweden)

    Karthic Raju

    2012-04-01

    Full Text Available A cell suspension culture was established from leaf explants of wild Gymnema sylvestre plants collected from Muniyankudisai,Tamilnadu, India. Murashige and Skoog medium supplemented with 9.0 μM l-1 of 2, 4- Dichlorophenoxy acetic acid and 2.1 μM l-1Benzyl adenine produced yellow friable callus with green patches.The cells were subcultured conscientiously twelve times to getconsistent growth of the cells in suspension. From the 10thsubculture onwards callus cells acclimatized to grow in suspensionwith aggregation and reached 168.6 g l-1 fw and 5.16 g l-1 dw of cellbiomass.

  16. Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension.

    Science.gov (United States)

    Kluge, Sabine; Benndorf, Dirk; Genzel, Yvonne; Scharfenberg, Klaus; Rapp, Erdmann; Reichl, Udo

    2015-08-20

    Adaptation of continuous cell lines to growth in suspension in a chemically defined medium has significant advantages for design and optimization in manufacturing of biologicals. In this work, changes in the protein expression level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth were analyzed. Therefore, three cell line adaptations were performed independently. Two adaptations were monitored closely to characterize short term changes in protein expression levels after serum deprivation. In addition, initial stages of suspension growth were analyzed for both adaptations. The third adaptation involved MDCK suspension cells (MDCKSUS2) grown over an extended time period to achieve robust growth characteristics. Here, cells of the final stage of adaptation were compared with their parental cell line (MDCKADH). A combination of two dimensional differential gel electrophoresis for relative protein quantification and tandem mass spectrometry for protein identification enabled insights into cellular physiology. The two closely monitored cell line adaptations followed different routes regarding specific changes in protein expression but resulted in similar proteome profiles at the initial stages of suspension growth analyzed. Compared to the MDCKADH cells more than 90% of all changes in the protein expression level were identified after serum deprivation and were related to cytoskeletal structure, genetic information processing and cellular metabolism. Myosin proteins, involved in cellular detachment by actin-myosin contractile mechanisms were also differentially expressed. Interestingly, for both of the two adaptations, proteins linked for tumorigenicity, like lactoylglutathione lyase and sulfotransferase 1A1 were differentially expressed. In contrast, none of these proteins were differentially expressed for the MDCKSUS2 cell line. Overall, proteomic monitoring allowed identification of key proteins involved in

  17. Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor.

    Science.gov (United States)

    McDonald, Karen A; Hong, Lo Ming; Trombly, David M; Xie, Qing; Jackman, Alan P

    2005-01-01

    Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.

  18. Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics.

    Science.gov (United States)

    Kauss, H.; Franke, R.; Krause, K.; Conrath, U.; Jeblick, W.; Grimmig, B.; Matern, U.

    1993-06-01

    The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic

  19. Lanthanum Prevents Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells by Controlling Early Induction Events

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In a previous study, a salt stress-induced programmed cell death (PCD) model was established in rice root tip cells. Here,by using Wuyunjing 8th rice seedlings, the effects of lanthanum on salt stress-induced PCD early events were studied. The peroxidase (APX). Imidazole (20 mmol/L), the inhibitor of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), could alleviate the occurrence of PCD obviously, and such alleviation could be enhanced by the addition of La3+,indicating the involvement of NADPH oxidase in the salt stress-induced PCD process. Taken together, lanthanum could prevent salt stress-induced PCD occurrence in the rice root tip cells by blocking the calcium influx under stress, which was followed by inhibiting calcium-dependent NADPH oxidase activity to prevent O2·-production and, enhancing the cytosolic antioxidative enzyme activities to scavenge the reactive oxygen species.

  20. Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening

    Science.gov (United States)

    Hilaire, E.; Young, S. A.; Willard, L. H.; McGee, J. D.; Sweat, T.; Chittoor, J. M.; Guikema, J. A.; Leach, J. E.

    2001-01-01

    The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

  1. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.

  2. Modulated differential photoacoustic cell to study the gelatinization in a starch-water suspension

    Directory of Open Access Journals (Sweden)

    J. A. Villada

    2014-06-01

    Full Text Available In this paper the design and implementation of a novel Differential Photoacoustic Cell (DPC system is presented. The system was used to study the thermo optic transition in water-starch suspension called gelatinization. The melting temperature of Gallium was used to calibrate the temperature of the system. Both temperature values for starch gelatinization and gallium melting were agreed with those obtained using differential scanning calorimetry (DSC. The results show that this system is suitable to study other thermal processes in food or any thermal transition at low temperature.

  3. Modulated differential photoacoustic cell to study the gelatinization in a starch-water suspension

    Science.gov (United States)

    Villada, J. A.; Herrera, W.; Espinosa-Arbeláez, D. G.; Mosquera, J. C.; Rodríguez-García, M. E.

    2014-06-01

    In this paper the design and implementation of a novel Differential Photoacoustic Cell (DPC) system is presented. The system was used to study the thermo optic transition in water-starch suspension called gelatinization. The melting temperature of Gallium was used to calibrate the temperature of the system. Both temperature values for starch gelatinization and gallium melting were agreed with those obtained using differential scanning calorimetry (DSC). The results show that this system is suitable to study other thermal processes in food or any thermal transition at low temperature.

  4. The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

    NARCIS (Netherlands)

    Schnettler, E.; Hemmes, J.C.; Goldbach, R.W.; Prins, M.W.

    2008-01-01

    The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is

  5. Developmental characteristics and aluminum resistance of root border cells in rice seedlings.

    Science.gov (United States)

    Cai, MiaoZhen; Zhang, ShuNa; Xing, ChengHua; Wang, FangMei; Ning, Wang; Lei, Zhu

    2011-05-01

    The developmental characteristics of root border cells (RBCs) and their role in protection of root apices of rice seedling from Al toxicity were evaluated in two rice (Oryza sativa L.) cultivars differing in Al tolerance. Root elongation and RBCs viability were used as indicators for Al effects. The formation of RBCs and the emergence of the root tip occurred almost simultaneously. Treatment of the root with Al inhibited root elongation and increased Al accumulation in the root tips. Physical removal of RBCs from root tips resulted in a more severe inhibition of root elongation and a higher Al accumulation in the root tips. These effects were more pronounced in the Al-sensitive rice cultivar (II You 6216) than that in the Al-tolerant rice cultivar (II You 838). The relative viability of attached and detached RBCs decreased with increasing Al concentrations. Al also induced a thicker mucilage layer surrounding attached RBCs of both cultivars, and detached RBCs did not. Maintaining the abundant live RBCs encapsulated root tip and enhancing their mucilage secretion, appear to be important in alleviating Al toxicity and in allowing exclusion of Al from the rice root apex.

  6. Solid oxide fuel cell electrolytes produced via very low pressure suspension plasma spray and electrophoretic deposition

    Science.gov (United States)

    Fleetwood, James D.

    Solid oxide fuel cells (SOFCs) are a promising element of comprehensive energy policies due to their direct mechanism for converting the oxidization of fuel, such as hydrogen, into electrical energy. Both very low pressure plasma spray and electrophoretic deposition allow working with high melting temperature SOFC suspension based feedstock on complex surfaces, such as in non-planar SOFC designs. Dense, thin electrolytes of ideal composition for SOFCs can be fabricated with each of these processes, while compositional control is achieved with dissolved dopant compounds that are incorporated into the coating during deposition. In the work reported, sub-micron 8 mole % Y2O3-ZrO2 (YSZ) and gadolinia-doped ceria (GDC), powders, including those in suspension with scandium-nitrate dopants, were deposited on NiO-YSZ anodes, via very low pressure suspension plasma spray (VLPSPS) at Sandia National Laboratories' Thermal Spray Research Laboratory and electrophoretic deposition (EPD) at Purdue University. Plasma spray was carried out in a chamber held at 320 - 1300 Pa, with the plasma composed of argon, hydrogen, and helium. EPD was characterized utilizing constant current deposition at 10 mm electrode separation, with deposits sintered from 1300 -- 1500 °C for 2 hours. The role of suspension constituents in EPD was analyzed based on a parametric study of powder loading, powder specific surface area, polyvinyl butyral (PVB) content, polyethyleneimine (PEI) content, and acetic acid content. Increasing PVB content and reduction of particle specific surface area were found to eliminate the formation of cracks when drying. PEI and acetic acid content were used to control suspension stability and the adhesion of deposits. Additionally, EPD was used to fabricate YSZ/GDC bilayer electrolyte systems. The resultant YSZ electrolytes were 2-27 microns thick and up to 97% dense. Electrolyte performance as part of a SOFC system with screen printed LSCF cathodes was evaluated with peak

  7. 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.

  8. Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    Zhang Dongyan; Yu Fang; Bai Fengwu; An Lijia

    2006-01-01

    The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension cell culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO-3/NH+4, and nitrate was favourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N source. The effect of total initial N on the cell cultures was also investigated with NO-3/NH+4 ratio of 1∶2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

  9. Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin.

    Science.gov (United States)

    Gilleta; Roisin; Fliniaux; Jacquin-Dubreuil; Barbotin; Nava-Saucedo

    2000-02-01

    Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.

  10. Suspension cell culture in microgravity and development of a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1987-01-01

    NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells.

  11. 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.

  12. [Correction of cronic liver failure by transplantation of liver cells suspension and cell-engineering designs (experimental investigation)].

    Science.gov (United States)

    Got'e, S V; Shagidulin, M Iu; Onishchenko, N A; Krasheninnikov, M E; Il'inskiĭ, I M; Mozheĭko, N P; Liundup, A V; Volkova, E A; Petrakov, K I; Avramov, P V; Perova, N V; Sevast'ianov, V I

    2013-01-01

    On an experimental model of chronic fibrotic liver damage (male rats Wistar (n-60), damage of CCl4, the duration of the experiment 90 days) it was studied the effectiveness of cell therapy for the correction of chronic liver failure. These rats were divided into 3 experimental groups: in the Ist-group (control, n=10) isotonic saline (650 mkl.) was injected; in the IInd-group (n=20) suspension of liver cells was applicated in a dose 8 - l0 x 10(6) cells; in the IIIrd-group (n=30) suspension of liver cells and bone marrow cells (mesenchymal stromal cells) in ratio 5:1 were used as cell associates on microparticles intjectable heterogeneous biopolymer hydrogel "SpheroGEL" (cell-engineering design) in common dose 8 - l0 x 10(6) It was ascertained that in the 2nd and in the 3rd groups the accelerated normalization of disturbed liver functional indices (ALT, AST, ALP) took place - to 30 days, but in the control group only to 90 days. The reliable differences in rats ofnormalization offunctional indices were absent between the IInd and the IIIrd groups. But in 90 days by using special histological dyeing it was found out that defibrotic processes in liver tissue were more expressed in the IIIrd group in comparison with the IIIrd group. Received results were consequence of prolonged vital activity of cells (liver cells and mesenchymal stromal bone marrow cells) into cell-engineering designs, which were transplanted in the IIIrd group. The obtained effect can be explained by that the developed cell-engineering designs provide adequate conditions for prolonged vital activity of the transplanted cells.

  13. Regeneration of transgenic rice plants from protoplasts following plasmid uptake

    Institute of Scientific and Technical Information of China (English)

    LiWenbin; SUNYongru

    1994-01-01

    Embryogenic cell suspension was obtained from the calli developed from mature seeds of riceRoncarolo (Oryza sativa L., a japonica cultivar from Italy ). The protoplasts were isolated from cell suspension by treatment of enzyme mixture and suspended in the solution containing 0.56%(w/v) MgCl2· 6H2O,0.10%(w/v) MES and 0.4 mol/L, pH 5.6 mannitol to a final density of 2×105/ml.

  14. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    Directory of Open Access Journals (Sweden)

    Kouki eYoshida

    2013-10-01

    Full Text Available Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs can regulate secondary wall formation in rice (Oryza sativa and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S has very low transcriptional activation ability, but the longer protein (OsSWN2L and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

  15. Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

    Science.gov (United States)

    Nagella, Praveen; Dandin, Vijayalaxmi S; Murthy, Hosakatte Niranjana

    2016-01-01

    Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described.

  16. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract.

    Science.gov (United States)

    Zhou, Liangyun; Yang, Jian; Yang, Guang; Kang, Chuanzhi; Xiao, Wenjuan; Lv, Chaogeng; Wang, Sheng; Tang, Jinfu; Guo, Lanping

    2016-09-14

    Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE) treatment. An ultra-performance liquid chromatography (UPLC) method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS) LC-MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2'-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 μg·g(-1)) at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-d-glucopyranoside. The content of 2'-Hydroxyaucuparin reached its highest (422.75 μg·g(-1)) at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

  17. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

    Directory of Open Access Journals (Sweden)

    Liangyun Zhou

    2016-09-01

    Full Text Available Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE treatment. An ultra-performance liquid chromatography (UPLC method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS LC−MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2′-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 μg·g−1 at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-d-glucopyranoside. The content of 2′-Hydroxyaucuparin reached its highest (422.75 μg·g−1 at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

  18. Ethanol production potential from fermented rice noodle wastewater treatment using entrapped yeast cell sequencing batch reactor

    Science.gov (United States)

    Siripattanakul-Ratpukdi, Sumana

    2012-03-01

    Fermented rice noodle production generates a large volume of starch-based wastewater. This study investigated the treatment of the fermented rice noodle wastewater using entrapped cell sequencing batch reactor (ECSBR) compared to traditional sequencing batch reactor (SBR). The yeast cells were applied because of their potential to convert reducing sugar in the wastewater to ethanol. In present study, preliminary treatment by acid hydrolysis was performed. A yeast culture, Saccharomyces cerevisiae, with calcium alginate cell entrapment was used. Optimum yeast cell loading in batch experiment and fermented rice noodle treatment performances using ECSBR and SBR systems were examined. In the first part, it was found that the cell loadings (0.6-2.7 × 108 cells/mL) did not play an important role in this study. Treatment reactions followed the second-order kinetics with the treatment efficiencies of 92-95%. In the second part, the result showed that ECSBR performed better than SBR in both treatment efficiency and system stability perspectives. ECSBR maintained glucose removal of 82.5 ± 10% for 5-cycle treatment while glucose removal by SBR declined from 96 to 40% within the 5-cycle treatment. Scanning electron microscopic images supported the treatment results. A number of yeast cells entrapped and attached onto the matrix grew in the entrapment matrix.

  19. Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Esteso, María J; Youssef, Sabry M; Casado-Vela, Juan; Vera-Urbina, Juan C; Sellés-Marchart, Susana; Bru-Martínez, Roque; Pedreño, María A

    2014-01-01

    Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

  20. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

    Science.gov (United States)

    Yakimova, E T; Kapchina-Toteva, V M; Laarhoven, L-J; Harren, F M; Woltering, E J

    2006-10-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.

  1. Suspension Culture Alters Insulin Secretion in Induced Human Umbilical Cord Matrix-Derived Mesenchymal Cells

    Directory of Open Access Journals (Sweden)

    Fatemeh Seyedi

    2016-04-01

    Full Text Available Objective: Worldwide, diabetes mellitus (DM is an ever-increasing metabolic disorder. A promising approach to the treatment of DM is the implantation of insulin producing cells (IPC that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells (hUCMs into IPCs and measured insulin production. Materials and Methods: In this experimental study, we exposed hUCMs cells to pancreatic medium and differentiated them into IPCs in monolayer and suspension cultures. Pancreatic medium consisted of serum-free Dulbecco’s modified eagle’s medium Nutrient mixture F12 (DMEM/F12 medium with 17.5 mM glucose supplemented by 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, and B-27 serum-free supplement. After differentiation, insulin content was analyzed by gene expression, immunocytochemistry (IHC and the chemiluminesence immunoassay (CLIA. Results: Reverse transcription-polymerase chain reaction (RT-PCR showed efficient expressions of NKX2.2, PDX1 and INSULIN genes in both groups. IHC analysis showed higher expression of insulin protein in the hanging drop group, and CLIA revealed a significant higher insulin production in hanging drops compared with the monolayer group following the glucose challenge test. Conclusion: We showed by this novel, simple technique that the suspension culture played an important role in differentiation of hUCMs into IPC. This culture was more efficient than the conventional culture method commonly used in IPC differentiation and cultivation.

  2. Gymnema sylvestre R. Br. suspension cell extract show antidiabetic potential in Alloxan induced diabetic albino male rats

    Institute of Scientific and Technical Information of China (English)

    R Karthic; S Nagaraj; P Arulmurugan; S Seshadri; R Rengasamy; K Kathiravan

    2012-01-01

    Objective: To study the antidiabetic effects of suspension cell extract of Gymnema sylvestre (G.sylvestre in vitro grown suspension cells of G. sylvestre along with field grown and wild plant leaves of G.sylvestre was tested on alloxan induced diabetic rats. Results: While oral administration of the extracts reduced the glucose content in blood and urine, sugar and lipids in serum significantly (P≤0.05), it also increased the body weight, total haemoglobin and plasma protein content.Conclusions:It can be concluded that G. sylvestre suspension cell extract show excellent) along with field grown and wild plants. Methods: The effect of ethanolic extracts of the antidiabetic potential against alloxan induced diabetic albino male rats therefore be considered as potent antidiabetic drug.

  3. More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments.

    Science.gov (United States)

    Rasche, Stefan; Herwartz, Denise; Schuster, Flora; Jablonka, Natalia; Weber, Andrea; Fischer, Rainer; Schillberg, Stefan

    2016-03-18

    Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from € 125 to € 45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization of plant cell suspension cultures.

  4. Cell wall changes involved in the automorphic curvature of rice coleoptiles under microgravity conditions in space.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Mori, Ryuji; Saiki, Mizue; Nakamura, Yukiko; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

    2004-12-01

    Seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) were cultivated on board the Space Shuttle STS-95 mission and changes in the morphology and the cell wall properties of coleoptiles were analyzed. In space, rice coleoptiles showed a spontaneous (automorphic) curvature toward the caryopsis in the elongating region. The angle of automorphic curvature was larger in Koshihikari than in a gibberellin-deficient dwarf cultivar, Tan-ginbozu, and the angle gradually decreased during the growth of coleoptiles in both cultivars. The more quickly expanding convex side of the bending region of the rice coleoptiles showed a greater extensibility of the cell wall than the opposite side. There was a significant correlation between the angle of curvature and the difference in the cell wall extensibility between the convex and the concave sides. Both the levels of the cell wall polysaccharides per unit length of coleoptile and the ratio of high-molecular-mass polysaccharides in the hemicellulose fraction were lower in the convex side than the concave one. Also, the activity of (1-->3),(1-->4)-beta-glucanases in the cell wall was higher in the convex side than the concave one. These results suggest that the uneven modifications of cell wall metabolism bring about the difference in the levels and the molecular size of the cell wall polysaccharides, thereby causing the difference in capacity of the cell wall to expand between the dorsal and the ventral sides, leading to the automorphic curvature of rice coleoptiles in space. The data also suggest the involvement of gibberellins in inducing the automorphic curvature under microgravity conditions.

  5. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells.

    Science.gov (United States)

    de, JongAnkeJ; Yakimova, Elena T; Kapchina, Veneta M; Woltering, Ernst J

    2002-02-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.

  6. Rice caryopsis development I:Dynamic changes in different cell layers

    Institute of Scientific and Technical Information of China (English)

    Xiaoba Wu; Jinxin Liu; Dongqi Li; Chun-Ming Liu

    2016-01-01

    Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a system-atic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30-day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination (DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.

  7. Rice Brittleness Mutants: A Way to Open the 'Black Box' of Monocot Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Baocai Zhang; Yihua Zhou

    2011-01-01

    Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae.Mechanical strength is an important agronomy trait of rice(Oryza sativa L.)plants that affects crop lodging and grain yield.As a prominent physical property of cell walls,mechanical strength reflects upon the structure of different wall polymers and how they interact.Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling.Our group focuses on the study of isolation of brittle culm(bc)mutants and characterization of their corresponding genes.To date,several bc mutants have been reported.The identified genes have covered several pathways of cell wall biosynthesis,revealing many secrets of monocot cell wall biosynthesis.Here,we review the progress achieved in this research field and also highlight the perspectives in expectancy.All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.

  8. Defense gene expression in elicitor-treated cell suspension cultures of french bean cv. Imuna.

    Science.gov (United States)

    Ellis, J S; Jennings, A C; Edwards, L A; Mavandad, M; Lamb, C J; Dixon, R A

    1989-12-01

    Cell suspension cultures of bean (Phaseolus vulgaris) cv. Imuna accumulated isoflavonoid phytoalexins on exposure to elicitor from the phytopathogenic fungus Colletotrichum lindemuthianum (CL). This was preceeded by rapid increases in the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). However, the patterns of expression of PAL and CHS genes differed from those observed in cultures of a previously studied bean cultivar. The relative levels of transcripts from individual members of the CHS multigene family differed significantly at 1.5 h compared to 22.5 h after elicitation. More strikingly, three PAL genes were expressed in cultivar Imuna in response to fungal elicitor, whereas two are expressed in elicitor-treated cell cultures of cultivar Canadian Wonder.

  9. The effect of CuO NPs on reactive oxygen species and cell cycle gene expression in roots of rice.

    Science.gov (United States)

    Wang, Shuling; Liu, Hanzhu; Zhang, Yuxi; Xin, Hua

    2015-03-01

    To evaluate the effect of CuO nanoparticles (NPs) on root growth, root reactive oxygen species (ROS) production, and the expression of 2 genes (OsCDC2 and OsCYCD) associated with root growth of Oryza sativa (rice), rice roots were treated with 5 mg/L CuO NP suspension, 5 mg/L CuO bulk particle suspension, and 0.27 mg/L CuSO4  · 5H2 O solution, with distilled water as control. The results indicated that CuO NPs and Cu(2+) severely inhibited the elongation and biomass of rice roots after 72-h exposure. Dyeing with 7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) showed that in all 3 treatment groups, the fluorescence was primarily located in the meristem zone, demonstrating that the meristem zone was where ROS were primarily generated. In addition, a significant increase in ROS was detected in the meristem zone of roots treated with the CuO NP suspension and the CuSO4  · 5H2 O solution, both of which greatly influenced the expression level of OsCDC2 and OsCYCD. The impact of Cu(2+) on these 2 genes was smaller than that of CuO NPs. The Cu content in roots of rice after treatment with CuO NPs was much higher than that found after the other treatments, which indicated that CuO NPs may have been absorbed into root tissue. Collectively, these data suggest that growth inhibition, higher ROS production, and gene expression inhibition may be caused not only by the ions themselves, but also the NPs. © 2014 SETAC.

