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Sample records for resting cell suspensions

  1. Characterization of cell suspensions from solid tumors

    Pallavicini, M.

    1985-01-01

    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

  2. Production of vanillic acid from vanillin by resting cells of Serratia marcescens.

    Perestelo, F; Dalcón, M A; de la Fuente, G

    1989-01-01

    Resting-cell suspensions of Serratia marcescens were able to convert, quantitatively, 0.3% vanillin to vanillic acid. The vanillic acid-producing activity reached a maximum after 28 h of incubation with 0.01% vanillin as an inducer. PMID:2669632

  3. Production of vanillic acid from vanillin by resting cells of Serratia marcescens.

    Perestelo, F; Dalcón, M A; de la Fuente, G

    1989-01-01

    Resting-cell suspensions of Serratia marcescens were able to convert, quantitatively, 0.3% vanillin to vanillic acid. The vanillic acid-producing activity reached a maximum after 28 h of incubation with 0.01% vanillin as an inducer.

  4. Can resting B cells present antigen to T cells

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1985-01-01

    Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

  5. Acoustic manipulation of bacteria cells suspensions

    GutiéRrez-Ramos, Salomé; Hoyos, Mauricio; Aider, Jean Luc; Ruiz, Carlos; Acoustofluidics Team Team; Soft; Bio Group Collaboration

    An acoustic contacless manipulation gives advantages in the exploration of the complex dynamics enviroment that active matter exhibits. Our works reports the control confinement and dispersion of Escherichia coliRP437-pZA3R-YFP suspensions (M9Glu-Ca) via acoustic levitation.The manipulation of the bacteria bath in a parallel plate resonator is achieved using the acoustic radiation force and the secondary radiation force. The primary radiation force generates levitation of the bacteria cells at the nodal plane of the ultrasonic standing wave generated inside the resonator. On the other side, secondary forces leads to the consolidation of stable aggregates. All the experiments were performed in the acoustic trap described, where we excite the emission plate with a continuous sinusoidal signal at a frequency in the order of MHz and a quartz slide as the reflector plate. In a typical experiment we observed that, before the input of the signal, the bacteria cells exhibit their typical run and tumble behavior and after the sound is turned on all of them displace towards the nodal plane, and instantaneously the aggregation begins in this region. CNRS French National Space Studies, CONACYT Mexico.

  6. Effects of Demersal Otter Trawls on the Re-suspension of Copepod Resting Eggs and its Potential Effects on Recruitment

    Hansen, Benni Winding; Drillet, Guillaume; Hay, G

    2014-01-01

    Resting eggs are important phases in the life strategy of many coastal and estuarine copepods. The egg mortality in the sediment layers where they are buried, as well as re-suspension from the sediment into the water column where eggs may hatch are factors that affect the success of this life str...... and recruit nauplii to the pelagic, egg viability was reduced by the passage of the gear components. This study also suggests that the viability is dependent on the gear component and accordingly potential effects must be considered for at this level....

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

    Chlorogenic acid is a free radical scavenger, antibacterial, anti- inflammatory, antiviral, hypoglycemic, and in addition to ... experiments, the effect of various strengths of B5 medium (1/4 .... Growth kinetics of L. macranthoides cell suspension ...

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

    PRAKASH KUMAR G

    proven effective in the control of agricultural pests in an environmentally ..... Prakash G and Srivastava A K 2005 Statistical media optimization for cell growth and ... Juss. suspension cultures; Process Biochemistry 40 3795–3800. Prakash G ...

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

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... Full Length Research Paper. Establishment of the callus ... study provided an efficient way for E. angustifolia cell suspension culture to produce secondary metabolite. .... was also observed that in these treatments the stem.

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

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Additionally, sorghum cell suspension cultures have been initiated from the friable ... proteomics technologies. The field of proteomics is .... air dried at room temperature and resuspended in 2 ml of urea buffer [9 M urea, 2 M ...

  11. Establishment and characterization of American elm cell suspension cultures

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

    2000-01-01

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

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

    Administrator

    2011-09-12

    Sep 12, 2011 ... This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata, an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production.

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

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

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

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

  15. Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension.

    Gillet; Mesnard; Fliniaux; Monti; Fliniaux

    1999-11-01

    A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension. This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography. Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA). The influence of culture conditions on the accumulation of this metabolite by N. plumbaginifolia cell suspensions has been studied. Darkness strongly inhibits the CA accumulation. Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content. This one was not influenced by a supplementation with quinic acid.

  16. Mapping and characterisation of the sorghum cell suspension ...

    Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step ...