  10. Ethylene signaling in salt stress- and salicylic acid-induced programmed cell death in tomato suspension cells.

    Science.gov (United States)

    Poór, Péter; Kovács, Judit; Szopkó, Dóra; Tari, Irma

    2013-02-01

    Salt stress- and salicylic acid (SA)-induced cell death can be activated by various signaling pathways including ethylene (ET) signaling in intact tomato plants. In tomato suspension cultures, a treatment with 250 mM NaCl increased the production of reactive oxygen species (ROS), nitric oxide (NO), and ET. The 10(-3) M SA-induced cell death was also accompanied by ROS and NO production, but ET emanation, the most characteristic difference between the two cell death programs, did not change. ET synthesis was enhanced by addition of ET precursor 1-aminocyclopropane-1-carboxylic acid, which, after 2 h, increased the ROS production in the case of both stressors and accelerated cell death under salt stress. However, it did not change the viability and NO levels in SA-treated samples. The effect of ET induced by salt stress could be blocked with silver thiosulfate (STS), an inhibitor of ET action. STS reduced the death of cells which is in accordance with the decrease in ROS production of cells exposed to high salinity. Unexpectedly, application of STS together with SA resulted in increasing ROS and reduced NO accumulation which led to a faster cell death. NaCl- and SA-induced cell death was blocked by Ca(2+) chelator EGTA and calmodulin inhibitor W-7, or with the inhibitors of ROS. The inhibitor of MAPKs, PD98059, and the cysteine protease inhibitor E-64 reduced cell death in both cases. These results show that NaCl induces cell death mainly by ET-induced ROS production, but ROS generated by SA was not controlled by ET in tomato cell suspension.

  11. Trueness-to-type and agronomic characteristics of Coffea arabica trees micropropagated by the embryogenic cell suspension technique.

    Science.gov (United States)

    Etienne, H; Bertrand, B

    2001-09-01

    Trueness-to-type and agronomic characteristics of trees of four coffee (Coffea arabica L.) F(1) hybrid clones derived from embryogenic cell suspensions were compared with those of trees produced from in vitro microcuttings. Three types of variants were observed among the 644 trees derived from embryogenic suspensions. Total frequency of the variants was 2.1% for trees originating from embryogenic cell suspensions, whereas no variant was found among the trees produced from microcuttings. The variant known as "thick leaf" had thick leaves, many abnormally starry flowers and low yields of large fruit. The "dwarf" variant was characterized by slow growth and small fruit. The "dwarf peaberry" variant had abnormal seeds in a single cavity, in addition to the "thick leaf" and "dwarf" characteristics. Compared with normal trees, the variants differed in leaf density and number of chloroplasts per guard cell. The variants aside, there were no differences in the main agronomic characteristics between trees produced from embryogenic suspensions and those produced from microcuttings. For all four clones, the trees had vegetative characteristics, productivity, fertility, and bean biochemical, mineral and organoleptic characteristics that were identical to those of the controls. We conclude that it is possible to generate coffee trees commercially with normal agronomic performance from embryogenic suspensions, because the frequency with which somaclonal variants occur is limited.

  12. A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells.

    Science.gov (United States)

    Yokoyama, T; Kobayashi, N; Kouchi, H; Minamisawa, K; Kaku, H; Tsuchiya, K

    2000-04-01

    Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.

  13. Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane.

    Science.gov (United States)

    Cesarino, Igor; Araújo, Pedro; Paes Leme, Adriana Franco; Creste, Silvana; Mazzafera, Paulo

    2013-01-01

    Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.

  14. Influences of Plant Growth Regulators,Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

    Institute of Scientific and Technical Information of China (English)

    TangWei; WuJiongyuan; 等

    1995-01-01

    A cell suspension culture of Panax ginseng which may be continuously subcultured has been established.Embryogenic callus derived from clutured young leaves was used to initiate the culture,Plant growth regulators,basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development ,The best selection of plant growth regulator,basal medium and carbohydrate level is 2mg/L 2,4-D:0.5mg/L KT,MS and 3% sucrose respectively.

  15. Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.).

    Science.gov (United States)

    Oung, Hui-Min; Lin, Ke-Chun; Wu, Tsung-Meng; Chandrika, Nulu Naga Prafulla; Hong, Chwan-Yang

    2015-12-01

    The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O2(·-), H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.

  16. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    OpenAIRE

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A; Minoru S.H. Ko; Motohashi, Tsutomu; KUNISADA, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the...

  17. Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF.

    Science.gov (United States)

    Luo, Junling; Ning, Tingting; Sun, Yunfang; Zhu, Jinghua; Zhu, Yingguo; Lin, Qishan; Yang, Daichang

    2009-02-01

    The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.

  18. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  19. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Flores-Sánchez, Isvett J; Ortega-López, Jaime; del Carmen Montes-Horcasitas, María; Ramos-Valdivia, Ana C

    2002-12-01

    Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

  20. Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension.

    Science.gov (United States)

    Chafai, Djamel Eddine; Mehle, Andraž; Tilmatine, Amar; Maouche, Bachir; Miklavčič, Damijan

    2015-12-01

    Electroporation of cells is successfully used in biology, biotechnology and medicine. Practical problems still arise in the electroporation of cells in suspension. For example, the determination of cell electroporation is still a demanding and time-consuming task. Electric pulses also cause contamination of the solution by the metal released from the electrodes and create local enhancements of the electric field, leading to the occurrence of electrochemical reactions at the electrode/electrolyte interface. In our study, we investigated the possibility of assessing modifications to the cell environment caused by pulsed electric fields using electrochemical impedance spectroscopy. We designed an experimental protocol to elucidate the mechanism by which a pulsed electric field affects the electrode state in relation to different electrolyte conductivities at the interface. The results show that a pulsed electric field affects electrodes and its degree depends on the electrolyte conductivity. Evolution of the electrochemical reaction rate depends on the initial free charges and those generated by the pulsed electric field. In the presence of biological cells, the initial free charges in the medium are reduced. The electrical current path at low frequency is longer, i.e., conductivity is decreased, even in the presence of increased permeability of the cell membrane created by the pulsed electric field.

  1. Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Elke Gabriel

    Full Text Available Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in

  2. Simultaneous reduction of nitrate and selenate by cell suspensions of selenium-respiring bacteria

    Science.gov (United States)

    Oremland, R.S.; Blum, J.S.; Bindi, A.B.; Dowdle, P.R.; Herbel, M.; Stolz, J.F.

    1999-01-01

    Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate- grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high- affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with 75Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.

  3. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    Science.gov (United States)

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  4. Microbial electricity generation in rice paddy fields: recent advances and perspectives in rhizosphere microbial fuel cells.

    Science.gov (United States)

    Kouzuma, Atsushi; Kaku, Nobuo; Watanabe, Kazuya

    2014-12-01

    Microbial fuel cells (MFCs) are devices that use living microbes for the conversion of organic matter into electricity. MFC systems can be applied to the generation of electricity at water/sediment interfaces in the environment, such as bay areas, wetlands, and rice paddy fields. Using these systems, electricity generation in paddy fields as high as ∼80 mW m(-2) (based on the projected anode area) has been demonstrated, and evidence suggests that rhizosphere microbes preferentially utilize organic exudates from rice roots for generating electricity. Phylogenetic and metagenomic analyses have been conducted to identify the microbial species and catabolic pathways that are involved in the conversion of root exudates into electricity, suggesting the importance of syntrophic interactions. In parallel, pot cultures of rice and other aquatic plants have been used for rhizosphere MFC experiments under controlled laboratory conditions. The findings from these studies have demonstrated the potential of electricity generation for mitigating methane emission from the rhizosphere. Notably, however, the presence of large amounts of organics in the rhizosphere drastically reduces the effect of electricity generation on methane production. Further studies are necessary to evaluate the potential of these systems for mitigating methane emission from rice paddy fields. We suggest that paddy-field MFCs represent a promising approach for harvesting latent energy of the natural world.

  5. Auxin and Cell Wall Invertase Related Signaling during Rice Grain Development

    Directory of Open Access Journals (Sweden)

    Sarah Russell French

    2014-02-01

    Full Text Available Indole-3-acetic acid (IAA synthesis is required for grain-fill in maize and appears to be regulated by cell-wall invertase (CWIN activity. OsYUC12 is one of three IAA biosynthesis genes we previously reported as expressed during early rice grain development, correlating with a large increase in IAA content of the grain. This work aimed to investigate further the role of OsYUC12 and its relationship to CWIN activity and invertase inhibitors (INVINH. The analysis shows a brief peak of OsYUC12 expression early in endosperm development. Meta-analysis of microarray data, confirmed by quantitative expression analysis, revealed that OsYUC12 is coexpressed with OsIAA29, which encodes an unusual AUX/IAA transcription factor previously reported as poorly expressed. Maximum expression of OsYUC12 and OsIAA29 coincided with maximum CWIN activity, but also with a peak in INVINH expression. Unlike ZmYUC1, OsYUC12 expression is not reduced in the rice CWIN mutant, gif1. Several reports have investigated CWIN expression in rice grains but none has reported on expression of INVINH in this species. We show that rice has 54 genes encoding putative invertase/pectin methylesterase inhibitors, seven of which are expressed exclusively during grain development. Our results suggest a more complex relationship between IAA, CWIN, and INVINH than previously proposed.

  6. Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells.

    Science.gov (United States)

    Stevenson, T T; McNeil, M; Darvill, A G; Albersheim, P

    1986-04-01

    The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.

  7. Effects of oligosaccharides from endophytic Fusarium oxysporum Dzf17 on activities of defense-related enzymes in Dioscorea zingiberensis suspension cell and seedling cultures

    Directory of Open Access Journals (Sweden)

    Peiqin Li

    2014-07-01

    Conclusions: Both EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.

  8. 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

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon......-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity...... 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...

  9. Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

    Science.gov (United States)

    Ledoux, Quentin; Van Cutsem, Pierre; Markό, Istvan E; Veys, Pascal

    2014-01-01

    H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

  10. Apoptosis Induced by High Concentrations of Nicotinamide in Tobacco Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    张贵友; 朱瑞宇; 戴尧仁

    2004-01-01

    As an inhibitor of poly(ADP-ribose) polymerase (PARP), nicotinamide has a restraining effect on apoptosis at certain low concentrations. In our present study, apoptosis induced by high concentrations of nicotinamide was observed in tobacco suspension cells. When cells were preincubated with 250 mmol/L nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180-200 bp, the condensation and peripheral distribution of nuclear chromatin, and a positive reaction to the TUNEL assay. At the same time, the degradation of PARP and the reduction in the potential of the inner membrane of mitochondria appeared in apoptotic cells induced by high concentrations of nicotinamide. This result indicates that apoptosis induced by high concentrations of nicotinamide is associated with caspase-3-like activity and with the opening of mitochondrial permeability pores. These results partially support the hypothesis that high concentrations of PARP inhibitor could force cells to enter an apoptotic pathway by delay of DNA repair in replicating cells.

  11. Serum-free spheroid suspension culture maintains high proliferation and differentiation potentials of mesenchymal stem cells

    Science.gov (United States)

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

    2016-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn’s disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this 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. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serum-containing adherent cultures. PMID:24616445

  12. Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology.

    Science.gov (United States)

    Lu, Chi-Hua; Engelmann, Nancy J; Lila, Mary Ann; Erdman, John W

    2008-09-10

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

  13. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  14. 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 (suspension culture experiments was lower than control. Increasing the elicitor concentrations lead to reduction in cell survival. In conclusion it is possible to produce linalool as a secondary metabolite and pharmaceutical agent in cell culture of cumin. It is necessary to determine the best combination of medium and elicitor.

  15. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

    Science.gov (United States)

    Onofre, J.; Baert, Y.; Faes, K.; Goossens, E.

    2016-01-01

    BACKGROUND Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23

  16. In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

    Indian Academy of Sciences (India)

    S Sujanya; B Poornasri Devi; Isha Sai

    2008-03-01

    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, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate:ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

  17. Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction.

    Science.gov (United States)

    Lee, Jae Seong; Lee, Gyun Min

    2012-12-01

    Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti-cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody-producing recombinant CHO (rCHO), in a serum-free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody-producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK-506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR-dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin-1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro-autophagy engineering in CHO cell cultures.

  18. 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.

  19. Effect of 211At treating pollen and stigma on generative cells and seed setting of rice

    Institute of Scientific and Technical Information of China (English)

    JinJian-Nan; ChenFang; 等

    1998-01-01

    Low specific radioactivity (7.4kBq/ml) 211At treating pollen and stigma can obviously affect morphological structures and physiological functions of pollen,stigma and ovule or embryo sac cells,and cause injury.Results showed that because of the radiation effects the seed setting rate of rice was decreased,and the development of some embryos were affected and others became abnormal.

  20. Metabolic cycles in primary metabolism of cell suspensions of Daucus carota L. analysed by C-NMR

    NARCIS (Netherlands)

    Krook, J.

    1999-01-01

    In the work described in this thesis, uptake and conversion of sugar by cells of batch-grown suspensions of Daucus carota L. were studied. Invasive techniques (measurements of enzyme activities and sugar and starch levels) and non-invasive techniques ( 13C-NMR) were used to

  1. PHIV-RootCell: a supervised image analysis tool for rice root anatomical parameter quantification

    Directory of Open Access Journals (Sweden)

    Marc eLartaud

    2015-01-01

    Full Text Available We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex and central metaxylem vessels, number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt-stress responses of root anatomical parameters in rice (Oryza sativa L.. Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-old stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex but a decrease in external (peripheral tissues (sclerenchyma, exodermis and epidermis. Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.

  2. Positional variation of antipodal cells in polyembryonic rice ApⅢ before and after fertilization

    Institute of Scientific and Technical Information of China (English)

    YUAN Liangchen; CHEN Yongzhe; MU Xijin; LIN Jinxing

    2003-01-01

    The variation in the position of antipodal cells before and after fertilization in polyembryonic rice strain ApⅢ was studied by using conventional sectioning technique. It was shown that initially the three antipodal cells lie at the chalazal end of the young embryo sacs. Along with the development of embryo sac, the antipodal cells proliferate into a multicellular structure containing up to about 20 cells. Most of the cells migrate to its dorsal side when the embryo sacs turn into mature. In a few embryo sacs the antipodal cells are situated in the positions adjacent to the egg apparatus at the micropylar end, while in other sacs, they spread from the midst of the embryo sac to the micropylar end clinging to the nucellar tissue. Furthermore, it is reported for the first time that cells of some egg apparatus or proembryos disorganize when antipodal cells lie at the micropylar end, indicating that the function of the antipodal cells may vary during the embryogenesis in rice ApⅢ.

  3. Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures.

    Directory of Open Access Journals (Sweden)

    Jincheng Wu

    Full Text Available Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC and human ESC (hESC clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

  4. A Study of Noncultured Extracted Hair Follicle Outer Root Sheath Cell Suspension for Transplantation in Vitiligo

    Science.gov (United States)

    Shah, Aarti N; Marfatia, Ritu K; Saikia, Siddhartha S

    2016-01-01

    Context: Vitiligo surgeries have come a long way from tissue grafts to cultured and non cultured cell transplantation. Extracted hair follicle outer root sheath cell transplantation (EHF ORS) suspension is more enriched with melanocyte. In a hair bulb, there is one melanocyte for every five keratinocytes which is much higher than the epidermal melanin unit. Aims: To analyse the effectiveness of cultured EHF ORS and to perform objective evaluation based on clinical improvement & photographic evidence. To observe any untoward events or side effects. Settings and Design: The study was open and uncontrolled. All the patients were screened at preliminary visit. Reviews were done every two weeks. The endpoint selected was six months post procedure. Materials and Methods: Twenty five patients of stable Vitiligo were included in the study and follicular unit were harvested by Follicular Unit Extraction method. Outer root sheath cells were extracted by trypsinization. The solution was transplanted over dermabraded recipient site. Pressure dressing was given. Patients were followed up regularly. Statistical Analysis Used: Descriptive Statistics, Chi-Square. Results: Mean ± SD repigmentation was 80.15% ± 22.9% with excellent repigmentation (90-100%) in 60% of patients. Conclusions: This method is safe, effective, and simpler than the other methods involving cell culturing and requiring a laboratory set-up but selection of patients is crucial for the success of the outcome. PMID:27601859

  5. Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

    Directory of Open Access Journals (Sweden)

    Nikolay Vasilev

    Full Text Available A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

  6. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    Science.gov (United States)

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

  7. Location of the Protein of RSG6,a Predominantly Expressed Gene in Rice Sperm Cells

    Institute of Scientific and Technical Information of China (English)

    Lan Li-qiong; Miao chen; Zeng yu; Wang Sheng-hua; Xu ying; Tang Lin; BAI Yu; Chen Fang

    2004-01-01

    Using Western blot and immunohistochemistry analysis, here the localization of RSG6 protein was determined in various tissues of rice. Western blot showed only a weak signal in mature pollen. Nevertheless, according to the result of immunohistochemistry with DAB and fluorescent staining, the expression of RSG6 protein appeared to begin at the bicellular microspore stage, and then keep activity in the sperm cells of mature pollens. The fluorescence pattern showed RSG6 polypeptide was present close to or attached to the surface of the isolated sperm cells. And this suggested that RSG6 might take an important part in the process of recognition of sperm cell and ovum.

  8. Establishment of Suspension Cell Culture from Agrobacterium-transformed Hairy Root Cells of Psammosilene tunicoides, an Endangered and Rare Medicinal Plant of China

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

    Full Text Available Psammosilene tunicoides is an important medicinal plant endemic in China. Its annual yield is severely limited due to slow growth, poor seed germination and excessive collection. To satisfy the growing market demands, it’s necessary to seek alternatives to field cultivation and wild resources of this endangered plant. Using Agrobacterium -transformed hairy roots as initial explants, here, we reported the development of a suspension cell culture system for P. tunicoides. Results showed the Agrobacterium -transformed hairy roots-derived suspension cells are fast in growth and strong in capacity for accumulation of bioactive metabolites. We established that 1/2MS was a suitable medium for culturing the hairy root-derived suspension cells and the optimal combination of phytohormones is 1.5 mg/L 2, 4-D+0.5 mg/L 6-BA+0.25 mg/L NAA+0.1 mg/L KT. Under this condition, the maximal biomass was achieved at the 20th day of culture with an average growth rate of 0.72 g/L/d; and the intracellular saponine content reached 0.92%, comparable to that of mother hairy roots. Compared with the normal P. tunicoides suspension cells, the hairy roots-derived suspension cells exhibited features of fast growth, short culture period and high concentration of saponines, suggesting that the large scale culture of hairy root-derived cells could be a feasible alternative to the wild resources of P. tunicoides.

  9. Production of taxoids in callus and cell suspensions of Taxus chinensis by using abiotic elicitors

    Directory of Open Access Journals (Sweden)

    Naivy Pérez

    2004-07-01

    Full Text Available Until now, several strategies have been developed to improve the production of secondary metabolites using plant cell cultures, manipulating the parameters of the environment, selecting high yielding cell clones and the use of elicitors. In the present work, callus and cell suspensions of Taxus chinensis lines SN and E15 were used, important specie for the production of metabolites with anticancer activity. The effect of different concentrations of the silver nitrate (1.0; 4.0 and 8.0mg.l-1 and the conditions of total dark and 12 h artificial light about the production of biomass and the taxoids accumulation were studied. The concentrations of AgNO3 used didn’t cause same effect under the different conditions of illumination to those that the cultivations were exposed. The AgNO3 increased the fresh weight under conditions of darkness in which superior values were obtained at 6.0g in all the treatments being bigger when using the concentration of 8.0mg (7.39g. In relation to the taxoids accumulation a bigger influence was observed with 4.0mg under conditions of light (0.93 mg/ gDW, treatment where smaller increment of biomass took place. Cell suspensions in both lines showed better growth that the cultivation of callus, while the biggest accumulation in Taxuyunnanine C was in the cultivation of callus in the line SN. It was observed that the cellular lines had a different behavior in conditions of light and darkness. As for the increment of the biomass the line E15 behaved from a superior way to the line SN as much in the darkness as to the light although the biggest value of fresh weight was obtained in the darkness (7.89g and the biggest accumulation in Taxuyunnanine C was under conditions of darkness with the line SN (0.98 mg/gDW. Keywords: cell cultures, elicitation, secondary metabolites, taxuyunnanine C Abbreviations: gDW - Grams of dry weight, gFW - grams of fresh weight, AgNO3 - Silver nitrate, Tc - Taxuyunnanine C

  10. Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.

  11. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  12. Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth.

    Science.gov (United States)

    Huang, N; Bethell, D; Card, C; Cornish, J; Marchbank, T; Wyatt, D; Mabery, K; Playford, R

    2008-01-01

    Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [(3)H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.

  13. High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells.

    Science.gov (United States)

    Genzel, Yvonne; Vogel, Thomas; Buck, Johannes; Behrendt, Ilona; Ramirez, Daniel Vazquez; Schiedner, Gudrun; Jordan, Ingo; Reichl, Udo

    2014-05-19

    High cell densities in animal cell culture can be obtained by continuous perfusion of fresh culture medium across hollow fiber membranes that retain the cells. Careful selection of the membrane type and cut-off allows to control accumulation of target molecules and removal of low molecular weight compounds. In this report, perfusion with the scalable ATF (alternating tangential filtration, Refine Technology) system was evaluated for two suspension cell lines, the avian cell line AGE1.CR and the human cell line CAP. Both were cultivated in chemically defined media optimized for batch cell growth in a 1L stirred tank bioreactor connected to the smallest ATF unit (ATF2) and infected with cell line-adapted human influenza A virus (A/PR/8/34 (H1N1), typical diameter: 80-100 nm). At concentrations of about 25 million cells/mL three different membrane cut-offs (50 kDa, 0.2 μm and 0.5 μm) were tested and compared to batch cultivations performed at 5 million cells/mL. For medium and large cut-offs no cell-density effect could be observed with cell-specific virus yields of 1428-1708 virions/AGE1.CR cell (infected with moi 0.001) and 1883-4086 virions/CAP cell (moi of 0.025) compared to 1292 virions/AGE1.CR cell and 3883 virions/CAP cell in batch cultures. Even at a concentration of 48 million AGE1.CR cells/mL (cut-off: 0.2 μm) a cell-specific yield of 1266 virions/cell was reached. Only for the small cut-off (50 kDa) used with AGE1.CR cells a decrease in cell-specific yield was measured with 518 virions/cell. Surprisingly, the ratio of infectious to total virions seemed to be increased in ATF compared to batch cultures. AGE1.CR cell-derived virus particles were present in the permeate (0.2 and 0.5 μm cut-off), whereas CAP cell-derived virions were not, suggesting possible differences in morphology, aggregation or membrane properties of the virions released by the two cell lines. To our knowledge, this is the first study that illustrates the potential of ATF

  14. 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-21SUS) 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-21SUS 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

  15. Investigation of Heavy Metal Hyperaccumulation at the Cellular Level: Development and Characterization of Thlaspi caerulescens Suspension Cell Lines1[OA

    Science.gov (United States)

    Klein, Melinda A.; Sekimoto, Hitoshi; Milner, Matthew J.; Kochian, Leon V.

    2008-01-01

    The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens. PMID:18550685

  16. Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines.

    Science.gov (United States)

    Klein, Melinda A; Sekimoto, Hitoshi; Milner, Matthew J; Kochian, Leon V

    2008-08-01

    The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.

  17. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures.

    Science.gov (United States)

    Dixon, R A; Gerrish, C; Lamb, C J; Robbins, M P

    1983-12-01

    Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with (2)H from (2)H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.

  19. Development of a low capital investment reactor system: application for plant cell suspension culture

    Science.gov (United States)

    Hsiao; Bacani; Carvalho; Curtis

    1999-01-01

    Growth of plant cell cultures is demonstrated in an uncontrolled, simple, and inexpensive plastic-lined vessel. Sustained specific growth rates of 0.22 day-1 for Hyoscyamus muticus cell suspension cultures are achieved in a low-cost gas-sparged bioreactor configuration (6.5 L working volume, wv) which is comparable to an "optimized" 5 L wv mechanically agitated fermentor. In an effort to reduce bioreactor costs, the need for an autoclavable vessel was eliminated. Sterilization is achieved by separate autoclaving of the plastic liner and by gas-phase sterilization using ethylene oxide. The initial run sterilized with ethylene oxide displayed a long lag, apparently due to residual sterilant gas. Because ethylene oxide could eliminate costs associated with autoclave rated vessels, a quantitative basis for aeration time was developed by experimental measurements and modeling of diffusion in the polymer liner. Operational techniques to eliminate toxicity are implemented to grow 0.2 kg dry weight of plant cells in 13 days in a 40 L (28.5 L wv) air-lift bioreactor without autoclave sterilization. The biomass yields for all reactors were statistically indistinguishable from shake flask culture.

  20. Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation.

    Science.gov (United States)

    Ben Amar, A; Cobanov, P; Boonrod, K; Krczal, G; Bouzid, S; Ghorbel, A; Reustle, G M

    2007-09-01

    An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.

  1. Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.

    Science.gov (United States)

    Luna-Palencia, Gabriela R; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

  2. Nitric oxide consumption through lipid peroxidation in brain cell suspensions and homogenates.

    Science.gov (United States)

    Keynes, Robert G; Griffiths, Charmaine H; Hall, Catherine; Garthwaite, John

    2005-05-01

    Mechanisms which inactivate NO (nitric oxide) are probably important in governing the physiological and pathological effects of this ubiquitous signalling molecule. Cells isolated from the cerebellum, a brain region rich in the NO signalling pathway, consume NO avidly. This property was preserved in brain homogenates and required both particulate and supernatant fractions. A purified fraction of the particulate component was rich in phospholipids, and NO consumption was inhibited by procedures that inhibited lipid peroxidation, namely a transition metal chelator, the vitamin E analogue Trolox and ascorbate oxidase. The requirement for the supernatant was accounted for by its content of ascorbate which catalyses metal-dependent lipid peroxidation. The NO-degrading activity of the homogenate was mimicked by a representative mixture of brain lipids together with ascorbate and, under these conditions, the lipids underwent peroxidation. In a suspension of cerebellar cells, there was a continuous low level of lipid peroxidation, and consumption of NO by the cells was decreased by approx. 50% by lipid-peroxidation inhibitors. Lipid peroxidation was also abolished when NO was supplied at a continuously low rate (approximately 100 nM/min), which explains why NO consumption by this process is saturable. Part of the activity remaining after the inhibition of lipid peroxidation was accounted for by contaminating red blood cells, but there was also another component whose activity was greatly enhanced when the cells were maintained under air-equilibrated conditions. A similar NO-consuming process was present in cerebellar glial cells grown in tissue culture but not in blood platelets or leucocytes, suggesting a specialized mechanism.

  3. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    Science.gov (United States)

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+).

  4. 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.

  5. Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

    Science.gov (United States)

    Yano, Kenji; Aya, Koichiro; Hirano, Ko; Ordonio, Reynante Lacsamana; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

    2015-02-01

    Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.

  6. 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; Grivel, Jean-Charles

    2009-12-01

    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.

  7. 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.

  8. Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

    Science.gov (United States)

    Zhou, Wenqi; Wang, Yuchuan; Wu, Zhongliang; Luo, Liang; Liu, Ping; Yan, Longfeng; Hou, Suiwen

    2016-07-01

    Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.

  9. Developmental characteristics and response to iron toxicity of root border cells in rice seedlings.

    Science.gov (United States)

    Xing, Cheng-hua; Zhu, Mei-hong; Cai, Miao-zhen; Liu, Peng; Xu, Gen-di; Wu, Shao-hui

    2008-03-01

    To investigate the Fe2+ effects on root tips in rice plant, experiments were carried out using border cells in vitro. The border cells were pre-planted in aeroponic culture and detached from root tips. Most border cells have a long elliptical shape. The number and the viability of border cells in situ reached the maxima of 1600 and 97.5%, respectively, at 20-25 mm root length. This mortality was more pronounced at the first 1-12 h exposure to 250 mg/L Fe2+ than at the last 12-36 h. After 36 h, the cell viability exposed to 250 mg/L Fe2+ decreased to nought, whereas it was 46.5% at 0 mg/L Fe2+. Increased Fe2+ dosage stimulated the death of detached border cells from rice cultivars. After 4 h Fe2+ treatment, the cell viabilities were > or =80% at 0 and 50 mg/L Fe2+ treatment and were border cells decreased by 10% when the Fe2+ concentration increased by 100 mg/L. After 24 h Fe2+ treatment, the viabilities of border cells at all the Fe2+ levels were border cells decreased by 20% when the Fe2+ concentration increased by 100 mg/L. The decreased viabilities of border cells indicated that Fe2+ dosage and treatment time would cause deadly effect on the border cells. The increased cell death could protect the root tips from toxic harm. Therefore, it may protect root from the damage caused by harmful iron toxicity.

  10. Developmental characteristics and response to iron toxicity of root border cells in rice seedlings

    Institute of Scientific and Technical Information of China (English)

    Cheng-hua XING; Mei-hong ZHU; Miao-zhen CAI; Peng LIU; Gen-di XU; Shao-hui WU

    2008-01-01

    To investigate the Fe2+ effects on root tips in rice plant, experiments were carded out using border cells in vitro. The border cells were pre-planted in aeroponic culture and detached from root tips. Most border cells have a long elliptical shape. The number and the viability of border cells in situ reached the maxima of 1600 and 97.5%, respectively, at 20~25 mm root length. This mortality was more pronounced at the first 1~12 h exposure to 250 mg/L Fe2+ than at the last 12~36 h. After 36 h, the cell viability exposed to 250 mg/L Fe2+ decreased to nought, whereas it was 46.5% at 0 mg/L Fe2+. Increased Fe2+ dosage stimulated the death of detached border cells from rice cultivars. After 4 h Fe2+ treatment, the cell viabilities were≥80% at 0 and 50 mg/L Fe2+ treatment and were <62% at 150, 250 and 350 mg/L Fe2+ treatment; The viability of border cells decreased by 10% when the Fe2+ concentration increased by 100 mg/L. After 24 h Fe2+ treatment, the viabilities of border cells at all the Fe2+ levels were <65%; The viability of border cells decreased by 20% when the Fe2+ concentration increased by 100 mg/L. The decreased viabilities of border cells indicated that Fe2+ dosage and treatment time would cause deadly effect on the border cells. The increased cell death could protect the root tips from toxic harm. Therefore, it may protect root from the damage caused by harmful iron toxicity.

  11. 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.

  12. Growth promotion and an increase in cell wall extensibility by silicon in rice and some other Poaceae seedlings.

    Science.gov (United States)

    Hossain, Mohammad Talim; Mori, Ryuji; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Fujii, Shuhei; Yamamoto, Ryoichi; Hoson, Takayuki

    2002-02-01

    The effect of silicon on organ growth and its mechanisms of action were studied in rice ( Oryza sativa L. cv. Koshihikari), oat ( Avena sativa L. cv. Victory), and wheat ( Triticum aestivum L. cv. Daichino-Minori) seedlings grown in the dark. Applying silicon in the form of silicic acid to these seedlings via culture solution resulted in growth promotion of third (rice) or second (oat and wheat) leaves. The optimal concentration of silicon was 5-10 mM. No growth promotion was observed in early organs, such as coleoptiles or first leaves. In silicon-treated rice third leaves, the epidermal cell length increased, especially in the basal regions, without any effect on the number of cells, showing that silicon promoted cell elongation but not cell division. Silicon also increased the cell wall extensibility significantly in the basal regions of rice third leaves. These results indicate that silicon stimulates growth of rice and some other Poaceae leaves by increasing cell wall extensibility.

  13. Synthesis of albumin via a precursor protein in cell suspensions from rat liver.

    Science.gov (United States)

    Edwards, K; Schreiber, G; Dryburgh, H; Urban, J; Inglis, A S

    1976-03-16

    The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optimized for the incorporation of amino acids into protein. Seven minutes after starting the incubation L-[1-14C]leucine was added, followed after 25 min by a 15 or 30-min chase with an 830-fold excess of non-radioactive L-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately of total protein was found to remain constant after addition of the non-radioactive L-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albumin-like protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.

  14. 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.

  15. Conjugation of the mycotoxins alternariol and alternariol monomethyl ether in tobacco suspension cells.

    Science.gov (United States)

    Hildebrand, Andreas A; Kohn, Beate N; Pfeiffer, Erika; Wefers, Daniel; Metzler, Manfred; Bunzel, Mirko

    2015-05-20

    The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as β-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

  16. Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures.

    Science.gov (United States)

    Dixon, R A; Dey, P M; Murphy, D L; Whitehead, I M

    1981-03-01

    The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 μg elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 μg ml(-1). The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; α-methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.

  17. Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures.

    Science.gov (United States)

    Chappell, J; Von Lanken, C; Vögeli, U; Bhatt, P

    1989-05-01

    The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.

  18. Neuroprotective effects of germinated brown rice against hydrogen peroxide induced cell death in human SH-SY5Y cells.

    Science.gov (United States)

    Ismail, Norsharina; Ismail, Maznah; Fathy, Siti Farhana; Musa, Siti Nor Asma; Imam, Mustapha Umar; Foo, Jhi Biau; Iqbal, Shahid

    2012-01-01

    The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H(2)O(2)) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H(2)O(2)-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.

  19. Evidence for a transketolase-mediated metabolic checkpoint governing biotrophic growth in rice cells by the blast fungus Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Jessie Fernandez

    2014-09-01

    Full Text Available The blast fungus Magnaporthe oryzae threatens global food security through the widespread destruction of cultivated rice. Foliar infection requires a specialized cell called an appressorium that generates turgor to force a thin penetration hypha through the rice cuticle and into the underlying epidermal cells, where the fungus grows for the first days of infection as a symptomless biotroph. Understanding what controls biotrophic growth could open new avenues for developing sustainable blast intervention programs. Here, using molecular genetics and live-cell imaging, we dismantled M. oryzae glucose-metabolizing pathways to reveal that the transketolase enzyme, encoded by TKL1, plays an essential role in facilitating host colonization during rice blast disease. In the absence of transketolase, Δtkl1 mutant strains formed functional appressoria that penetrated rice cuticles successfully and developed invasive hyphae (IH in rice cells from primary hyphae. However, Δtkl1 could not undertake sustained biotrophic growth or cell-to-cell movement. Transcript data and observations using fluorescently labeled histone H1:RFP fusion proteins indicated Δtkl1 mutant strains were alive in host cells but were delayed in mitosis. Mitotic delay could be reversed and IH growth restored by the addition of exogenous ATP, a metabolite depleted in Δtkl1 mutant strains. We show that ATP might act via the TOR signaling pathway, and TOR is likely a downstream target of activation for TKL1. TKL1 is also involved in controlling the migration of appressorial nuclei into primary hyphae in host cells. When taken together, our results indicate transketolase has a novel role in mediating--via ATP and TOR signaling--an in planta-specific metabolic checkpoint that controls nuclear migration from appressoria into primary hyphae, prevents mitotic delay in early IH and promotes biotrophic growth. This work thus provides new information about the metabolic strategies employed by M

  20. Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

    Science.gov (United States)

    Kouzuma, Atsushi; Kasai, Takuya; Nakagawa, Gen; Yamamuro, Ayaka; Abe, Takashi; Watanabe, Kazuya

    2013-01-01

    In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors. PMID:24223712

  1. Comparative metagenomics of anode-associated microbiomes developed in rice paddy-field microbial fuel cells.

    Science.gov (United States)

    Kouzuma, Atsushi; Kasai, Takuya; Nakagawa, Gen; Yamamuro, Ayaka; Abe, Takashi; Watanabe, Kazuya

    2013-01-01

    In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.

  2. Comparative metagenomics of anode-associated microbiomes developed in rice paddy-field microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Atsushi Kouzuma

    Full Text Available In sediment-type microbial fuel cells (sMFCs operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.

  3. Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings.

    Science.gov (United States)

    Nakamura, Yukiko; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Hoson, Takayuki

    2002-12-01

    The mechanism inducing the difference in growth rate under various temperature (10-50 degrees C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 degrees C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 degrees C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 degrees C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 degrees C were transferred to the initial temperature (30 degrees C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall.

  4. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    Science.gov (United States)

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  5. Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

    Institute of Scientific and Technical Information of China (English)

    李景川; 马忠海; 元英进; 孙安慈; 胡昌序

    2001-01-01

    The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of “partition” and “bifurcation” were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

  6. 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.

  7. 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) × 10(6) 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.

  8. Fermented Brown Rice and Rice Bran with Aspergillus oryzae (FBRA Prevents Inflammation-Related Carcinogenesis in Mice, through Inhibition of Inflammatory Cell Infiltration

    Directory of Open Access Journals (Sweden)

    Kunishige Onuma

    2015-12-01

    Full Text Available We have established an inflammation-related carcinogenesis model in mouse, in which regressive QR-32 cells subcutaneously co-implanted with a foreign body—gelatin sponge—convert themselves into lethal tumors due to massive infiltration of inflammatory cells into the sponge. Animals were fed with a diet containing 5% or 10% fermented brown rice and rice bran with Aspergillus oryzae (FBRA. In 5% and 10% FBRA diet groups, tumor incidences were lower (35% and 20%, respectively than in the non-treated group (70%. We found that FBRA reduced the number of inflammatory cells infiltrating into the sponge. FBRA administration did not cause myelosuppression, which indicated that the anti-inflammatory effects of FBRA took place at the inflammatory lesion. FBRA did not have antitumor effects on the implanted QRsP-11 tumor cells, which is a tumorigenic cell line established from a tumor arisen after co-implantation of QR-32 cells with sponge. FBRA did not reduce formation of 8-hydroxy-2′-deoxyguanine adducts, a marker of oxidative DNA damage in the inflammatory lesion; however, it reduced expression of inflammation-related genes such as TNF-α, Mac-1, CCL3 and CXCL2. These results suggest that FBRA will be an effective chemopreventive agent against inflammation-related carcinogenesis that acts by inhibiting inflammatory cell infiltration into inflammatory lesions.

  9. Antioxidant metabolism of coffee cell suspension cultures in response to cadmium.

    Science.gov (United States)

    Gomes-Junior, Rui A; Moldes, Carlos A; Delite, Fabricio S; Pompeu, Georgia B; Gratão, Priscila L; Mazzafera, Paulo; Lea, Peter J; Azevedo, Ricardo A

    2006-11-01

    The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.

  10. Single-Beam Acoustic Trapping of Red Blood Cells and Polystyrene Microspheres in Flowing Red Blood Cell Saline and Plasma Suspensions.

    Science.gov (United States)

    Liu, Hsiao-Chuan; Li, Ying; Chen, Ruimin; Jung, Hayong; Shung, K Kirk

    2017-02-21

    Single-beam acoustic tweezers (SBATs) represent a new technology for particle and cell trapping. The advantages of SBATs are their deep penetration into tissues, reduction of tissue damage and ease of application to in vivo studies. The use of these tools for applications in drug delivery in vivo must meet the following conditions: large penetration depth, strong trapping force and tissue safety. A reasonable penetration depth for SBATs in the development of in vivo applications was established in a previous study conducted in water with zero velocity. However, capturing objects in flowing fluid can provide more meaningful results. In this study, we investigated the capability of SBATs to trap red blood cells (RBCs) and polystyrene microspheres in flowing RBC suspensions. Two different types of RBC suspension were prepared in this work: an RBC phosphate-buffered saline (PBS) suspension and an RBC plasma suspension. The results indicated that SBATs successfully trapped RBCs and polystyrene microspheres in a flowing RBC PBS suspension with an average steady velocity of 1.6 cm/s in a 2-mm-diameter polyimide. Furthermore, SBATs were found able to trap RBCs in a flowing RBC PBS suspension at speeds as high as 7.9 cm/s in a polyimide tube, which is higher than the velocity in capillaries (0.03 cm/s) and approaches the velocity in arterioles and venules. Moreover, the results also indicated that polystyrene microspheres can be trapped in an RBC plasma suspension, where aggregation is observed. This work represents a step forward in using this tool in actual in vivo experimentation.

  11. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  12. 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.

  13. Fabrication and electrochemical performance of solid oxide fuel cell components by atmospheric and suspension plasma spray

    Institute of Scientific and Technical Information of China (English)

    XIA Wei-sheng; YANG Yun-zhen; ZHANG Hai-ou; WANG Gui-lan

    2009-01-01

    The theory of functionally graded material (FGM) was applied in the fabrication process of PEN (Positive- Electrolyte-Negative),the core component of solid oxide fuel cell (SOFC).To enhance its electrochemical performance,the functionally graded PEN of planar SOFC was prepared by atmospheric plasma spray (APS).The cross-sectional SEM micrograph and element energy spectrum of the resultant PEN were analyzed.Its interface resistance was also compared with that without the graded layers to investigate the electrochemical performance enhanced by the functionally graded layers.Moreover,a new process,suspension plasma spray (SPS) was applied to preparing the SOFC electrolyte.Higher densification of the coating by SPS,1.61%,is observed,which is helpful to effectively improve its electrical conductivity.The grain size of the electrolyte coating fabricated by SPS is also smaller than that by APS,which is more favourable to obtain the dense electrolyte coatings.To sum up,all mentioned above can prove that the hybrid process of APS and SPS could be a better approach to fabricate the PEN of SOFC stacks,in which APS is for porous electrodes and SPS for dense electrolyte.

  14. The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines.

    Science.gov (United States)

    Ferreira, Stephanus J; Kossmann, Jens; Lloyd, James R; Groenewald, Jan-Hendrik

    2008-11-01

    Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.

  15. Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.

    Science.gov (United States)

    Hanley, K; Chappell, J

    1992-01-01

    Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

  16. Acaricidal activities of whole cell suspension, cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

    Institute of Scientific and Technical Information of China (English)

    Prapassom BUSSAMAN; Chirayu SA-UTH; Paweena RATTANAS ENA; Angsumarn CHANDRAPATYA

    2012-01-01

    Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp.,a mushroom mite endemic in Thailand.To develop an effective formulation of Xenorhabdus stokiae,treatments using different parts of X.stokiae isolate PB09 culture,including whole cell suspension,cell-free supernatant,and crude cell extract,were performed.The results show that different parts ofX.stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity.Application with cell-free supernatant of X.stokiae culture resulted in both the highest mite mortality rate [(89.00+3.60)%] and the lowest mite fecundity [(41.33+23.69) eggs/gravid female].Whole cell suspension of X.stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant,suggesting that X.stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media.Crude cell extract of X.stokiae was not effective against mites.Cell-free supernatant of X.stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

  17. Nucleotide sequences of chloroplast 5S ribosomal RNA from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata.

    OpenAIRE

    Yamano, Y; Ohyama, K; Komano, T

    1984-01-01

    The nucleotide sequences of chloroplast 5S rRNAs from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata were determined. Their nucleotide sequences, 119 nucleotides long, were highly homologous to each other (96% identity) and had high homology with those from chloroplast 5S rRNAs of two higher plants, tobacco (92% identity) and spinach (92-91% identity), but less homology (87-85% identity) with that from a lower plant, the fern Dryopteris acuminata.

  18. The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    2000-01-01

    A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma...... cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse...... an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found...

  19. Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function.

    Science.gov (United States)

    Li, Yi; Bao, Yi M; Wei, Chun H; Kang, Zhen S; Zhong, Yong W; Mao, Peng; Wu, Gang; Chen, Zhang L; Schiemann, Joachim; Nelson, Richard S

    2004-05-01

    Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.