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

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

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

    Parshad, Henrik; Frydenvang, Karla; Liljefors, Tommy

    2003-01-01

    buffer is used as release media. Generally, the initial release of the drug salt from in situ suspensions occurred faster as compared to conventional suspensions, probably due to incomplete precipitation of the drug salt, and hence formation of supersaturated solutions where the rate of release......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...... indicated that the release of drug salt in deionized water was predominantly limited by the diffusion across the membrane whereas it is essentially dissolution rate controlled in 0.05 M phosphate buffer (pH 7.40). Thus, the in vitro model appears to have a potential in formulation screening when phosphate...

  19. Different characteristics between menadione and menadione sodium bisulfite as redox mediator in yeast cell suspension

    Yamashoji, Shiro

    2016-01-01

    Menadione promoted the production of active oxygen species (AOS) in both yeast cell suspension and the crude enzymes from the cells, but menadione sodium bisulfite (MSB) had little effect on the production of AOS in the cell suspension. MSB kept the stable increase in the electron transfer from intact yeast cells to anode compared to menadione, but the electron transfer promoted by MSB was inhibited in permeabilized yeast cell suspension. Menadione promoted oxidation of NAD(P)H much faster th...

  20. AgNOR Count in Resting Cells (Resting NOR Is a New Prognostic Marker in Invasive Bladder Tumor

    Mitsuro Tomobe

    2001-01-01

    Full Text Available Purpose: We have previously demonstrated that the AgNOR count in proliferating cells is a predictor of tumor recurrence in superficial bladder tumor (J. Urol. 162 (1999, 63–68. In the present study, we evaluate the type of AgNOR associated with cell cycles as a prognostic factor in invasive bladder tumor using a double staining technique employing both AgNOR and MIB-1 labelling. Materials and methods: Forty-four paraffin sections of invasive bladder tumors were stained simultaneously with AgNOR and MIB-1. The number of AgNORs in proliferating (MIB-1 positive or resting (MIB-1 negative cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. Results: The AgNOR count in proliferating cells (proliferating NOR was significantly higher than that in resting cells (resting NOR (p < 0.01. The resting NOR in tumors with distant metastases was significantly higher than that in tumors without metastases (p < 0.05. Patients with a low resting NOR tumor had a better prognosis than those with a high resting NOR tumor, whereas the proliferating NOR was not associated with survival. Survival analysis revealed that the resting NOR was the most powerful prognostic marker in patients with invasive bladder tumor (p < 0.05. Conclusions: Resting NOR had a predictive value in the prognosis of patients with invasive bladder tumor. Keywords: Transitional cell carcinoma, invasive, resting cell, AgNORs, MIB-1

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

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

    2006-01-01

    The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension-cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence...... and fluorochrome labelling of resin-embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls...

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

    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.

  3. Convection in a colloidal suspension in a closed horizontal cell

    Smorodin, B. L.; Cherepanov, I. N.

    2015-01-01

    The experimentally detected [1] oscillatory regimes of convection in a colloidal suspension of nanoparticles with a large anomalous thermal diffusivity in a closed horizontal cell heated from below have been simulated numerically. The concentration inhomogeneity near the vertical cavity boundaries arising from the interaction of thermal-diffusion separation and convective mixing has been proven to serve as a source of oscillatory regimes (traveling waves). The dependence of the Rayleigh number at the boundary of existence of the traveling-wave regime on the aspect ratio of the closed cavity has been established. The spatial characteristics of the emerging traveling waves have been determined

  4. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    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.

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

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

    1992-12-05

    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. (c) 1992 John Wiley & Sons, Inc.

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

    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.

  7. Characterization of bone marrow derived mesenchymal stem cells in suspension

    2012-01-01

    Introduction Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. PMID:23083975

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

    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...... not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10–6M) or NAA (10−5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10−7M) and 5.1 days on supraoptimal levels of 2,4-D (10−5M). Cultures grew on NH4+-N alone (from ammonium...... 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...

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

    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.

  10. Stereotaxic implantation of dispersed cell suspensions into brain. A systematic appraisal of cell placement and survival

    Plunkett, R.J.; Weber, R.J.; Oldfield, E.H.

    1988-01-01

    The application of several recent advances in cell biology, brain implantation, and cell-mediated tumor immunotherapy requires successful and reproducible placement of viable cell suspensions into brain. Stereotaxic implantation is being used to inject cytotoxic lymphocytes into gliomas and to replace dopaminergic cells in parkinsonian models. Systematic assessment of the factors that influence success in implantation of cell suspensions into solid tissues is needed. A model was developed for investigation of stereotaxic implantation using radiolabeled rat lymphokine-activated killer (LAK) cells. Anesthetized rats received microliter injections of cell suspension into the right caudate nucleus. The injection volume, cell concentration, infusion rate, and needle size were varied systematically. The animals were sacrificed 1 hour after injection; the brain was removed and sectioned, and the radioactivity was counted. Three aliquots of the suspension were injected into counting tubes for control analysis. Recovery of radioactivity was expressed as the percent of mean counts per minute (cpm) in the right frontal lobe/mean cpm in the three control tubes. To assess the viability of implanted cells, the right frontal region was mechanically dissociated in media and centrifuged, and the pellet and supernatant were counted. By using small needles and slow infusion of volumes of 10 microliters or less, 85% to 90% of the radioactivity was recovered in the caudate nucleus. At least half of the implanted cells were viable. Consistent, accurate implantation of dispersed cells into brain over a range of volumes, cell concentrations, infusion rates, and needle sizes was achieved