  20. Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    Yang Shengchang

    2006-01-01

    A rapid and concentration-dependent generation of superoxide anion (·O-2), measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts (LaCl3 and GdCl3) were added to tobacco (Nicotiana tabacum) cell suspension culture.Addition of superoxide dismutase (480 U·ml-1) and Tiron (5 μmol·L-1) to cell culture suspension decreases the level of lanthanide cation-induced ·O-2 generation, suggesting that ·O-2 generation is extra-cellular.Pretreatment of the cell culture suspension with diphenyleneiodonium (10 and 50 μmol·L-1), quinacrine (1 and 5 mmol·L-1) and imidazol (10 mmol·L-1), inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase.In addition, addition of SHAM (1 and 5 mmol·L-1), azide (0.2 and 1 mmol·L-1), inhibitor of peroxidase, has no influence on ·O-2 generation.

  1. Elevated Carbon Dioxide Alleviates Aluminum Toxicity by Decreasing Cell Wall Hemicellulose in Rice (Oryza sativa

    Directory of Open Access Journals (Sweden)

    Xiao Fang Zhu

    2017-07-01

    Full Text Available Carbon dioxide (CO2 is involved in plant growth as well as plant responses to abiotic stresses; however, it remains unclear whether CO2 is involved in the response of rice (Oryza sativa to aluminum (Al toxicity. In the current study, we discovered that elevated CO2 (600 μL·L−1 significantly alleviated Al-induced inhibition of root elongation that occurred in ambient CO2 (400 μL·L−1. This protective effect was accompanied by a reduced Al accumulation in root apex. Al significantly induced citrate efflux and the expression of OsALS1, but elevated CO2 had no further effect. By contrast, elevated CO2 significantly decreased Al-induced accumulation of hemicellulose, as well as its Al retention. As a result, the amount of Al fixed in the cell wall was reduced, indicating an alleviation of Al-induced damage to cell wall function. Furthermore, elevated CO2 decreased the Al-induced root nitric oxide (NO accumulation, and the addition of the NO scavenger c-PTIO (2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide abolished this alleviation effect, indicating that NO maybe involved in the CO2-alleviated Al toxicity. Taken together, these results demonstrate that the alleviation of Al toxicity in rice by elevated CO2 is mediated by decreasing hemicellulose content and the Al fixation in the cell wall, possibly via the NO pathway.

  2. Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris.

    Science.gov (United States)

    Dixon, R A; Dey, P M; Lawton, M A; Lamb, C J

    1983-02-01

    Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.

  3. A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation

    Directory of Open Access Journals (Sweden)

    Dong Hai-Tao

    2012-04-01

    Full Text Available Abstract Background Miniature inverted repeat transposable element (MITE is one type of transposable element (TE, which is largely found in eukaryotic genomes and involved in a wide variety of biological events. However, only few MITEs were proved to be currently active and their physiological function remains largely unknown. Results We found that the amplicon discrepancy of a gene locus LOC_Os01g0420 in different rice cultivar genomes was resulted from the existence of a member of Gaijin-like MITEs (mGing. This result indicated that mGing transposition was occurred at this gene locus. By using a modified transposon display (TD analysis, the active transpositions of mGing were detected in rice Jiahua No. 1 genome under three conditions: in seedlings germinated from the seeds received a high dose γ-ray irradiation, in plantlets regenerated from anther-derived calli and from scutellum-derived calli, and were confirmed by PCR validation and sequencing. Sequence analysis revealed that single nucleotide polymorphisms (SNPs or short additional DNA sequences at transposition sites post mGing transposition. It suggested that sequence modification was possibly taken place during mGing transposition. Furthermore, cell re-differentiation experiment showed that active transpositions of both mGing and mPing (another well studied MITE were identified only in regenerated plantlets. Conclusions It is for the first time that mGing active transposition was demonstrated under γ-ray irradiation or in cell re-differentiation process in rice. This newly identified active MITE will provide a foundation for further analysis of the roles of MITEs in biological process.

  4. LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

    OpenAIRE

    Staszków, Anna; Swarcewicz, Barbara; Banasiak, Joanna; Muth, Dorota; Jasiński, Michał; Stobiecki, Maciej

    2011-01-01

    Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant...

  5. Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

    DEFF Research Database (Denmark)

    Garcia-Angulo, P.; Willats, W. G. T.; Encina, A. E.

    2006-01-01

    analysed showed calcofluor-stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4-ß-glucan in these cell lines...

  6. 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.

  7. The durative use of suspension cells and callus for volatile oil by comparative with seeds and fruits in Capparis spinosa L.

    Directory of Open Access Journals (Sweden)

    Yongtai Yin

    Full Text Available Capparis spinosa is one of the most important eremophytes among the medicinal plants, and continued destruction of these plants poses a major threat to species survival. The development of methods to extract compounds, especially those of medicinal value, without harvesting the whole plant is an issue of considerable socioeconomic importance. On the basis of an established system for culture of suspension cells and callus in vitro, Gas Chromatograph-Mass Spectrometer (GC-MS was used for the volatile oil composition analyzing in seed, fruit, suspension cells and callus. Fatty acids were the major component, and the highest content of alkanes was detected in seed, with <1.0% in suspension cells and callus. Esters, olefins and heterocyclic compounds were significantly higher in fruit than in the other materials. The content of acid esters in the suspension cells and callus was significantly higher than in seed and fruit. This indicated that the suspension cells and callus could be helpful for increasing the value of volatile oil and replacing seeds and fruit partially as a source of some compounds of the volatile oil and may also produce some new medical compounds. The above results give valuable information for sustainable use of C. spinosa and provide a foundation for use of the C. spinosa suspension cells and callus as an ongoing medical resource.

  8. Effect of pH on acid production from sorbitol in washed cell suspensions of oral bacteria.

    Science.gov (United States)

    Kalfas, S; Maki, Y; Birkhed, D; Edwardsson, S

    1990-01-01

    The acid production from sorbitol and glucose was studied under anaerobic conditions in resting cell suspensions of bacteria from the predominant sorbitol-fermenting human dental plaque flora, belonging to the genera Streptococcus, Lactobacillus and Actinomyces. The acid production activity of the bacterial cells was followed by titration with alkali, at environmental pH 7.0, 6.0 and 5.0 after addition of carbohydrate solution. The metabolic end products formed in the suspensions were analyzed thereafter by isotachophoretic and enzymatic methods. The results showed that sorbitol was fermented at a slower rate than glucose. Lowering the environmental pH decreased the acid production activity from the two carbohydrates. Compared with glucose, the catabolism of sorbitol was affected to greater extent by the pH conditions. The total amount of acids formed from sorbitol was considerably less than from glucose. Lactic acid, which was the major end product in glucose-challenged suspensions, was produced only in low concentrations from sorbitol by all strains tested. The ratio strong (formic + lactic)/weak acids was moreover lower for sorbitol than for glucose. The present results further illustrate some of the mechanisms behind the low cariogenic potential of this sugar substitute.

  9. Cell-wall Invertases from Rice are Differentially Expressed in Caryopsis during the Grain Filling Stage

    Institute of Scientific and Technical Information of China (English)

    Yong-Qin Wang; Gui-Fang Xiao; Zhen Zhu; Xiao-Li Wei; Hong-Lin Xu; Cheng-Lin Chai; Kun Meng; Hong-Li Zhai; Ai-Jun Sun; Yong-Gang Peng; Bin Wu

    2008-01-01

    Cell-wall Invertase plays an important role in sucrose partitioning between source and sink organs in higher plants. To Investigate the role of cell-wall invertases for seed development In rice (Oryza sativa L.), cDNAs of three putative cell-wall invertase genes OsCIN1, OsCIN2 and OsCIN3 were Isolated. Semi-quantitative reverse transcdption-polymerase chain reaction analysis revealed different expression patterns of the three genes in various rice tissues/organs. In developing ceryopses, they exhibited similar temporal expression patterns, expressed highly at the early and middle grain filling stages and gradually declined to low levels afterward. However, the spatial expression patterns of them were very different, with OsCIN1 primarily expressed in the ceryopsis coat, OsCIN2 in embryo and endosperm, and OsCIN3 In embryo. Further RNA in situ hybridization analysis revealed that a strong signal of OsCIN2 mRNA was detected In the vascular parenchyma surrounding the xylem of the chalazal vein and the aleurone layer, whereas OsCIN3 transcdpt was strongly detected in the vascular parenchyma surrounding the phloem of the chalazal vein, cross.cells, the aleurone layer and the nucellar tissue.These data indicate that the three cell-wall invertase genes play complementary/synergetic roles in assimilate unloading during the grain filling stage. In addition, the cell type-specific expression patterns of OsClN3 In source leaf blades end anthers were also Investigated, and its corresponding physiological roles were discussed.

  10. 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.

  11. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  12. Bioactive compounds in pigmented rice bran inhibit growth of human cancer cells

    Science.gov (United States)

    Rice bran contains both lipophilic and hydrophilic antioxidants. Our previous studies have shown that pigmented rice cultivars contained several-fold higher total phenolic concentrations and antioxidant capacities than non-pigmented cultivars. We investigated three rice brans (purple, red and light-...

  13. Rice varietal differences in bioactive bran components for inhibition of colorectal cancer cell growth

    Science.gov (United States)

    Studies support that the bran fraction of rice contains bioactive compounds capable of inhibiting the formation of colonic tumors. Screening bran extracts from diverse rice varieties represents a novel approach to assessing the colon cancer chemopreventive properties of rice bran. We analyzed a pane...

  14. Mitosis and microtubule organizational changes in rice root-tip cells

    Institute of Scientific and Technical Information of China (English)

    XUSHIXIONG(SYZEE); CHUNGUILI; CHENGZHU

    1993-01-01

    The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.

  15. Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero).

    Science.gov (United States)

    Paillet, Cristian; Forno, Guillermina; Soldano, Nicolas; Kratje, Ricardo; Etcheverrigaray, Marina

    2011-09-22

    Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5μg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Hatanaka, Rie; Sugawara, Yasutake

    2010-03-01

    Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H(2)O g(-1) dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H(2)O g(-1) DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H(2)O g(-1) DW, desiccation-tolerant cells that had been precultured were vitrified above 0 degrees C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.

  17. Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation.

    Science.gov (United States)

    Bolwell, G P

    1984-09-01

    Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.

  18. In vitro induction of α-pinene, pulegone, menthol, menthone and limonene in cell suspension culture of pennyroyal (Mentha pulegium).

    Science.gov (United States)

    Darvishi, E; Kahrizi, D; Bahraminejad, S; Mansouri, M

    2016-03-20

    Medicinal plants are known as important sources of secondary metabolites. Because of the economic value of pennyroyal [Mentha pulegium L. (Lamiaceae)] in food industries, propagation of this valuable plant has special importance. Plant cell suspension culture can increase some produced components. The aim of this research was performing cell culture for induction of some secondary metabolites of M. pulegium and compares it with native one. The MS medium was used for suspension culture. To investigate quantitative materials, 4 levels of yeast extract elicitor (20, 40, 60 and 80 mg/L) and salicylic acid in 4 levels (2, 4, 6 and 8 mg/L) were used. Obtained extracts were analyzed by GC-MS. Statistical analysis showed that the amount of limonene, menthone, menthol and α-pinene were more than mentioned compounds in natural plant as control. The maximum amount of this metabolites were obtained as limonene (in 60 mg/l yeast extract), menthone (in 40 mg/l yeast extract and 2 mg/l salicylic acid), menthol (in 6 mg/l salicylic acid) and α-pinene (in 4 mg/l salicylic acid) in the M. pulegium cell culture. The Pulegone was fond more in natural plants than cell culture mass. The most important secondary metabolites were increased by cell culture containing of salicylic acid and yeast extract elicitors in M. pulegume.

  19. Effect of phenylalanine on Taxol production and antioxidant activity of extracts of suspension-cultured hazel (Corylus avellana L.) cells.

    Science.gov (United States)

    Bemani, Ebrahim; Ghanati, Faezeh; Rezaei, Ayatollah; Jamshidi, Mitra

    2013-07-01

    Taxol is produced by a few microorganisms and plants such as yew (Taxus sp.). Recent researches have shown that hazel (Corylus avellana L.) is also able to produce Taxol. In the present study, effects of different concentrations of phenylalanine (Phe) on the production of Taxol, antioxidant activity, and cytotoxic effects of extracts of suspension-cultured hazel cells were investigated. The cells were treated with different concentrations of Phe on day 7 of subculture and were harvested on day 14. The results showed that the amounts of Taxol and antioxidant activity were increased by increasing the phenylalanine supply. Interestingly, the cytotoxic effects of hazel cell extract were even stronger than that of pure Taxol (standard), suggesting hazel cell extract as a novel and suitable probe for treating human cancer. Application of phenylalanine to hazel cells exaggerates their effects.

  20. OsRAN2, essential for mitosis, enhances cold tolerance in rice by promoting export of intranuclear tubulin and maintaining cell division under cold stress.

    Science.gov (United States)

    Chen, Na; Xu, Yunyuan; Wang, Xin; DU, Cheng; DU, Jizhou; Yuan, Ming; Xu, Zhihong; Chong, Kang

    2011-01-01

    With global climate change, abnormally low temperatures have affected the world's rice production. Many genes have been shown to be essential for molecular improvement of rice cold-tolerance traits. However, less is known about the molecular cellular mechanism of their response to cold stress. Here, we investigated OsRAN2 involved in regulation of cell division during cold stress in rice. Expression of OsRAN2 was increased under cold treatment, but not during salt and drought stress. The mean root mitotic index was closely related to the expression level of OsRAN2. Knockdown transgenic rice lines showed an aberrant organization of spindles during mitosis and stunted growth during development. Overexpression of OsRAN2 enhanced cold tolerance in rice. The transgenic rice overexpressing OsRAN2 showed maintained cell division, decreased proportion of cells with intranuclear tubulin and formation of a normal nuclear envelope under the cold condition. Our study suggests a mechanism for OsRAN2 in regulating cold resistance in rice by maintaining cell division through promoting the normal export of intranuclear tubulin at the end of mitosis. This insight could help improve the cold-tolerance trait in rice.

  1. Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

    Science.gov (United States)

    Hodge, Alexander J; Zhong, Juming; Lipke, Elizabeth A

    2016-04-01

    The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased βMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors

  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: AgNO3 (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. Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Muzaffar, Suhail; Bose, Chinchu; Banerji, Ashok; Nair, Bipin G; Chattoo, Bharat B

    2016-01-01

    Anacardic acid (6-pentadecylsalicylic acid), extracted from cashew nut shell liquid, is a natural phenolic lipid well known for its strong antibacterial, antioxidant, and anticancer activities. Its effect has been well studied in bacterial and mammalian systems but remains largely unexplored in fungi. The present study identifies antifungal, cytotoxic, and antioxidant activities of anacardic acid in the rice blast fungus Magnaporthe oryzae. It was found that anacardic acid causes inhibition of conidial germination and mycelial growth in this ascomycetous fungus. Phosphatidylserine externalization, chromatin condensation, DNA degradation, and loss of mitochondrial membrane potential suggest that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that anacardic acid induces caspase-independent apoptosis in M. oryzae. Expression of a predicted ortholog of apoptosis-inducing factor (AIF) was upregulated during the process of apoptosis, suggesting the possibility of mitochondria dependent apoptosis via activation of apoptosis-inducing factor. Anacardic acid treatment leads to decrease in reactive oxygen species rather than increase in reactive oxygen species (ROS) accumulation normally observed during apoptosis, confirming the antioxidant properties of anacardic acid as suggested by earlier reports. Our study also shows that anacardic acid renders the fungus highly sensitive to DNA damaging agents like ethyl methanesulfonate (EMS). Treatment of rice leaves with anacardic acid prevents M. oryzae from infecting the plant without affecting the leaf, suggesting that anacardic acid can be an effective antifungal agent.

  4. A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnanajothi; Jeyaraj, Murugaraj; Rajesh, Manoharan; Muthuselvam, Manickam; Selvaraj, Natesan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-08-01

    Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.

  5. RNA-sequencing reveals previously unannotated protein- and microRNA-coding genes expressed in aleurone cells of rice seeds.

    Science.gov (United States)

    Watanabe, Kenneth A; Ringler, Patricia; Gu, Lingkun; Shen, Qingxi J

    2014-01-01

    The rice genome annotation has been greatly improved in recent years, largely due to the availability of full length cDNA sequences derived from many tissues. Among those yet to be studied is the aleurone layer, which produces hydrolases for mobilization of seed storage reserves during seed germination and post germination growth. Herein, we report transcriptomes of aleurone cells treated with the hormones abscisic acid, gibberellic acid, or both. Using a comprehensive approach, we identified hundreds of novel genes. To minimize the number of false positives, only transcripts that did not overlap with existing annotations, had a high level of expression, and showed a high level of uniqueness within the rice genome were considered to be novel genes. This approach led to the identification of 553 novel genes that encode proteins and/or microRNAs. The transcriptome data reported here will help to further improve the annotation of the rice genome.

  6. Opposite extremes in ethylene/nitric oxide ratio induce cell death in suspension culture and root apices of tomato exposed to salt stress.

    Science.gov (United States)

    Poór, P; Borbély, P; Kovács, Judit; Papp, Anita; Szepesi, Ágnes; Takács, Z; Tari, Irma

    2014-12-01

    The plant hormone ethylene or the gaseous signalling molecule nitric oxide (NO) may enhance salt stress tolerance by maintaining ion homeostasis, first of all K+/Na+ ratio of tissues. Ethylene and NO accumulation increased in the root apices and suspension culture cells of tomato at sublethal salt stress caused by 100 mM NaCl, however, the induction phase of programmed cell death (PCD) was different at lethal salt concentration. The production of ethylene by root apices and the accumulation of NO in the cells of suspension culture did not increase during the initiation of PCD after 250 mM NaCl treatment. Moreover, cells in suspension culture accumulated higher amount of reactive oxygen species which, along with NO deficiency contributed to cell death induction. The absence of ethylene in the apical root segments and the absence of NO accumulation in the cell suspension resulted in similar ion disequilibrium, namely K+/Na+ ratio of 1.41 ± 0.1 and 1.68 ± 0.3 in intact plant tissues and suspension culture cells, respectively that was not tolerated by tomato.

  7. Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

    Science.gov (United States)

    Dores, C; Rancourt, D; Dobrinski, I

    2015-05-01

    To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

  8. Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

    Science.gov (United States)

    Cavez, Damien; Hachez, Charles; Chaumont, François

    2009-09-01

    Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

  9. Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins.

    Science.gov (United States)

    Yamazaki, N; Fry, S C; Darvill, A G; Albersheim, P

    1983-07-01

    A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

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

    Science.gov (United States)

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

    2016-04-01

    Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions

  11. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

    Science.gov (United States)

    Broussau, Sophie; Jabbour, Nadine; Lachapelle, Guillaume; Durocher, Yves; Tom, Rosanne; Transfiguracion, Julia; Gilbert, Rénald; Massie, Bernard

    2008-03-01

    We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

  12. Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

    Science.gov (United States)

    Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

    2011-07-01

    The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

  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. ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

    Science.gov (United States)

    David Gara, Pedro M.; Garabano, Natalia I.; Llansola Portoles, Manuel J.; Moreno, M. Sergio; Dodat, Diego; Casas, Oscar R.; Gonzalez, Mónica C.; Kotler, Mónica L.

    2012-03-01

    The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O2 •-/HO2 •, HO•, and H2O2 generated upon 4-MeV X-ray irradiation of 6.4 μM silicon nanoparticle aqueous suspensions were on the order of 10 μM per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic 1O2 was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO2 and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of design of therapy strategies.

  15. Suspension model for blood flow through a catheterized arterial stenosis with peripheral layer of plasma free from cells

    Science.gov (United States)

    Ponalagusamy, R.

    2016-06-01

    The present article describes the blood flow in a catheterized artery with radially symmetric and axially asymmetric stenosis. To understand the effects of red cell concentration, plasma layer thickness and catheter size simultaneously, blood is considered by a two-layered model comprising a core region of suspension of all the erythrocytes (particles) supposed to be a particle-fluid mixture and a peripheral zone of cell-free plasma. The analytical expressions for flow features, such as fluid phase and particle phase velocities, flow rate, wall shear stress and resistive force are obtained. It is witnessed that the presence of the catheter causes a substantial increase in the frictional forces on the walls of arterial stenosis and catheter, shear stress and flow resistance, in addition to that, have occurred due to the presence of red cells concentration (volume fraction density of the particles) and the absence of peripheral plasma layer near the wall of the stenosed artery. The introduction of an axially asymmetric nature of stenosis and plasma layer thickness causes significant reduction in flow resistance. One can notice that the two-phase fluid (suspension model) is more profound to the thickness of peripheral plasma layer and catheter than the single-phase fluid.

  16. Processing and intracellular localization of rice stripe virus Pc2 protein in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Shuling; Zhang, Gaozhan; Dai, Xuejuan; Hou, Yanling; Li, Min [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Liang, Jiansheng, E-mail: jsliang@yzu.edu.cn [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Liang, Changyong, E-mail: cyliang@yzu.edu.cn [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China)

    2012-08-01

    Rice stripe virus (RSV) belongs to the genus Tenuivirus and its genome consists of four single-stranded RNAs encoding seven proteins. Here, we have analyzed the processing and membrane association of Pc2 encoded by vcRNA2 in insect cells. The enhanced green fluorescent protein (eGFP) was fused to the Pc2 and used for the detection of Pc2 fusion proteins. The results showed that Pc2 was cleaved to produce two proteins named Pc2-N and Pc2-C. When expressed alone, either Pc2-N or Pc2-C could transport to the Endoplasmic reticulum (ER) membranes independently. Further mutagenesis studies revealed that Pc2 contained three ER-targeting domains. The results led us to propose a model for the topology of the Pc2 in which an internal signal peptide immediately followed a cleavage site, and two transmembrane regions are contained.

  17. Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

    Science.gov (United States)

    Fan, Guo-cheng; Gao, Fang-luan; Wei, Tai-yun; Huang, Mei-ying; Xie, Li-yan; Wu, Zu-jian; Lin, Qi-ying; Xie, Lian-hui

    2010-12-01

    To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

  18. Brownian Dynamics of a Suspension of Particles with Constrained Voronoi Cell Volumes

    KAUST Repository

    Singh, John P.

    2015-06-23

    © 2015 American Chemical Society. Solvent-free polymer-grafted nanoparticle fluids consist of inorganic core particles fluidized by polymers tethered to their surfaces. The attachment of the suspending fluid to the particle surface creates a strong penalty for local variations in the fluid volume surrounding the particles. As a model of such a suspension we perform Brownian dynamics of an equilibrium system consisting of hard spheres which experience a many-particle potential proportional to the variance of the Voronoi volumes surrounding each particle (E = α(Vi-V0)2). The coefficient of proportionality α can be varied such that pure hard sphere dynamics is recovered as α → 0, while an incompressible array of hairy particles is obtained as α →. As α is increased the distribution of Voronoi volumes becomes narrower, the mean coordination number of the particle increases and the variance in the number of nearest neighbors decreases. The nearest neighbor peaks in the pair distribution function are suppressed and shifted to larger radial separations as the constraint acts to maintain relatively uniform interstitial regions. The structure factor of the model suspension satisfies S(k=0) → 0 as α → in accordance with expectation for a single component (particle plus tethered fluid) incompressible system. The tracer diffusivity of the particles is reduced by the volume constraint and goes to zero at φ 0.52, indicating an earlier glass transition than has been observed in hard sphere suspensions. The total pressure of the suspension grows in proportion to (αkBT)1/2 as the strength of the volume-constraint potential grows. This stress arises primarily from the interparticle potential forces, while the hard-sphere collisional contribution to the stress is suppressed by the volume constraint.

  19. Brownian Dynamics of a Suspension of Particles with Constrained Voronoi Cell Volumes.

    Science.gov (United States)

    Singh, John P; Walsh, Stuart D C; Koch, Donald L

    2015-06-23

    Solvent-free polymer-grafted nanoparticle fluids consist of inorganic core particles fluidized by polymers tethered to their surfaces. The attachment of the suspending fluid to the particle surface creates a strong penalty for local variations in the fluid volume surrounding the particles. As a model of such a suspension we perform Brownian dynamics of an equilibrium system consisting of hard spheres which experience a many-particle potential proportional to the variance of the Voronoi volumes surrounding each particle (E = α(Vi-V0)(2)). The coefficient of proportionality α can be varied such that pure hard sphere dynamics is recovered as α → 0, while an incompressible array of hairy particles is obtained as α → ∞. As α is increased the distribution of Voronoi volumes becomes narrower, the mean coordination number of the particle increases and the variance in the number of nearest neighbors decreases. The nearest neighbor peaks in the pair distribution function are suppressed and shifted to larger radial separations as the constraint acts to maintain relatively uniform interstitial regions. The structure factor of the model suspension satisfies S(k=0) → 0 as α → ∞ in accordance with expectation for a single component (particle plus tethered fluid) incompressible system. The tracer diffusivity of the particles is reduced by the volume constraint and goes to zero at ϕ ∼ 0.52, indicating an earlier glass transition than has been observed in hard sphere suspensions. The total pressure of the suspension grows in proportion to (αkBT)(1/2) as the strength of the volume-constraint potential grows. This stress arises primarily from the interparticle potential forces, while the hard-sphere collisional contribution to the stress is suppressed by the volume constraint.

  20. Elicitor induced activation of the methylerythritol phosphate pathway toward phytoalexins biosynthesis in rice.

    Science.gov (United States)

    Okada, Atsushi; Shimizu, Takafumi; Okada, Kazunori; Kuzuyama, Tomohisa; Koga, Jinichiro; Shibuya, Naoto; Nojiri, Hideaki; Yamane, Hisakazu

    2007-09-01

    Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

  1. Suppression of cell wall-related genes associated with stunting of Oryza glaberrima infected with Rice tungro spherical virus

    Directory of Open Access Journals (Sweden)

    Bernard O. Budot

    2014-02-01

    Full Text Available Rice tungro disease is a complex disease caused by the interaction between Rice tungro bacilliform virus and Rice tungro spherical virus (RTSV. RTSV alone does not cause recognizable symptoms in most Asian rice (Oryza sativa plants, whereas some African rice (O. glaberrima plants were found to become stunted by RTSV. Stunting of rice plants by virus infections usually accompanies the suppression of various cell wall-related genes. The expression of cell wall-related genes was examined in O. glaberrima and O. sativa infected with RTSV to see the relationship between the severity of stunting and the suppression of cell wall-related genes by RTSV. The heights of four accessions of O. glaberrima were found to decline by 14 to 34% at 28 days post-inoculation (dpi with RTSV, whereas the height reduction of O. sativa plants by RTSV was not significant. RTSV accumulated more in O. glaberrima plants than in O. sativa plants, but the level of RTSV accumulation was not correlated with the degree of height reduction among the four accessions of O. glaberrima. Examination for expression of genes for cellulose synthase A5 (CESA5 and A6 (CESA6, cellulose synthase-like A9 (CSLA9 and C7, and -expansin 1 (expansin 1 and 15 precursors in O. glaberrima and O. sativa plants between 7 and 28 dpi with RTSV showed that the genes such as those for CESA5, CESA6, CSLA9, and expansin 1were more significantly suppressed in stunted plants of O. glaberrima at 14 dpi with RTSV than in O. sativa, suggesting that stunting of O. glaberrima might be associated with these cell wall-related genes suppressed by RTSV. Examination for expression of these genes in O. sativa plants infected with other rice viruses in previous studies indicated that the suppression of the expansin 1 gene is likely to be a signature response commonly associated with virus-induced stunting of Oryza species. These results suggest that stunting of O. glaberrima by RTSV infection might be associated with

  2. The Rice RMR1 Associates with a Distinct Prevacuolar Compartment for the Protein Storage Vacuole Pathway

    Institute of Scientific and Technical Information of China (English)

    Yun Shen; Junqi Wang; Yu Ding; SzeWan Lo; Guillaume Gouzerh; Jean-Marc Neuhaus; Liwen Jiang

    2011-01-01

    Transport of vacuolar proteins from Golgi apparatus or trans-Golgi network (TGN) to vacuoles is a receptormediated process via an intermediate membrane-bound prevacuolar compartment (PVC) in plant cells.Both vacuolar sorting receptor (VSR) and receptor homology region-transmembrane domain-RING-H2 (RMR) proteins have been shown to function in transporting storage proteins to protein storage vacuole (PSV),but little is known about the nature of the PVC for the PSV pathway.Here,we use the rice RMR1 (OsRMR1) as a probe to study the PSV pathway in plants.Immunogold electron microscopy (EM) with specific OsRMR1 antibodies showed that OsRMR1 proteins were found in the Golgi apparatus,TGN,and a distinct organelle with characteristics of PVC in both rice culture cells and developing rice seeds,as well as the protein body type Ⅱ (PBII) or PSV in developing rice seeds.This organelle,also found in both tobacco BY-2 and Arabidopsis suspension cultured cells,is morphologically distinct from the VSR-positive multivesicular lytic PVC or multivesicular body (MVB) and thus represent a PVC for the PSV pathway that we name storage PVC (sPVC).Further in vivo and in vitro interaction studies using truncated OsRMR1 proteins secreted into the culture media of transgenic BY-2 suspension cells demonstrated that OsRMR1 functions as a sorting receptor in transporting vicilin-like storage proteins.

  3. Cell Wall Targeted in planta Iron Accumulation Enhances Biomass Conversion and Seed Iron Concentration in Arabidopsis and Rice

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Haibing; Wei, Hui; Ma, Guojie; Antunes, Mauricio S.; Vogt, Stefan; Cox, Joseph; Zhang, Xiao; Liu, Xiping; Bu, Lintao; Gleber, S. Charlotte; Carpita, Nicholas C.; Makowski, Lee; Himmel, Michael E.; Tucker, Melvin P.; McCann, Maureen C.; Murphy, Angus S.; Peer, Wendy A.

    2016-10-01

    Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusion polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.

  4. Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Voegeli, U.; Bhatt, P.N.; Chappell, J.

    1987-04-01

    Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. (/sup 14/C)-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using (/sup 3/H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results.

  5. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  6. Hydrodynamic interaction between two red blood cells in simple shear flow: its impact on the rheology of a semi-dilute suspension

    Science.gov (United States)

    Omori, Toshihiro; Ishikawa, Takuji; Imai, Yohsuke; Yamaguchi, Takami

    2014-10-01

    Blood is a suspension of red blood cells (RBCs) and its rheology is important when discussing the physiology of the cardiovascular system. In this study, we performed a numerical investigation of the rheological properties of an RBC suspension from the dilute to semi-dilute regime. RBCs were modelled as a capsule with a two-dimensional hyperelastic membrane. Large deformation of the thin membrane was calculated by a finite element method. Due to the small size of the RBC, fluid motion around the RBC was assumed to follow Stokes flow and was solved by a boundary element method. In the dilute limit, cell-cell interactions were omitted and the bulk stress of the suspension was calculated by the stresslet generated on a single RBC. Interestingly, the effective shear viscosity of the dilute suspension decreased with increasing viscosity of the internal liquid. In the semi-dilute regime, cells can be considered as showing pairwise interactions. The effective shear viscosity of the semi-dilute suspension shows a quadratic increase with respect to the volume fraction. These findings are important for understanding the complex phenomena of blood rheology.

  7. Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

    Science.gov (United States)

    Klein, Tino; Heremans, Yves; Heimberg, Harry; Pipeleers, Daniel; Madsen, Ole D; Serup, Palle; Heller, R Scott

    2009-01-01

    We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.

  8. 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.

  9. Development, characterization, and optimization of a new suspension chicken-induced pluripotent stem cell line for the production of Newcastle disease vaccine

    Science.gov (United States)

    Traditionally, substrates for production of vaccines have been embryonated eggs or adherent cell culture. The daunting challenge of scaling up these technologies in the face of an outbreak has been a limitation for industrial applicability. Suspension cell lines are better suited in many ways to e...

  10. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by

  11. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi

    2015-01-01

    Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.

  12. Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space.

    Directory of Open Access Journals (Sweden)

    Kazuyuki Wakabayashi

    Full Text Available Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA and p-coumaric acid, but it suppressed increases in diferulic acid (DFA isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL and cell wall-bound peroxidase (CW-PRX in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.

  13. Load-cell based characterization system for a “Violin-Mode” shadow-sensor in advanced LIGO suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Lockerbie, N. A.; Tokmakov, K. V. [SUPA (Scottish Universities Physics Alliance) Department of Physics, University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG (United Kingdom)

    2016-07-15

    The background to this work was a prototype shadow sensor, which was designed for retro-fitting to an advanced LIGO (Laser Interferometer Gravitational wave Observatory) test-mass/mirror suspension, in which 40 kg test-mass/mirrors are each suspended by four approximately 600 mm long by 0.4 mm diameter fused-silica suspension fibres. The shadow sensor comprised a LED source of Near InfraRed (NIR) radiation and a rectangular silicon photodiode detector, which, together, were to bracket the fibre under test. The aim was to detect transverse Violin-Mode resonances in the suspension fibres. Part of the testing procedure involved tensioning a silica fibre sample and translating it transversely through the illuminating NIR beam, so as to measure the DC responsivity of the detection system to fibre displacement. However, an equally important part of the procedure, reported here, was to keep the fibre under test stationary within the beam, whilst trying to detect low-level AC Violin-Mode resonances excited on the fibre, in order to confirm the primary function of the sensor. Therefore, a tensioning system, incorporating a load-cell readout, was built into the test fibre’s holder. The fibre then was excited by a signal generator, audio power amplifier, and distant loudspeaker, and clear resonances were detected. A theory for the expected fundamental resonant frequency as a function of fibre tension was developed and is reported here, and this theory was found to match closely with the detected resonant frequencies as they varied with tension. Consequently, the resonances seen were identified as being proper Violin-Mode fundamental resonances of the fibre, and the operation of the Violin-Mode detection system was validated.

  14. Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation.

    Science.gov (United States)

    Liu, Xiaofei; Li, Ming; Liu, Kai; Tang, Ding; Sun, Mingfa; Li, Yafei; Shen, Yi; Du, Guijie; Cheng, Zhukuan

    2016-04-01

    Moderate leaf rolling maintains the erectness of leaves and minimizes the shadowing between leaves which is helpful to establish ideal plant architecture. Here, we describe asrl2(semi-rolled leaf2) rice mutant, which has incurved leaves due to the presence of defective sclerenchymatous cells on the abaxial side of the leaf and displays narrow leaves and reduced plant height. Map-based cloning revealed that SRL2 encodes a novel plant-specific protein of unknown biochemical function.SRL2 was mainly expressed in the vascular bundles of leaf blades, leaf sheaths, and roots, especially in their sclerenchymatous cells. The transcriptional activities of several leaf development-related YABBY genes were significantly altered in the srl2 mutant. Double mutant analysis suggested that SRL2 and SHALLOT-LIKE1(SLL1)/ROLLED LEAF9(RL9) function in distinct pathways that regulate abaxial-side leaf development. Hence, SRL2 plays an important role in regulating leaf development, particularly during sclerenchymatous cell differentiation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang; ZHANG XinBo

    2008-01-01

    Heat shock (HS, 40℃, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H2O2) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO spe-cific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H2O2 generation and NO burst, but also the HS-triggered hypericin produc-tion. Hypericin contents of the cells treated with both NO and H2O2 are significantly higher than those of the cells treated with NO alone, although H2O2 per se has no effects on hypericin production of the cells, which suggests the synergistic action between H2O2 and NO on hypericin production. NO treatmentenhances H2O2 levels of H. perforatum cells, while external application of H2O2 induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H2O2 and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H2O2 accumulation and NO generation, while cPTIO can also suppress H2O2 levels of the heat shocked cells. The results imply that H2O2 and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H2O2 and NO but also abolish the synergistic effects of H2O2 and NO on hypericin production, showing that the synergism of H2O2 and NO on secondary metsbolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H2O2 and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.

  16. Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b6/f Complex

    Institute of Scientific and Technical Information of China (English)

    张贵友; 李萍; 朱瑞宇; 田瑞华; 戴尧仁

    2004-01-01

    An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco suspension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei,evidenced by DNA electrophoresis and fluorescence microscopy observations.Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still exhibited the apoptosis-inducing activity,but the supernatant had no effect,which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble.The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO,the specific inhibitor of Caspase-3,implying involvement of a Caspase-3-like protease during the process.Furthermore,extensive apoptosis in nuclei was induced by cytochrome b6/f on the thylakoid membrane,indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.

  17. Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures.

    Science.gov (United States)

    Robinson, S A; Stewart, G R; Phillips, R

    1992-03-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

  18. The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss.

    Science.gov (United States)

    Bekkaoui, F; Saxena, P K; Attree, S M; Fowke, L C; Dunstan, D I

    1987-12-01

    Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 10(6) protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10(5) protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

  19. Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Baroja-Fernández, E; Zandueta-Criado, A; Rodríguez-López, M; Akazawa, T; Pozueta-Romero, J

    2000-09-01

    The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.

  20. Im"plant"ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation.

    Science.gov (United States)

    Kajiura, Hiroyuki; Fujiyama, Kazuhito

    2015-01-01

    Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

  1. A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    张卫; Shintaro; Furusaki; Chris; Franco

    1999-01-01

    A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30℃ (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthoeyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was deereased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influeneed by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg(?)g-fresh cell-1 was obtained on day 9 by a temperature-

  2. Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi.

    Science.gov (United States)

    Netala, Vasudeva Reddy; Kotakadi, Venkata Subbaiah; Gaddam, Susmila Aparna; Ghosh, Sukhendu Bikash; Tartte, Vijaya

    2016-12-01

    The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

  3. Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

    Science.gov (United States)

    Nakamura, Yukiko; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2003-01-01

    The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice (Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10-50 degrees C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30-40 degrees C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40 degrees C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30-40 degrees C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40 degrees C were substantially higher than those grown at 10, 20 and 50 degrees C. Furthermore, the activities of (1-->3),(1-->4)-beta-glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1-->3),(1-->4)-beta-glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30-40 degrees C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of beta -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of beta-glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.

  4. Role of Rice stripe virus NSvc4 in cell-to-cell movement and symptom development in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Yi eXu

    2012-12-01

    Full Text Available Our previous work has demonstrated that the NSvc4 protein of Rice stripe virus (RSV functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD are unknown. Here we provide evidence that the NSvc4 moves on the actin filament and endoplasmic reticulum (ER network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4 localization to PD, thus impeding RSV infection of Nicotiana benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal 125 amino acids (AAs of the NSvc4 determine PD targeting and that a transmembrane domain spanning AAs 106 to 125 is critical for PD localization. We also found that the NSvc4 protein can localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal 73 AAs are essential for chloroplast localization. Furthermore, expression of NSvc4 from a Potato virus X (PVX vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4 in Spodoptera frugiperda 9 (Sf-9 cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants.

  5. 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%.

  6. The location of aluminium in protoplasts and suspension cells taken from Coffea arabica L. with different tolerance of Al.

    Science.gov (United States)

    Ramírez-Benítez, J Efraín; Hernández-Sotomayor, S M Teresa; Muñoz-Sánchez, J Armando

    2009-11-01

    Biotechnological advances in coffee research (in vitro manipulation, multiplication, generation and development of transgenic coffee plants with specific traits like high yield and good quality) have contributed to description of the metabolic pathways involved in the response mechanisms to environmental factors like abiotic stress. Coffea arabica L. plants grow in acidic soils, and therefore aluminium (Al) toxicity is a major negative impact on crop productivity. To understand Al toxicity mechanisms in cells via the Al absorption kinetic, we isolated protoplasts from two C. arabica L. suspension cell lines: Al-sensitive (L2) and Al-tolerant (LAMt). Protoplasts of LAMt line exhibited lower Al absorption levels than protoplasts of the L2 line. Use of two fluorescent tracers (morin and calcofluor white) indicated that Al interacts with internal cell structures, such as the plasma membrane and nucleus, with differences in both cell lines. Al-tolerance in the LAMt is probably associated with the cell wall as well as intracellular structures. These data will help to better understand Al toxicity in C. arabica, and Al toxicity mechanisms in plant cells.

  7. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

    Directory of Open Access Journals (Sweden)

    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  8. Salicylic acid antagonizes abscisic acid inhibition of shoot growth and cell cycle progression in rice

    Science.gov (United States)

    Meguro, Ayano; Sato, Yutaka

    2014-04-01

    We analysed effects of abscisic acid (ABA, a negative regulatory hormone), alone and in combination with positive or neutral hormones, including salicylic acid (SA), on rice growth and expression of cell cycle-related genes. ABA significantly inhibited shoot growth and induced expression of OsKRP4, OsKRP5, and OsKRP6. A yeast two-hybrid assay showed that OsKRP4, OsKRP5, and OsKRP6 interacted with OsCDKA;1 and/or OsCDKA;2. When SA was simultaneously supplied with ABA, the antagonistic effect of SA completely blocked ABA inhibition. SA also blocked ABA inhibition of DNA replication and thymidine incorporation in the shoot apical meristem. These results suggest that ABA arrests cell cycle progression by inducing expression of OsKRP4, OsKRP5, and OsKRP6, which inhibit the G1/S transition, and that SA antagonizes ABA by blocking expression of OsKRP genes.

  9. The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

    Directory of Open Access Journals (Sweden)

    Maycock Christopher

    2010-10-01

    Full Text Available Abstract Background Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

  10. 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.

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

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    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(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 [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone 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 (Abutilon theophrasti Medic.) 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.

  12. Uptake and Metabolism of Clomazone in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    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 I50 values for growth, chlorophyll (Chl), β-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 [14C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone 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 (Abutilon theophrasti Medic.) 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. PMID:16667349

  13. 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.

  14. 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)

    Abolghasem Abbasi Kajani

    2012-10-01

    Full Text Available 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.MethodsWe 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.ResultsThe 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.

  15. ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

    Energy Technology Data Exchange (ETDEWEB)

    David Gara, Pedro M. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Garabano, Natalia I. [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina); Llansola Portoles, Manuel J. [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Moreno, M. Sergio [Centro Atomico Bariloche (Argentina); Dodat, Diego; Casas, Oscar R. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Gonzalez, Monica C., E-mail: gonzalez@inifta.unlp.edu.ar [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Kotler, Monica L., E-mail: kotler@qb.fcen.uba.ar [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina)

    2012-03-15

    The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O{sub 2}{sup Bullet -}/HO{sub 2}{sup Bullet}, HO{sup Bullet}, and H{sub 2}O{sub 2} generated upon 4-MeV X-ray irradiation of 6.4 {mu}M silicon nanoparticle aqueous suspensions were on the order of 10 {mu}M per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic {sup 1}O{sub 2} was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO{sub 2} and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of <5 nm size have the potential to be used as radiosensitizers for improving the outcomes of cancer radiotherapy. Their capability of producing {sup 1}O{sub 2} upon X-ray irradiation opens novel approaches in the design of therapy strategies.

  16. "Fungal elicitors combined with a sucrose feed significantly enhance triterpene production of a Salvia fruticosa cell suspension".

    Science.gov (United States)

    Kümmritz, Sibylle; Louis, Marilena; Haas, Christiane; Oehmichen, Franz; Gantz, Stephanie; Delenk, Hubertus; Steudler, Susanne; Bley, Thomas; Steingroewer, Juliane

    2016-08-01

    Oleanolic (OA) and ursolic acid (UA) are plant secondary metabolites with diverse pharmacological properties. To reach reasonable productivities with plant cell suspension cultures, elicitation is a widely used strategy. Within the presented work, the effects of different elicitors on growth and production of OA and UA in a Salvia fruticosa cell suspension culture were examined. Beside commonly used elicitors like jasmonic acid (JA) and yeast extract, the influence of medium filtrates of the endophytic fungi Aspergillus niger and Trichoderma virens was investigated. The best eliciting effects were achieved with JA and fungal medium filtrates. Both increased the triterpene content by approximately 70 %. Since JA showed significant growth inhibition, the volumetric triterpene yield did not increase. But, adding fungal filtrates increased the volumetric triterpene yield by approximately 70 % to 32.6 mgOA l(-1) and 65.9 mgUA l(-1) for T. virens compared to the control with 19.4 mgOA l(-1) and 33.3 mgUA l(-1). An elicitation strategy combining fungal medium filtrate of T. virens with sucrose feeding significantly enhanced cell dry weight concentration to 22.2 g l(-1) as well as triterpene content by approximately 140 %. In total, this led to an approximately 500 % increase of volumetric triterpene yield referring to the control with final values of 112.9 mgOA l(-1) and 210.4 mgUA l(-1). Despite the doubled cultivation duration, productivities of 6.7 mgOA l(-1) day(-1) and 12.4 mgUA l(-1) day(-1) were reached. These results demonstrate methods by which increased productivities of triterpenes can be achieved to attain yields competing with intact plants.

  17. Zinc up-regulated the expression of the rice metallonthionein gene family and enhanced the zinc tolerance of yeast cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Northern blot and functional complementation assay were employed to analyze the effects of zinc on expression of ten rice metallothionein genes (OsMT-Is) in rice seedlings and the growth of yeast cells transformed with OsMT-Is. Northern blot revealed that in shoots of the rice seedlings treated with different Zn2+ concentrations, expression of most members of OsMT-I family was increased, except the type 4 OsMT-Is (OsMT-I-4a, 4b and 4c). In roots, Zn2+ significantly increased the transcription of OsMT-I-1b and OsMT-I-2c, but reduced the trascription of OsMT-I-1a and OsMT-I-3a. When these ten cDNAs were heterologously expressed in zinc sensitive yeast mutant, all transgenic yeasts showed increased tolerance to Zn2+, and zinc accumulation in these yeast cells also increased.These indicated that OsMT-I family members might respond to extra Zn2+, and they could enhance Zn2+ tolerance of cells by direct binding Zn2+.

  18. [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

  19. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

  20. The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application

    DEFF Research Database (Denmark)

    Dong, Jing; Bowra, Steve; Vincze, Éva

    2010-01-01

    suspension culture from both species. Results We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we......Background The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell...... chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed...

  1. Anti-Cancer Activity of Resveratrol and Derivatives Produced by Grapevine Cell Suspensions in a 14 L Stirred Bioreactor.

    Science.gov (United States)

    Nivelle, Laetitia; Hubert, Jane; Courot, Eric; Jeandet, Philippe; Aziz, Aziz; Nuzillard, Jean-Marc; Renault, Jean-Hugues; Clément, Christophe; Martiny, Laurent; Delmas, Dominique; Tarpin, Michel

    2017-03-16

    In the present study, resveratrol and various oligomeric derivatives were obtained from a 14 L bioreactor culture of elicited grapevine cell suspensions (Vitis labrusca L.). The crude ethyl acetate stilbene extract obtained from the culture medium was fractionated by centrifugal partition chromatography (CPC) using a gradient elution method and the major stilbenes contained in the fractions were subsequently identified by using a (13)C-NMR-based dereplication procedure and further 2D NMR analyses including HSQC, HMBC, and COSY. Beside δ-viniferin (2), leachianol F (4) and G (4'), four stilbenes (resveratrol (1), ε-viniferin (5), pallidol (3) and a newly characterized dimer (6)) were recovered as pure compounds in sufficient amounts to allow assessment of their biological activity on the cell growth of three different cell lines, including two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28) and a healthy human dermal fibroblast HDF line. Among the dimers obtained in this study, the newly characterized resveratrol dimer (6) has never been described in nature and its biological potential was evaluated here for the first time. ε-viniferin as well as dimer (6) showed IC50 values on the three tested cell lines lower than the ones exerted by resveratrol and pallidol. However, activities of the first two compounds were significantly decreased in the presence of fetal bovine serum although that of resveratrol and pallidol was not. The differential tumor activity exerted by resveratrol on healthy and cancer lines was also discussed.

  2. Anti-Cancer Activity of Resveratrol and Derivatives Produced by Grapevine Cell Suspensions in a 14 L Stirred Bioreactor

    Directory of Open Access Journals (Sweden)

    Laetitia Nivelle

    2017-03-01

    Full Text Available In the present study, resveratrol and various oligomeric derivatives were obtained from a 14 L bioreactor culture of elicited grapevine cell suspensions (Vitis labrusca L.. The crude ethyl acetate stilbene extract obtained from the culture medium was fractionated by centrifugal partition chromatography (CPC using a gradient elution method and the major stilbenes contained in the fractions were subsequently identified by using a 13C-NMR-based dereplication procedure and further 2D NMR analyses including HSQC, HMBC, and COSY. Beside δ-viniferin (2, leachianol F (4 and G (4′, four stilbenes (resveratrol (1, ε-viniferin (5, pallidol (3 and a newly characterized dimer (6 were recovered as pure compounds in sufficient amounts to allow assessment of their biological activity on the cell growth of three different cell lines, including two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28 and a healthy human dermal fibroblast HDF line. Among the dimers obtained in this study, the newly characterized resveratrol dimer (6 has never been described in nature and its biological potential was evaluated here for the first time. ε-viniferin as well as dimer (6 showed IC50 values on the three tested cell lines lower than the ones exerted by resveratrol and pallidol. However, activities of the first two compounds were significantly decreased in the presence of fetal bovine serum although that of resveratrol and pallidol was not. The differential tumor activity exerted by resveratrol on healthy and cancer lines was also discussed.

  3. Ultrasonic manipulation of yeast cells in suspension for absorption spectroscopy with an immersible mid-infrared fiberoptic probe.

    Science.gov (United States)

    Koch, Cosima; Brandstetter, Markus; Lendl, Bernhard; Radel, Stefan

    2013-06-01

    Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.

  4. Comparison of hindlimb unloading and partial weight suspension models for spaceflight-type condition induced effects on white blood cells

    Science.gov (United States)

    Wilson, Jolaine M; Krigsfeld, Gabriel S.; Sanzari, Jenine K.; Wagner, Erika B.; Mick, Rosemarie; Kennedy, Ann R.

    2013-01-01

    Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems. PMID:23766550

  5. Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm cells.

    Science.gov (United States)

    Popper, Zoë A; Fry, Stephen C

    2005-07-01

    Covalent linkages between xyloglucan and rhamnogalacturonan-I (RG-I) have been reported in the primary cell walls of cultured Rosa cells and may contribute to wall architecture. This study investigated whether this chemical feature is general to angiosperms or whether Rosa is unusual. * Xyloglucan was alkali-extracted from the walls of l-[1-3H]arabinose-fed suspension-cultured cells of Arabidopsis, sycamore, rose, tomato, spinach, maize and barley. The polysaccharide was precipitated with 50 % ethanol and subjected to anion-exchange chromatography in 8 m urea. Eluted fractions were Driselase-digested, yielding [3H]isoprimeverose (diagnostic of [3H]xyloglucan). The Arabidopsis cells were also fed [6-14C]glucuronic acid, and radiolabelled pectins were extracted with ammonium oxalate. * [3H]Xyloglucan was detected in acidic (galacturonate-containing) as well as non-anionic polysaccharide fractions. The proportion of the [3H]isoprimeverose units that were in anionic fractions was: Arabidopsis, 45 %; sycamore, 60 %; rose, 44 %; tomato, 75 %; spinach, 70 %; maize, 50 %; barley, 70 %. In Arabidopsis cultures fed d-[6-(14)C]glucuronate, 20 % of the (galacturonate-14C)-labelled pectins were found to hydrogen-bond to cellulose, a characteristic normally restricted to hemicelluloses such as xyloglucan. * Alkali-stable, anionic complexes of xyloglucan (reported in the case of Rosa to be xyloglucan-RG-I covalent complexes) are widespread in the cell walls of angiosperms, including gramineous monocots.

  6. [Analysis of the mechanism of intensification of fermentation process using yeast cells in a suspension of high-dispersed oxides].

    Science.gov (United States)

    Bagatskaya, A N; Mazurenko, R V; Makhno, S N; Gorbik, P P

    2014-01-01

    The differential microcalorimetry was used to explore an influence of particles of silicon dioxide, and also other high-dispersed oxides (0.05% of masses.) in water suspension of yeast cells on intensification of the process of their fermentation in endogenous metabolic conditions. It was shown that intensification of the processes of the vital activity of yeast microorganisms was observed in the specified interval of the concentration of silicon dioxide hydrosol particles. Mechanisms of interaction between SiO2 particles and a surface of a cellular organism, as well as interaction between SiO2 particles and one of metabolism products--carbon dioxide were studied. It was found out, that Al2O3, TiO2 hydrosols also had a stimulating effect, but it is lower compared to that of SiO2.

  7. 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.

  8. Rice Hypersensitive Induced Reaction Protein 1 (OsHIR1 associates with plasma membrane and triggers hypersensitive cell death

    Directory of Open Access Journals (Sweden)

    Sun Sai-Ming

    2010-12-01

    Full Text Available Abstract Background In plants, HIR (Hypersensitive Induced Reaction proteins, members of the PID (Proliferation, Ion and Death superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1 as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1. Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. Result Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. Conclusion The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant

  9. Discrimination of intra- and extracellular 23Na + signals in yeast cell suspensions using longitudinal magnetic resonance relaxography

    Science.gov (United States)

    Zhang, Yajie; Poirer-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2010-07-01

    This study tested the ability of MR relaxography (MRR) to discriminate intra- (Nai+) and extracellular (Nae+)23Na + signals using their longitudinal relaxation time constant ( T1) values. Na +-loaded yeast cell ( Saccharomyces cerevisiae) suspensions were investigated. Two types of compartmental 23Na +T1 differences were examined: a selective Nae+T1 decrease induced by an extracellular relaxation reagent (RR e), GdDOTP 5-; and, an intrinsic T1 difference. Parallel studies using the established method of 23Na MRS with an extracellular shift reagent (SR e), TmDOTP 5-, were used to validate the MRR measurements. With 12.8 mM RR e, the 23Nae+T1 was 2.4 ms and the 23Nai+T1 was 9.5 ms (9.4T, 24 °C). The Na + amounts and spontaneous efflux rate constants were found to be identical within experimental error whether measured by MRR/RR e or by MRS/SR e. Without RR e, the Na +-loaded yeast cell suspension 23Na MR signal exhibited two T1 values, 9.1 (±0.3) ms and 32.7 (±2.3) ms, assigned to 23Nai+ and 23Nae+, respectively. The Nai+ content measured was lower, 0.88 (±0.06); while Nae+ was higher, 1.43 (±0.12) compared with MRS/SR e measures on the same samples. However, the measured efflux rate constant was identical. T1 MRR potentially may be used for Nai+ determination in vivo and Na + flux measurements; with RR e for animal studies and without RR e for humans.

  10. Proteomics Coupled with Metabolite and Cell Wall Profiling Reveal Metabolic Processes of a Developing Rice Stem Internode

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Williams, Brad J.; Thangella, Padmavathi A. V.; Ladak, Adam; Schepmoes, Athena A.; Olivos, Hernando J.; Zhao, Kangmei; Callister, Stephen J.; Bartley, Laura E.

    2017-07-13

    Internodes of grass stems function in mechanical support, transport, and, in some species, are a major sink organ for carbon in the form of cell wall polymers. This study reports cell wall composition, proteomic and metabolite analyses of the rice elongating internode. Along eight segments of the second rice internode (internode II) at booting stage, cellulose, lignin, and xylose increase as a percentage of cell wall material from the younger to the older internode segments, indicating active cell wall synthesis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) of trypsin-digested peptides of size-fractionated proteins extracted from this internode at booting reveals 2547proteins with at least two unique peptides. The dataset includes many glycosyltransferases, acyltransferases, glycosyl hydrolases, cell wall-localized proteins, and protein kinases that have or may have functions in cell wall biosynthesis or remodeling. Phospho-enrichment of the internode II peptides identified 21 unique phosphopeptides belonging to 20 phosphoproteins including an LRR-III family receptor like kinase. GO over-representation and KEGG pathway analyses highlight the abundances of internode proteins involved in biosynthetic processes, especially the synthesis of secondary metabolites such as phenylpropanoids and flavonoids. LC-MS of hot methanol-extracted secondary metabolites from internode II at four stages (elongation, early mature, mature and post mature) indicates that secondary metabolites in stems are distinct from those of roots and leaves, and differ during stem maturation. This work fills a void of knowledge of proteomics and metabolomics data for grass stems, specifically for rice, and provides baseline knowledge for more detailed studies of cell wall synthesis and other biological processes during internode development, toward improving grass agronomic properties.

  11. Proteomics Coupled with Metabolite and Cell Wall Profiling Reveal Metabolic Processes of a Developing Rice Stem Internode

    Directory of Open Access Journals (Sweden)

    Fan Lin

    2017-07-01

    Full Text Available Internodes of grass stems function in mechanical support, transport, and, in some species, are a major sink organ for carbon in the form of cell wall polymers. This study reports cell wall composition, proteomic, and metabolite analyses of the rice elongating internode. Cellulose, lignin, and xylose increase as a percentage of cell wall material along eight segments of the second rice internode (internode II at booting stage, from the younger to the older internode segments, indicating active cell wall synthesis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS of trypsin-digested proteins from this internode at booting reveals 2,547 proteins with at least two unique peptides in two biological replicates. The dataset includes many glycosyltransferases, acyltransferases, glycosyl hydrolases, cell wall-localized proteins, and protein kinases that have or may have functions in cell wall biosynthesis or remodeling. Phospho-enrichment of internode II peptides identified 21 unique phosphopeptides belonging to 20 phosphoproteins including a leucine rich repeat-III family receptor like kinase. GO over-representation and KEGG pathway analyses highlight the abundances of proteins involved in biosynthetic processes, especially the synthesis of secondary metabolites such as phenylpropanoids and flavonoids. LC-MS/MS of hot methanol-extracted secondary metabolites from internode II at four stages (booting/elongation, early mature, mature, and post mature indicates that internode secondary metabolites are distinct from those of roots and leaves, and differ across stem maturation. This work fills a void of in-depth proteomics and metabolomics data for grass stems, specifically for rice, and provides baseline knowledge for more detailed studies of cell wall synthesis and other biological processes characteristic of internode development, toward improving grass agronomic properties.

  12. Rice Bran Feruloylated Oligosaccharides Activate Dendritic Cells via Toll-Like Receptor 2 and 4 Signaling

    Directory of Open Access Journals (Sweden)

    Chi Chen Lin

    2014-04-01

    Full Text Available This work presents the effects of feruloylated oligosaccharides (FOs of rice bran on murine bone marrow-derived dendritic cells (BMDCs and the potential pathway through which the effects are mediated. We found that FOs induced phenotypic maturation of DCs, as shown by the increased expression of CD40, CD80/CD86 and MHC-I/II molecules. FOs efficiently induced maturation of DCs generated from C3H/HeN or C57BL/6 mice with normal toll-like receptor 4 (TLR-4 or TLR-2 but not DCs from mice with mutated TLR4 or TLR2. The mechanism of action of FOs may be mediated by increased phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPKs and increased NF-kB activity, which are important signaling molecules downstream of TLR-4 and TLR-2. These data suggest that FOs induce DCs maturation through TLR-4 and/or TLR-2 and that FOs might have potential efficacy against tumor or virus infection or represent a candidate-adjuvant approach for application in immunotherapy and vaccination.

  13. 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.

  14. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/toxicity in human cell lines (Caco-2/HL-7702).

    Science.gov (United States)

    Aziz, Rukhsanda; Rafiq, Muhammad Tariq; Li, Tingqiang; Liu, Di; He, Zhenli; Stoffella, P J; Sun, Kewang; Xiaoe, Yang

    2015-04-01

    Cadmium (Cd) enters the food chain from polluted soils via contaminated cereals and vegetables; therefore, an understanding of Cd bioaccessibility, bioavailability, and toxicity in humans through rice grain is needed. This study assessed the Cd bioaccessibility, bioavailability, and toxicity to humans from rice grown on Cd-contaminated soils using an in vitro digestion method combined with a Caco-2/HL-7702 cell model. Cadmium bioaccessibility (18.45-30.41%) and bioavailability (4.04-8.62%) were found to be significantly higher in yellow soil (YS) rice than calcareous soil (CS) rice with the corresponding values of 6.89-11.43 and 1.77-2.25%, respectively. Toxicity assays showed an initial toxicity in YS rice at 6 mg kg(-1) Cd, whereas CS rice did not show any significant change due to low Cd concentrations. The acidic soils of Cd-contaminated areas can contribute to a higher dietary intake of Cd. Therefore, it is imperative to monitor Cd concentration in rice to minimize human health risk.

  15. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effect of carbon dioxide and ethylene on alkaloid production.

    Science.gov (United States)

    Kim, D I; Pedersen, H; Chin, C K

    1991-08-05

    Airlift bioreactor operations have been studied for the growth-associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas-stripping also played a significant role and it was discovered that CO(2) and ethylene were important for product formation. By supplying a mixture of CO(2) and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases.

  16. Testing automated liquid-based cytology samples with a manual liquid-based cytology method using residual cell suspensions from 500 ThinPrep cases.

    Science.gov (United States)

    Maksem, John A; Dhanwada, Vijaya; Trueblood, Joy E; Weidmann, James; Kane, Bruce; Bolick, David R; Bedrossian, Carlos W M; Kurtycz, Daniel F I; Stewart, Jim

    2006-06-01

    We report a technical improvement upon a previously disclosed manual liquid-based cytology (MLBC) method; and, we use the improved method to prepare slides from residual ThinPrep specimens in order to see how often ThinPrep diagnoses correspond to diagnoses derived from exhaustive examination of their parent sample suspensions. Residual cell suspensions from 500 ThinPrep cases comprising (1) 20 low-grade squamous intraepithelial lesions (LSILs); (2) 200 high risk (HR) negatives and 20 ASC-US; and (3) 260 screening cytology specimens were studied. Institutional review committee guidelines allowed us to know diagnoses by groups of specimens, but did not allow us to know individual patient diagnoses, so we could not perform case-by-case matched outcome-comparisons. Cells were concentrated by conventional centrifugation and sedimented into a polymer gel that was then vortex-mixed and converted into a viscous cell-rich suspension. The cell suspension was smeared between two clean glass slides, which were air-dried and stained with the Papanicolaou stain. Two study-sets were created, comprising one slide from each case. Each of the two study sets was examined by two cytopathologists, and discordant diagnoses were adjudicated. Because of the ambiguity involved in the "atypical" (ASC-US, ASC-H, AGC) diagnosis categories, only outcomes at the level of LSIL or greater were recorded. All MLBC SILs were digitally imaged and abnormal slides plus digital images were sent to the laboratory that provided the residual automated liquid-based cytology (ALBC) suspensions. The final diagnoses were confirmed by the laboratory that provided the residual ALBC specimens. MLBC slides of the 20 LSIL cases afforded 2 high-grade squamous intraepithelial lesions (HSILs) and 18 LSILs. Those of the 200 HR-Negatives showed 3 HSILs and 30 LSILs; and those of the 20 HR-ASC-US showed 3 HSILs and 9 LSILs. MLBC slides of the 260 screening cytology specimens showed 1 Carcinoma, 3 HSILs and 20 LSILs

  17. Habituation to thaxtomin A in hybrid poplar cell suspensions provides enhanced and durable resistance to inhibitors of cellulose synthesis

    Directory of Open Access Journals (Sweden)

    Beaulieu Carole

    2010-12-01

    Full Text Available Abstract Background Thaxtomin A (TA, a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA. Results Habituation to TA was performed by adding increasing levels of TA to cell cultures at the time of subculture over a period of 12 months. TA-habituated cells were then cultured in the absence of TA for more than three years. These cells displayed a reduced size and growth compared to control cells and had fragmented vacuoles filled with electron-dense material. Habituation to TA was associated with changes in the cell wall composition, with a reduction in cellulose and an increase in pectin levels. Remarkably, high level of resistance to TA was maintained in TA-habituated cells even after being cultured in the absence of TA. Moreover, these cells exhibited enhanced resistance to two other inhibitors of cellulose biosynthesis, dichlobenil and isoxaben. Analysis of gene expression in TA-habituated cells using an Affymetrix GeneChip Poplar Genome Array revealed that durable resistance to TA is associated with a major and complex reprogramming of gene expression implicating processes such as cell wall synthesis and modification, lignin and flavonoid synthesis, as well as DNA and chromatin modifications. Conclusions We have shown that habituation to TA induced durable resistance to the bacterial toxin in poplar cells. TA-habituation also enhanced resistance to two other structurally

  18. Expression patterns of the rice class I metallothionein gene family in response to lead stress in rice seedlings and functional complementation of its members in lead-sensitive yeast cells

    Institute of Scientific and Technical Information of China (English)

    XU YuFeng; ZHOU GongKe; ZHOU Lu; LI YiQin; LIU JinYuan

    2007-01-01

    Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions.In this paper, Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings.With the exception of OsMT-I-3b, the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots.Of these genes, transcription of OsMT-I-1a, OsMT-I-1b, OsMT-I-2c, OsMT-I-4a, OsMT-I-4b and OsMT-I-4c increased significantly, while transcription of OsMT-I-2a and OsMT-I-3a increased marginally.In contrast, the expression of OsMT-I-2b was inhibited.Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots, but had no obvious effects on the expression of OsMT-I-1a, OsMT-I-1b, OsMT-I-4a and OsMT-I-4b.All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells.These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate increased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is.This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress.

  19. THE CHANGE OF KINETIK PARAMETERS OF THE WEAK-ASSOCIATED WITH WALL CELL PEROXIDASE IN THE SUSPENSION CULTURE OF POTATO CELLS IN THE BEGINNING OF INFECTION

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2006-03-01

    Full Text Available The change in kinetic parameters of extracellular peroxidase of suspension cells of resistant potato variety (Lugovskoi and sensitive variety (Luk,ynovskii in the initial period of infection by 5369 Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. pathogen was examined. Extracellular peroxidases of resistant and sensitive potato variety without pathogens were shown to be concurrently inhibited. At the beginning of infection enzyme activity was extremely increased due to enhanced affinity to substrate as a result of reducing of competitive inhibiting. In increasing enzyme activity is sensitive potato variant evidently caused by other mechanism.

  20. Site of Clomazone Action in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [14C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [14C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID:16667768

  1. Adaptational changes in the lipids and fatty acid profile of the cell and thylakoid membrane of rice plants exposed to sunlight.

    Science.gov (United States)

    Vaz, Janet F; Sharma, Prabhat Kumar

    2010-07-01

    Adaptational changes occurring in the lipids and fatty acids of the cell and the thylakoid membrane in response to high light treatment, was studied in 30 days old rice (Oryza sativa L. cv. Jyothi) plants grown under low (150-200 μmol m(-2) s(-1)) or moderate (600-800 μmol m(-2) s(-1)) light conditions. Results were compared with rice plants grown in high (1200-2200 μmol m(-2) s(-1)) light conditions. Exposure of rice plants and isolated chloroplast to high light, resulted in an increase in the amount of malonaldehyde, indicating oxidation of membrane lipids. Qualitative and quantitative changes in the phosphoglycolipids and quantitative changes in neutral lipids were observed in rice plants grown under the different growth conditions. A few of the phosphoglycolipids and neutral lipids were present exclusively in plants grown at low or moderate or high light, indicating requirement of different type of lipid composition of rice plants in response to their different growth irradiances. However, no significant quantitative changes were observed in the different saturated and unsaturated fatty acid groups of total lipids in low, moderate and high light grown rice plants, as a result of exposure to high light. No qualitative changes in the fatty acid composition due to difference in growth irradiance or high light treatment were seen. The changes observed in the phosphoglycolipids and neutral lipid composition of cell and thylakoid membrane of low, moderate and high light grown rice plants in response to high light, are probably the result of physiological changes in the rice plants, to sustain optimum structure and function of the cell and thylakoid membrane to maintain active physiological functions to endure high light conditions.

  2. RICE CROSSES

    African Journals Online (AJOL)

    Spikelet sterility is the greatest barrier to rice hybridization. ... sterility, Hybridization, Duplicate recessive epis- tasis ' ... hybrid fertility of intra-subspecific crosses, i.e., indica by ..... 'Germ 59(1) 293$ ' ' male sterility and fertility restoration in rice.

  3. [Enhanced production of taxuyunnanine c in cell suspension cultures of Taxus chinensis by methyl jasmonate elicitation and in situ absorption].

    Science.gov (United States)

    Gao, Mingbo; Zhang, Wei; Yu, Xingju

    2010-02-01

    A bioprocess intensification strategy that combines both elicitation and in situ absorption was developed to improve the production of taxuyunnanine c (Tc) in cell suspension cultures of Taxus chinensis. When 100 micromol/L methyl jasmonate was added as an elicitor on Day 7, the Tc content and yield increased 3.6 and 3.3 times respectively, however the cell growth was reduced by 10%-30%. Significant improvement in Tc yield was observed when an absorbent XAD-7 was added on different time of the culture period. The optimum Tc yield was achieved when 100 g/L XAD-7 was added simultaneously with 100 micromol/L methyl jasmonate on Day 7. The maximum Tc yield of 477.4 mg/L was obtained on Day 21 of the culture, being 6.3-fold of the control and 1.9-fold of the 100 micromol/L methyl jasmonate treatment alone. In the combined treatment, 94% of the Tc produced was secreted outside of the cells and absorbed on XAD-7 absorbents. The results demonstrated that the process strategy combining elicitation and in situ absorption was effective to intensify the Tc biosynthesis via elicitation with the removal of product feedback inhibition via absorption, presenting a great potential in commercial applications.

  4. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Sepúlveda-Jiménez, Gabriela; Trejo-Espino, José Luis; Cerda-García-Rojas, Carlos M; de la Torre, Mayra; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2007-09-01

    Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

  5. Characterization of Nanoparticle Dispersion in Red Blood Cell Suspension by the Lattice Boltzmann-Immersed Boundary Method

    Directory of Open Access Journals (Sweden)

    Jifu Tan

    2016-02-01

    Full Text Available Nanodrug-carrier delivery in the blood stream is strongly influenced by nanoparticle (NP dispersion. This paper presents a numerical study on NP transport and dispersion in red blood cell (RBC suspensions under shear and channel flow conditions, utilizing an immersed boundary fluid-structure interaction model with a lattice Boltzmann fluid solver, an elastic cell membrane model and a particle motion model driven by both hydrodynamic loading and Brownian dynamics. The model can capture the multiphase features of the blood flow. Simulations were performed to obtain an empirical formula to predict NP dispersion rate for a range of shear rates and cell concentrations. NP dispersion rate predictions from the formula were then compared to observations from previous experimental and numerical studies. The proposed formula is shown to accurately predict the NP dispersion rate. The simulation results also confirm previous findings that the NP dispersion rate is strongly influenced by local disturbances in the flow due to RBC motion and deformation. The proposed formula provides an efficient method for estimating the NP dispersion rate in modeling NP transport in large-scale vascular networks without explicit RBC and NP models.

  6. Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

    Science.gov (United States)

    Hamerski, D; Schmitt, D; Matern, U

    1990-01-01

    Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

  7. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-09

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  8. An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells.

    Science.gov (United States)

    Bown, A W

    1982-09-01

    Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H(+) efflux. Rates varied between 0.12 and 1.99 nanomoles H(+) per 10(6) cells per minute or 3 and 40 nanomoles H(+) per milligram chlorophyll per minute. The mean rate of H(+) efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H(+) efflux from illuminated cells. Bubbling with N(2) or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H(+) efflux in light or dark. In the absence of aeration, H(+) efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO(2)-free air, rates of H(+) efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO(2) fixation but not photosynthetic CO(2) fixation. It is suggested that H(+) efflux is dependent on respiration and dark CO(2) fixation, but independent of photosynthesis.

  9. Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins.

    Science.gov (United States)

    Roowi, Siti Habsah; Ho, Chai-Ling; Alwee, Sharifah Shahrul Rabiah Syed; Abdullah, Meilina Ong; Napis, Suhaimi

    2010-09-01

    Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.

  10. SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

    Science.gov (United States)

    Li, Jintao; Zhao, Yu; Chu, Huangwei; Wang, Likai; Fu, Yanru; Liu, Ping; Upadhyaya, Narayana; Chen, Chunli; Mou, Tongmin; Feng, Yuqi; Kumar, Prakash; Xu, Jian

    2015-08-01

    Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb), a mild gibberellin (GA) deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

  11. SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

    Directory of Open Access Journals (Sweden)

    Jintao Li

    2015-08-01

    Full Text Available Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb, a mild gibberellin (GA deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

  12. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  13. Treating Cutaneous T-cell Lymphoma with Highly Irregular Surfaces with Photon Irradiation Using Rice as Tissue Compensator

    Directory of Open Access Journals (Sweden)

    Lonika eMajithia

    2015-02-01

    Full Text Available Purpose: Cutaneous T-cell lymphoma (CTCL is known to have an excellent response to radiotherapy, an important treatment modality for this disease. In patients with extremity and digit involvement, the irregular surface and depth variations create difficulty in delivering a homogenous dose using electrons. We sought to evaluate photon irradiation with rice packing as tissue equivalence and determine clinical tolerance and response. Materials and Methods: Three consecutive CTCL patients with extensive lower extremity involvement including the digits were treated using external beam photon therapy with rice packing for tissue compensation. The entire foot was treated to 30-40 Gy in 2-3 Gy per fraction using 6 MV photons prescribed to the mid-plane of an indexed box filled with rice in which the foot was placed. Optically stimulated luminescence dosimeter (OSLD was used for dose measurement to determine the dose deposition to the skin surface. Treatment tolerance and response were monitored with clinical evaluation. Results: All patients tolerated the treatment without treatment breaks. Toxicities included grade 3 erythema and desquamation with resolution within 4 weeks. No late toxicities were observed. All four treated sites had partial response (PR by the end of the treatment course. All patients reported improved functionality after treatment, with less pain, drainage, or swelling. No local recurrence has been observed in these patients with a median follow-up time of 14 months. Conclusion: Tissue compensation with rice packing offers a convenient, inexpensive and reproducible method for the treatment of CTCL with highly irregular surfaces.

  14. Effect of Plant Growth Regulators on Callus, Cell Suspension and Cell Line Selection for Flavonoid Production from Pegaga (centella asiatica L. urban

    Directory of Open Access Journals (Sweden)

    Suat H. Tan

    2010-01-01

    Full Text Available Problem statement: Considering pegaga medicinal properties and over-exploitation, the requirement for a tissue culture technique as an alternative production system was crucial. Approach: Investigation of cell suspension culture response to different plant growth regulators (PRGs for flavonoid production from elite cell line was carried out. Callus cultures were initiated from the leaf explants of Centella asiatica on Murashige and Skoog (MS medium containing B5 vitamins and 30 g L−1 sucrose supplemented with different concentrations (0.5-2.5 mg L−1 of 2,4-D, NAA, Dicamba, Picloram and IBA supplied singly and in combination with different concentrations (0.5-1.5 mg L−1 of kinetin, BAP and TDZ. Results: Callus induction was observed for all the PGRs tested. The highest callus induction frequency (86.67% was observed in MS medium containing 2.0 mg L−1 2,4-D while the combination of 2.0 mg L−1 2,4-D and 1 mg L−1 kinetin in MS medium gave the highest biomass yield (0.27 g dry weight culture−1. This combination was also found to be best for callus proliferation for all the accessions investigated. Among the four accessions tested, UPM03 was found to have the highest biomass yield (0.041 g DW culture−1 and hydrolysed flavonoid content (10.75 mg g−1 DW after the 12th day of culture. The flavonoids present in the four accessions were quercetin, kaempherol, luteolin and rutin based on high performance liquid chromatography (HPLC analysis. These results indicated that C. asiatica accession UPM03 was the potential elite cell line in mass production of flavonoid, especially luteolin. Coclusions/Recommendations: In the establishment of cell suspension culture, 2 mg L−1 2,4-D and 1 mg L−1 kinetin were the best PGRs in supporting the cell growth and flavonoid production. This is the first report on the use of PRGs on the establishment of cell suspension cultures in flavonoid production of C. asiatica.

  15. 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.

  16. INSECTICIDAL POTENTIALITY OF FLAVONOIDS FROM CELL SUSPENSION CULTURE OF MARCHANTIA LINEARIS LEHM. & LINDENB AGAINST SPODOPTERA LITURA F.

    Directory of Open Access Journals (Sweden)

    Remya Krishnan

    2015-05-01

    Full Text Available Bryophytes were diverse, primitive non vascular am phibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing gr owth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lowe r rpm bring about lower leve l of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quer cetin, luteolin, apigenin , rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedan t, larvicidal and pupicidal activities at all the concentrations against 5 th instar larvae of Spodoptera litura . The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food and ECD (Efficiency of conversion of digested food and increased AD (Approximate digestibility and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the co nsumption of the control diet by the larvae. Faecal production reduced proportionally with

  17. Aluminium-induced phospholipid signal transduction pathway in Coffea arabica suspension cells and its amelioration by silicic acid.

    Science.gov (United States)

    Quintal-Tun, Fausto; Muñoz-Sánchez, J Armando; Ramos-Díaz, Ana; Escamilla-Bencomo, Armando; Martínez-Estévez, Manuel; Exley, Christopher; Hernández-Sotomayor, S M Teresa

    2007-02-01

    Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.

  18. Flow-Through Electroporation of HL-60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes.

    Science.gov (United States)

    Chen, Zhiqiang; Akenhead, Michael A; Sun, Xinghua; Sapper, Harrison; Shin, Hainsworth Y; Hinds, Bruce J

    2016-08-01

    A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10μm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. 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.

  20. 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.

  1. Identification of Quantitative Trait Loci Affecting Hemicellulose Characteristics Based on Cell Wall Composition in a Wild and Cultivated Rice Species

    Institute of Scientific and Technical Information of China (English)

    Si-Ju Zhang; Xue-Qin Song; Bai-Sheng Yu; Bao-Cai Zhang; Chuan-Qing Sun; J. Paul Knox; Yi-Hua Zhou

    2012-01-01

    Cell wall hemicellulosic polysaccharides are structurally complex and diverse.Knowledge about the synthesisof cell wall hemicelluloses and their biological roles is limited.Quantitative trait loci (QTL) mapping is a helpful tool for the dissection of complex phenotypes for gene identification.In this study,we exploited the natural variation in cell wall monosaccharide levels between a common wild rice,Yuanj,and an elite indica cultivar,Teqing,and performed QTL mapping with their introgression lines (ILs).Chemical analyses conducted on the culms of Yuanj and Teqing showed that the major alterations are found in glucose and xylose levels,which are correlated with specific hemicellulosic polymers.Glycosidic linkage examination revealed that,in Yuanj,an increase in glucose content results from a higher level of mixed linkage β-glucan (MLG),whereas a reduction in xylose content reflects a low level of xylan backbone and a varied arabinoxylan (AX) structure.Seventeen QTLs for monosaccharides have been identified through composition analysis of the culm residues of 95 core ILs.Four major QTLs affecting xylose and glucose levels are responsible for 19 and 21% of the phenotypic variance,respectively.This study provides a unique resource for the genetic dissection of rice cell wall formation and remodeling in the vegetative organs.

  2. Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice

    Institute of Scientific and Technical Information of China (English)

    Tae-Il Jeon; Chang-Hwa Jung; Jeong-Yong Cho; Dong Ki Park; Jae-Hak Moon

    2013-01-01

    Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). Methods: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. Results: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one-and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.

  3. First insights in the Eu(III) speciation in plant cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Moll, Henry; Sachs, Susanne [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biogeochemistry

    2017-06-01

    More than 80 % of the initial Eu(III) amount was associated on Brassica napus cells after an incubation time of 24 h. The spectroscopic speciation of the cell-bound Eu(III) is characterized by an increased intensity of the {sup 7}F{sub 2} transition and prolonged luminescence lifetimes.

  4. Measurement of rapid membrane permeation in cell suspensions by application of a generalized capillary method

    DEFF Research Database (Denmark)

    Klösgen, Beate; Schönert, Hansjürgen; Deuticke, Bernhard

    1988-01-01

    the diffusion process of a solute in a composite system was derived using a series-parallel-pathway model with explicit consideration of the diffusion pathways inside and between the cells. This renders the technique insensitive to unstirred layer effects. Any single cell population of known size distribution...

  5. Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis

    NARCIS (Netherlands)

    Munoz-Pinedo, Cristina; Green, Douglas R.; Berg, van den Albert

    2005-01-01

    We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF- and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phospha

  6. Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis.

    Science.gov (United States)

    Muñoz-Pinedo, Cristina; Green, Douglas R; van den Berg, Albert

    2005-06-01

    We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF-alpha and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phosphatidyl-serine, membrane blebbing and permeabilization of the cell membrane have been observed in real time with different individual cells. From the results, an interesting heterogeneicity in the apoptotic phenotype has been observed. The CRISC method is easy to use and provides biologist with a powerful additional tool to study in real-time processes of several hours of duration such as apoptosis. We predict that the period of cell viability obtained after protein coating of the PDMS devices (>80 h) will also allow monitoring of other biological processes of longer duration or long onset time, such as mitosis, phagocytosis and differentiation.

  7. Shading Contributes to the Reduction of Stem Mechanical Strength by Decreasing Cell Wall Synthesis in Japonica Rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Longmei Wu

    2017-05-01

    Full Text Available Low solar radiation caused by industrial development and solar dimming has become a limitation in crop production in China. It is widely accepted that low solar radiation influences many aspects of plant development, including slender, weak stems and susceptibility to lodging. However, the underlying mechanisms are not well understood. To clarify how low solar radiation affects stem mechanical strength formation and lodging resistance, the japonica rice cultivars Wuyunjing23 (lodging-resistant and W3668 (lodging-susceptible were grown under field conditions with normal light (Control and shading (the incident light was reduced by 60% with a black nylon net. The yield and yield components, plant morphological characteristics, the stem mechanical strength, cell wall components, culm microstructure, gene expression correlated with cellulose and lignin biosynthesis were measured. The results showed that shading significantly reduced grain yield attributed to reduction of spikelets per panicles and grain weight. The stem-breaking strength decreased significantly under shading treatment; consequently, resulting in higher lodging index in rice plant in both varieties, as revealed by decreased by culm diameter, culm wall thickness and increased plant height, gravity center height. Compared with control, cell wall components including non-structural carbohydrate, sucrose, cellulose, and lignin reduced quite higher. With histochemical straining, shading largely reduced lignin deposition in the sclerenchyma cells and vascular bundle cells compared with control, and decreased cellulose deposition in the parenchyma cells of culm tissue in both Wuyunjing23 and W3668. And under shading condition, gene expression involved in secondary cell wall synthesis, OsPAL, OsCOMT, OsCCoAOMT, OsCCR, and OsCAD2, and primary cell wall synthesis, OsCesA1, OsCesA3, and OsCesA8 were decreased significantly. These results suggest that gene expression involved in the reduction of

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

    Science.gov (United States)

    Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

    2016-01-01

    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.

  9. Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Macherel, D; Kobayashi, H; Akazawa, T

    1988-01-01

    Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

  10. Studies of Frequency–Dependent Changes under Modulated Ultrasound Exposure on Cells in Suspension

    Directory of Open Access Journals (Sweden)

    Anna A. Oleshkevich

    2015-03-01

    Full Text Available Characteristics of the modulated ultrasound effect (range of modulation frequencies 10Hz–1000Hz intensity 0.2 W/cm2 on white blood cells (WBCs from different animals were studied. The quantitative ratio of WBCs was the most strongly altered by ultrasonic modulation frequency 1000 Hz. This frequency led to degenerative changes of cells. The presence of non-typeable or destroyed cells in smears was indicated. The results obtained demonstrated the possibility of directed impact on different WBC forms.

  11. Unifying suspension and granular rheology.

    Science.gov (United States)

    Boyer, François; Guazzelli, Élisabeth; Pouliquen, Olivier

    2011-10-28

    Using an original pressure-imposed shear cell, we study the rheology of dense suspensions. We show that they exhibit a viscoplastic behavior similarly to granular media successfully described by a frictional rheology and fully characterized by the evolution of the friction coefficient μ and the volume fraction ϕ with a dimensionless viscous number I(v). Dense suspension and granular media are thus unified under a common framework. These results are shown to be compatible with classical empirical models of suspension rheology and provide a clear determination of constitutive laws close to the jamming transition.

  12. Microbial Community Analysis of Anodes from Sediment Microbial Fuel Cells Powered by Rhizodeposits of Living Rice Plants ▿ †

    Science.gov (United States)

    De Schamphelaire, Liesje; Cabezas, Angela; Marzorati, Massimo; Friedrich, Michael W.; Boon, Nico; Verstraete, Willy

    2010-01-01

    By placing the anode of a sediment microbial fuel cell (SMFC) in the rhizosphere of a rice plant, root-excreted rhizodeposits can be microbially oxidized with concomitant current generation. Here, various molecular techniques were used to characterize the composition of bacterial and archaeal communities on such anodes, as influenced by electrical circuitry, sediment matrix, and the presence of plants. Closed-circuit anodes in potting soil were enriched with Desulfobulbus-like species, members of the family Geobacteraceae, and as yet uncultured representatives of the domain Archaea. PMID:20097806

  13. Geobacter, Anaeromyxobacter and Anaerolineae populations are enriched on anodes of root exudate-driven microbial fuel cells in rice field soil.

    Science.gov (United States)

    Cabezas, Angela; Pommerenke, Bianca; Boon, Nico; Friedrich, Michael W

    2015-06-01

    Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC.

  14. Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells

    Science.gov (United States)

    Alfano, Randall; Youngblood, Bradford A; Zhang, Deshui; Huang, Ning; MacDonald, Clinton C

    2014-01-01

    Stem cell-based therapy has the potential to treat an array of human diseases. However, to study the therapeutic potential and safety of these cells, a scalable cell culture medium is needed that is free of human or bovine-derived serum proteins. Thus, cost-effective recombinant serum proteins and cytokines are needed to produce such mediums. One such cytokine, leukemia inhibitory factor (LIF), has been shown to be a critical paracrine factor that maintains stem cell pluripotency in murine embryonic stem cells and human naïve stem cells while simultaneously inhibiting differentiation. We recently produced recombinant human LIF (rhLIF) in a rice-based protein expression system known as ExpressTec.12 We described expression of rice-derived rhLIF and demonstrated its biological equivalency to E. coli-derived rhLIF in traditional and embryonic mouse stem cell systems. Here we describe the expression yield of rice-derived rhLIF and the scale up production capacity. We provide further evidence of the efficacy of rice-derived rhLIF in additional stem cell systems including human neural stem cells and mouse induced pluripotent stem (iPS) cells. The expression level, biological activity, and potential for production at commercial scale of rice-derived rhLIF provides a proof-of-principal for ExpressTec-derived proteins to produce regulatory-friendly, high performance, and dependable stem cell media. PMID:24776984

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

    National Research Council Canada - National Science Library

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

    .... 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...

  16. Electrical Impedance Spectroscopy for Detection of Cells in Suspensions Using Microfluidic Device with Integrated Microneedles

    National Research Council Canada - National Science Library

    Mansor, Muhammad; Takeuchi, Masaru; Nakajima, Masahiro; Hasegawa, Yasuhisa; Ahmad, Mohd

    2017-01-01

    .... The state of the art method for impedance flow cytometry detection utilizes an embedded electrode in the microfluidic to perform measurement of electrical impedance of the presence of cells at the sensing area...

  17. Novel O-D-Galacturonoyl Esters in the Pectic Polysaccharides of Suspension-Cultured Plant Cells

    National Research Council Canada - National Science Library

    John A. Brown; Stephen C. Fry

    1993-01-01

    Driselase digestion of uronate-6- C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo...

  18. Numerical simulation of red blood cell suspensions behind a moving interface in a capillary

    CERN Document Server

    Zhao, Shihai

    2013-01-01

    Computational modeling and simulation are presented on the motion of red blood cells behind a moving interface in a capillary. The methodology is based on an immersed boundary method and the skeleton structure of the red blood cell (RBC) membrane is modeled as a spring network. The computational domain is moving with either a designated RBC or an interface in an infinitely long two-dimensional channel with an undisturbed flow field in front of the domain. The tanking-treading and the inclination angle of a cell in a simple shear flow are briefly discussed for the validation purpose. We then present the results of the motion of red blood cells behind a moving interface in a capillary, which show that the RBCs with higher velocity than the interface speed form a concentrated slug behind the interface.

  19. Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

    Science.gov (United States)

    Kagenishi, Tomoko; Yokawa, Ken; Kuse, Masaki; Isobe, Minoru; Bouteau, François; Kawano, Tomonori

    2009-01-01

    Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

  20. Optimizing the production of suspension cells using the G-Rex “M” series

    Directory of Open Access Journals (Sweden)

    Pradip Bajgain

    2014-01-01

    Full Text Available Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex “M” series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

  1. Rice Homeodomain Protein WOX11 Recruits a Histone Acetyltransferase Complex to Establish Programs of Cell Proliferation of Crown Root Meristem.

    Science.gov (United States)

    Zhou, Shaoli; Jiang, Wei; Long, Fei; Cheng, Saifeng; Yang, Wenjing; Zhao, Yu; Zhou, Dao-Xiu

    2017-05-01

    Shoot-borne crown roots are the major root system in cereals. Previous work has shown that the Wuschel-related homeobox gene WOX11 is necessary and sufficient to promote rice (Oryza sativa) crown root emergence and elongation. Here, we show that WOX11 recruits the ADA2-GCN5 histone acetyltransferase module to activate downstream target genes in crown root meristem. Rice ADA2 and GCN5 genes are highly expressed in root meristem and are shown to be essential for cell division and growth. WOX11 and ADA2-GCN5 commonly target and regulate a set of root-specific genes involved in energy metabolism, cell wall biosynthesis, and hormone response, some of which are known to be important for root development. The results indicate that the recruitment of ADA2-GCN5 by WOX11 establishes gene expression programs of crown root meristem cell division and suggest that permissive chromatin modification involving histone acetylation is a strategy for WOX11 to stimulate root meristem development. © 2017 American Society of Plant Biologists. All rights reserved.

  2. A conserved function for Arabidopsis SUPERMAN in regulating floral-whorl cell proliferation in rice, a monocotyledonous plant.

    Science.gov (United States)

    Nandi, A K; Kushalappa, K; Prasad, K; Vijayraghavan, U

    2000-02-24

    Studies of floral organ development in two dicotyledonous plants, Arabidopsis thaliana and Antirrhinum majus, have shown that three sets of genes (A, B and C) can pattern sepals, petals, stamens and carpels [1] [2]. Mechanisms that define boundaries between these floral whorls are unclear, however. The Arabidopsis gene SUPERMAN (SUP), which encodes a putative transcription factor, maintains the boundary between stamens and carpels [3] [4] [5], possibly by regulating cell proliferation. By overexpressing SUP cDNA in rice, we examined whether its effects on whorl boundaries are conserved in a divergent monocotyledonous species. High-level ectopic SUP expression in transgenic rice resulted in juvenile death or dwarf plants with decreased axillary growth. Plants with lower levels of SUP RNA were vegetatively normal, but the flowers showed ubiquitous ventral carpel expansion. This was often coupled with reduced stamen number, or occurrence of third-whorl stamen-carpel mosaic organs. Additionally, proliferation of second-whorl ventral cells produced adventitious lodicules, and flowers lost the asymmetry that is normally inherent to this whorl. We predict that SUP is a conserved regulator of floral whorl boundaries and that it affects cell proliferation.

  3. 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.

  4. 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.

  5. Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

    Directory of Open Access Journals (Sweden)

    Alaín González Pose

    2014-09-01

    Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

  6. Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

    Science.gov (United States)

    Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

    2009-03-01

    Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

  7. Lift and down-gradient shear-induced diffusion in Red Blood Cell suspensions

    CERN Document Server

    Grandchamp, Xavier; Srivastav, Aparna; Minetti, Christophe; Podgorski, Thomas

    2013-01-01

    The distribution of Red Blood Cells in a confined channel flow is inhomogeneous and shows a marked depletion near the walls due to a competition between migration away from the walls and shear-induced diffusion resulting from interactions between particles. We investigated the lift of RBCs in a shear flow near a wall and measured a significant lift velocity despite the tumbling motion of cells. We also provide values for the collective and anisotropic shear-induced diffusion of a cloud of RBCs, both in the direction of shear and in the direction of vorticity. A generic down-gradient subdiffusion characterized by an exponent 1/3 is highlighted.

  8. Identification and quantitative determination of pinoresinol in Taxus ×media Rehder needles, cell suspension and shoot cultures

    Directory of Open Access Journals (Sweden)

    Paulina Mistrzak

    2015-01-01

    Full Text Available The aim of our study was to investigate the presence and quantitative contents of lignans in the tissues of Taxus ×media. The presence of the lignans: pinoresinol, matairesinol and secoisolariciresinol was assessed in needles, shoots cultures and suspension culture. Pinoresinol was the only lignan found in the tissue of T. ×media. The total pinoresinol content in the needles and in the shoots was 1.24 mg/g dry weight (dw and 0.69 mg/g dw, respectively. Most of the pinoresinol identified was appeared glycosidically bound. In needles, the amount of glycosidically bound pinoresinol (0.81 mg/g dw was about twice as high as that of free pinoresinol (0.43 mg/g dw. The content of free and glycosidically bound pinoresinol showed the level of 0.18 mg/g dw and 0.51 mg/g dw, respectively in the in vitro shoot cultures. In the cell culture, no pinoresinol was found.

  9. Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Mitsui, T; Akazawa, T

    1986-12-01

    Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

  10. Electrical Impedance Spectroscopy for Detection of Cells in Suspensions Using Microfluidic Device with Integrated Microneedles

    Directory of Open Access Journals (Sweden)

    Muhammad Asraf Mansor

    2017-02-01

    Full Text Available In this study, we introduce novel method of flow cytometry for cell detection based on impedance measurements. The state of the art method for impedance flow cytometry detection utilizes an embedded electrode in the microfluidic to perform measurement of electrical impedance of the presence of cells at the sensing area. Nonetheless, this method requires an expensive and complicated electrode fabrication process. Furthermore, reuse of the fabricated electrode also requires an intensive and tedious cleaning process. Due to that, we present a microfluidic device with integrated microneedles. The two microneedles are placed at the half height of the microchannel for cell detection and electrical measurement. A commercially-available Tungsten needle was utilized for the microneedles. The microneedles are easily removed from the disposable PDMS (Polydimethylsiloxane microchannel and can be reused with a simple cleaning process, such as washing by ultrasonic cleaning. Although this device was low cost, it preserves the core functionality of the sensor, which is capable of detecting passing cells at the sensing area. Therefore, this device is suitable for low-cost medical and food safety screening and testing process in developing countries.

  11. Evaluation of simulated microgravity environments induced by diamagnetic levitation of plant cell suspension cultures

    NARCIS (Netherlands)

    Kamal, K.Y.; Herranz, R.; van Loon, J.J.W.A.; Christianen, P.C.M.; Medina, F.J.

    2016-01-01

    Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell prolifer

  12. Effect of phytic acid from rice and corn on morphology, cell proliferation, apoptosis and cyclooxygenase-2 expression in swine jejunal explants

    Directory of Open Access Journals (Sweden)

    Elisângela Olegário da Silva

    2014-06-01

    Full Text Available Phytic acid (IP6 is a potent antioxidant present in several natural foods. Beneficial effects on colon cancer and inflammation have been associated to IP6 in several studies, however, scarce data about the effect on small intestine are available. The aim of the present study was to evaluate the effect of different doses of IP6 from rice and corn on intestinal morphology, cellular proliferation, apoptosis and cyclooxygenase-2 (Cox-2 expression using swine jejunal explants as experimental model. This report demonstrated that explants treated with 0.5 mM, 2.5 mM and 5 mM of IP6 from rice and 2.5 mM and 5 mM from corn showed higher villi height compared to control. Explants treated with 2.5 mM and 5 mM IP6 from rice exhibited a significant reduction on intestinal histological changes (villi atrophy and fusion, edema, lymphatic vessel dilation, loss of apical enterocytes, cell vacuolation, necrotic debris, morphology of enterocytes and microvilli and number of villi. The cellular proliferation decreased in the explants treated with the dosages of 2.5 mM and 5 mM from rice and a significant decrease in cell apoptosis was observed in the treatments with 2.5 mM IP6 from rice and 5 mM IP6 from corn compared to the control. The explants treated with 2.5 mM and 5 mM IP6 from rice and corn showed a significant reduction of the Cox-2 expression. Higher dosages of IP6 from rice and corn used in this experiment increased the viability and preservation of intestinal tissue as evidenced by morphological and immunohistochemical assays.

  13. Phenylpropanoids, Phenylalanine Ammonia Lyase and Peroxidases in Elicitor‐challenged Cassava (Manihot esculenta) Suspension Cells and Leaves

    Science.gov (United States)

    GÓMEZ‐VÁSQUEZ, ROCÍO; DAY, ROBERT; BUSCHMANN, HOLGER; RANDLES, SOPHIE; BEECHING, JOHN R.; COOPER, RICHARD M.

    2004-01-01

    • Background and aims Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. • Methods Phenylalanine ammonia‐lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. • Key results A single and rapid (≥2–3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four‐fold in cells, 48 h post‐elicitation. POD isoforms (2–7 isoforms, pI 3·1–8·8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post‐elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3·6, present in all samples. Unidentified phenolics and possibly scopolin increased post‐elicitation, but there was no enhancement of scopoletin, rutin or kaempferol‐3‐O‐rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by ≥50 µg mL–1 of ferulic acid and quercetin and ≥10 µg mL–1 of scopoletin. • Conclusions Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and

  14. Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry.

    Science.gov (United States)

    Kvitvang, Hans F N; Bruheim, Per

    2015-08-15

    Capillary ion chromatography (capIC) is the premium separation technology for low molecular phosphometabolites and nucleotides in biological extracts. Removal of excessive amounts of salt during sample preparation stages is a prerequisite to enable high quality capIC separation in combination with reproducible and sensitive MS detection. Existing sampling protocols for mammalian cells used for GC-MS and LC-MS metabolic profiling can therefore not be directly applied to capIC separations. Here, the development of a fast filtration sampling protocol for mammalian suspension cells tailored for quantitative profiling of the phosphometabolome on capIC-MS/MS is presented. The whole procedure from sampling the culture to transfer of filter to quenching and extraction solution takes less than 10s. To prevent leakage it is critical that a low vacuum pressure is applied, and satisfactorily reproducibility was only obtained by usage of a vacuum pressure controlling device. A vacuum of 60mbar was optimal for filtration of multiple myeloma Jjn-3 cell cultures through 5μm polyvinylidene (PVDF) filters. A quick deionized water (DI-water) rinse step prior to extraction was tested, and significantly higher metabolite yields were obtained during capIC-MS/MS analyses in this extract compared to extracts prepared by saline and reduced saline (25%) washing steps only. In addition, chromatographic performance was dramatically improved. Thus, it was verified that a quick DI-water rinse is tolerated by the cells and can be included as the final stage during filtration. Over 30 metabolites were quantitated in JJN-3 cell extracts by using the optimized sampling protocol with subsequent capIC-MS/MS analysis, and up to 2 million cells can be used in a single filtration step for the chosen filter and vacuum pressure. The technical set-up is also highly advantageous for microbial metabolome filtration protocols after optimization of vacuum pressure and washing solutions, and the reduced salt

  15. Microbial cell disruption for improving lipid recovery using pressurized CO2 : Role of CO2 solubility in cell suspension, sugar broth, and spent media.

    Science.gov (United States)

    Howlader, Md Shamim; French, William Todd; Shields-Menard, Sara A; Amirsadeghi, Marta; Green, Magan; Rai, Neeraj

    2017-05-01

    The study of in situ gas explosion to lyse the triglyceride-rich cells involves the solubilization of gas (e.g., carbon dioxide, CO2 ) in lipid-rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO2 as well as to determine the solubility of CO2 in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2,000 and 3,500 kPa, respectively, at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO2 . We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3,500 kPa compared to control (no pressure) and P = 2,000 kPa, which implied that more intracellular lipid was released due to the pressurized CO2 . Solubility of CO2 in R. glutinis cell suspension was found to be higher than the solubility of CO2 in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO2 were found to be -14.22 kJ mol(-1) and 48.10 kJ mol(-1)  K(-1) , 9.64 kJ mol(-1) and 32.52 kJ mol(-1)  K(-1) , and 7.50 kJ mol(-1) and 25.22 kJ mol(-1)  K(-1) in R. glutinis, spent media, and sugar broth media, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:737-748, 2017. © 2017 American Institute of Chemical Engineers.

  16. Rice ubiquitin ligase EL5 prevents root meristematic cell death under high nitrogen conditions and interacts with a cytosolic GAPDH.

    Science.gov (United States)

    Nishizawa, Yoko; Mochizuki, Susumu; Koiwai, Hanae; Kondo, Katsuhiko; Kishimoto, Kyutaro; Katoh, Etsuko; Minami, Eiichi

    2015-01-01

    Root formation in rice transformants overexpressing mutated EL5 (mEL5) was severely inhibited because of meristematic cell death. Cell death was caused by nitrogen sources, particularly nitrate forms, in the culture medium. Nitrite treatment increased the cytokinin contents in roots, but mEL5 contained more cytokinins than non-transformants. Transcriptome profiling showed overlaps between nitrite-responsive genes in non-transformants and genes with altered expression in untreated mEL5. These results indicate that impairment of EL5 function activates nitrogen signaling despite the absence of a nitrogen source. Physical interaction between the EL5 C-terminal region and a cytosolic glyceraldehyde-3-phosphate dehydrogenase, OsGapC2, was demonstrated in vitro and in vivo. Elucidation of the role of glyceraldehyde-3-phosphate dehydrogenase in oxidative cell death in plants is expected in future.

  17. Pro-apoptotic effect of rice bran inositol hexaphosphate (IP6) on HT-29 colorectal cancer cells.

    Science.gov (United States)

    Shafie, Nurul Husna; Esa, Norhaizan Mohd; Ithnin, Hairuszah; Saad, Norazalina; Pandurangan, Ashok Kumar

    2013-12-02

    Inositol hexaphosphate (IP6), or phytic acid is a natural dietary ingredient and has been described as a "natural cancer fighter", being an essential component of nutritional diets. The marked anti-cancer effect of IP6 has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP6. Collectively, this present study has shown that rice bran IP6 induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8).

  18. 25 % Pymetrozine and thiamethoxam suspension residues detection and degradation in the rice ecosystem%25%吡蚜酮·噻虫嗉悬浮剂在水福生态系统中的残留检测和消解动态

    Institute of Scientific and Technical Information of China (English)

    夏志; 胡德禹; 张钰萍

    2012-01-01

    In this work, we developed a novel method of simuhaneous determination of residues and dynam its degradation for both pymetrozine and thiamethoxam in soil, rice straw, paddy water and brown rice. The ki netics study of pymetrozine residue in water, soil, and rice straw wereC=0.134e^0.12,C=1.377e^0.11t, and C = 0.741^0.10t, respectively. And the kinetics study of thiamethoxam in water, soil, and rice straw were C = 0. 114e 0. 12t , C 10 11Be 0. ,0,, and C - 0. 626e 0. 12t, respectively. The results show that a dosage of 0. 0525 0. 0788 g/m2 of 25~ pymetrozine and thiamethoxam suspension could be used in rice field ecosystem with har vest interval of 21 days.%运用高效液相色谱分析技术测定25%吡蚜酮·噻虫嗪悬浮剂在稻田水、土壤、植株和糙米中的消解动态和最终残留。吡蚜酮在稻田水、土壤和植株中的消解动态方程分别为C=0.134e^0.12,C=1.377e^0.11t及C=0.741e^0.101。噻虫嗪存稻田水、土壤和植株巾的消解动态方程分别为C=0.114e^-0.20,C=1.118^0.10t。及C=0.626e^-0.12t。最终残留结果显月i,25%吡蚜酮·噻虫嗪悬浮剂施用剂量为0.0525~0.0788g/m^2时,施药距水稻的安全收获间隔期为21d。

  19. Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats.

    Science.gov (United States)

    Ismail, Hammad; Mirza, Bushra

    2015-06-27

    Lactuca sativa (lettuce) has been traditionally used for relieving pain, inflammation, stomach problems including indigestion and lack of appetite. Moreover, the therapeutic significance of L. sativa includes its anticonvulsant, sedative-hypnotic and antioxidant properties. In the present study, the MC (methanol and chloroform; 1:1) and aqueous extracts of seed and leaf along with cell suspension exudate were prepared. These extracts were explored for their analgesic, anti-inflammatory, antidepressant and anticoagulant effects by hot plate analgesic assay; carrageenan induced hind paw edema test, forced swimming test and capillary method for blood clotting respectively in a rat model. The results were analyzed using one-way Analysis of Variance (ANOVA) followed by Turkey multiple comparison test. Interestingly, the extracts and the cell suspension exudate showed dual inhibition by reducing pain and inflammation. The results indicated that the aqueous extracts of leaf exhibited highest analgesic and anti-inflammatory activities followed by leaf MC, cell suspension exudate, seed aqueous and seed MC extracts. The current findings show that aqueous and MC extracts of seed have the least immobility time in the forced swimming test, which could act as an anti-depressant on the central nervous system. The leaf extracts and cell suspension exudate also expressed moderate anti-depressant activities. In anticoagulant assay, the coagulation time of aspirin (positive control) and MC extract of leaf was comparable, suggesting strong anti-coagulant effect. Additionally, no abnormal behavior or lethality was observed in any animal tested. Taken together, L. sativa can potentially act as a strong herbal drug due to its multiple pharmaceutical effects and is therefore of interest in drug discovery and development of formulations.

  20. LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula.

    Science.gov (United States)

    Staszków, Anna; Swarcewicz, Barbara; Banasiak, Joanna; Muth, Dorota; Jasiński, Michał; Stobiecki, Maciej

    2011-12-01

    Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H](+). Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS(3) experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

  1. Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls.

    Science.gov (United States)

    Kotake, Toshihisa; Aohara, Tsutomu; Hirano, Ko; Sato, Ami; Kaneko, Yasuko; Tsumuraya, Yoichi; Takatsuji, Hiroshi; Kawasaki, Shinji

    2011-03-01

    The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

  2. Biochemical and morphological responses to abiotc elicitor chitin in suspension-cultured sugarcane cells

    Directory of Open Access Journals (Sweden)

    Maria Izabel Gallão

    2010-04-01

    Full Text Available Cells of Saccharum officinarum submitted to hydrolyzated chitin for 1 to 8h produced phenolic compounds. These alterations were observed through cytochemical methods using Toluidine Blue and Phloroglucinol/HCl. After 4 h, besides cell wall change, there was a change in nuclear pattern of chitin treated cells. There was a 96% increase in nuclear area in 6 h chitin treated material, as observed by Feulgen reaction. The treated cells showed chromatin compacted regions and a degeneration process of nucleoli. In the outer areas of cell wall, there was a polysaccharide desagregation, confirming results obtained for different plants with the use of other elicitors. Peroxidase activity was maximal after 4 h and decreased progressively. PAL activity started to increase at 4 h of incubation. These results showed that chitin hydrolyzate stimulated a defense response in sugarcane cells.Células de Saccharum officinarum quando submetidas a quitina hidrolisada por 1 a 8h produziram material fenólico. Essas alterações foram observadas por meio de métodos citoquímicos como o Azul de Toluidina e Floroglucinol/HCl. Após 4 h, além das mudanças nas paredes celulares houve uma mudança no padrão nuclear das células tratadas com quitina. Por observação da reação de Feulgen, houve um aumento de 96% na área nuclear no material em 6h. Para as células tratadas foram observadas regiões de cromatina compactada e um processo de degeneração do nucléolo. Nas áreas externas da parede celular existia uma desagregação dos polisacarídios confirmando os resultados obtidos para diferentes plantas com o uso de outros elicitores. A atividade da peroxidase foi maxima após 4 h e então decresceu progressivamente. A atividade da PAL aumentou a partir de 4 h de incubação. Estes resultados mostram que o hidrolisado de quitina estimula as respostas de defesa em células de cana.

  3. Stuies on histological changing rice during aging

    Institute of Scientific and Technical Information of China (English)

    Qian haifeng; Houyiming; Yao huiyuan

    2001-01-01

    The changing of rice endosperm cell during aging was inspected and analyzed by tissue section method in this paper, which was considered as the main causation of the descending of the eating quality of aged rice. A new effective method of improving the eating quality of aged rice was also carried out through enzymatic treatment which was based on the changing of histological structure of aged rice.

  4. Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production

    OpenAIRE

    Behbahani, Mandana; Shanehsazzadeh, Mehrnaz; Hessami,Mohamad Javad

    2011-01-01

    Lycopene is present in a range of fresh fruits and vegetables, especially in the leaves of Barringtonia racemosa. The traditional lycopene extraction from the plant is being employed instead of an easy propagation technique like cell culture process from the leaf explants. We intend to assess how lycopene could be extracted via tissue culture under light (illuminance: 8,200 lux under white fluorescent lamps, photoperiod 16 h per day at 25ºC) and dark. Leaf explants of Barringtonia racemosa we...

  5. Novel O-D-galacturonoyl esters in the pectic polysaccharides of suspension-cultured plant cells.

    Science.gov (United States)

    Brown, J A; Fry, S C

    1993-11-01

    Driselase digestion of uronate-6-14C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo-[14C]galacturonides. One typical Driselase digestion product (compound 17) yielded alpha-(1-->4)-D-[14C]galacturonotriose(GalA3) upon very mild treatment with alkali (50% yield of GalA3 in 7.2 min at pH 11 and 25 degrees C). One of the three galacturonate residues in compound 17 was reducible to a galactose residue wit