  11. ( Linum usitatissimum L. cv. Modran cell suspension culture

    Aleksandra Seta-Koselska

    2018-01-01

    Full Text Available Flax ( Linum usitatissimum L. is an ancient crop that is widely cultivated as a source of oil, fiber, and bioactive compounds. Flax fiber is traditionally used in textile industry, linseed oil is processed for industrial oils, paints, varnishes and bio-petroleum. Flaxseeds are also rich in α-linolenic acid and phytochemicals such as lignans. In addition to the commercial aspects, this species has been used widely and readily in biotechnological, developmental, and plant-pathogen interaction studies. Differences in the levels of endogenous hormones in various cultivars of flax significantly affected the intensity of callogenesis and determined the type and concentration of growth regulators necessary for callus production. The aim of our investigation was to optimize the culture conditions for callus formation and cell proliferation in liquid medium of the Polish cultivar of fiber flax – Modran. In the first step, 4 combinations of phytohormones in the medium were tested to obtain established callus tissue suitable for initiation of suspension culture. Next, we investigated the effect of chosen plant growth regulators on cell divisions, fresh and dry weight, and dispersal of callus cells in liquid medium. Fast growing and friable callus was obtained in a modified MS medium supplemented with 0.5 mg/l BAP and 0.1 mg/l NAA. We determined that for the initiation of cell suspension supplementation with 0.5 mg/l BAP and 0.5 mg/l NAA is optimal. The results obtained indicated that high concentration of cytokinin (BAP in liquid medium limited cell proliferation and decreased biomass formation.

  12. Regio-selective deglycosylation of icariin by cell suspension cultures of Glycyrrhiza uralensis and Morus alba.

    Zhang, De-Wu; Tao, Xiao-Yu; Chen, Ri-Dao; Yu, Li-Yan; Dai, Jun-Gui

    2015-01-01

    Biotransformations of icariin (1) by cell suspension cultures of Glycyrrhiza uralensis and Morus alba yielded two new metabolites, icaruralins A and B (2 and 3), and one known metabolite, baohuoside I (4). Their structures were determined on the basis of extensive spectroscopic analysis. This is the first report that the cell suspension cultures of G. uralensis and M. alba possess deglycosylation functionality.

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

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

    1996-01-01

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

  14. Reflection coefficients of permeant molecules in human red cell suspensions.

    Owen, J D; Eyring, E M

    1975-08-01

    The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.

  15. Enrichment of unlabeled human Langerhans cells from epidermal cell suspensions by discontinuous density gradient centrifugation

    Teunissen, M. B.; Wormmeester, J.; Kapsenberg, M. L.; Bos, J. D.

    1988-01-01

    In this report we introduce an alternative procedure for enrichment of human epidermal Langerhans cells (LC) from epidermal cell suspensions of normal skin. By means of discontinuous Ficoll-Metrizoate density gradient centrifugation, a fraction containing high numbers of viable, more than 80% pure

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

    Smorodin, Boris; Ishutov, Sergey [Department of Physics of Phase Transitions, Perm State University, Perm (Russian Federation); Cherepanov, Ivan, E-mail: bsmorodin@yandex.ru [Department of Radio Electronics and Information Security, Perm State University, Perm (Russian Federation)

    2016-12-15

    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). (paper)

  17. Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

    Zhang, Xiaomei; Lv, Yonggang

    2017-12-01

    Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

    Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C

    1994-01-01

    We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

  19. Iron induction of ferritin synthesis in soybean cell suspensions.

    Proudhon, D; Briat, J F; Lescure, A M

    1989-06-01

    In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes.

  20. Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

    Matsuno, Koya; Fujimura, Tatsuhito

    2015-08-01

    Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

  1. Lignans from cell suspension cultures of Phyllanthus niruri, an Indonesian medicinal plant

    Elfahmi, [No Value; Batterman, S; Koulman, A; Hackl, T; Bos, R; Kayser, O; Woerdenbag, HJ; Quax, WJ

    Cell suspension cultures of Phyllanthus niruri were used to study the lignan profiles and biosynthesis. Suspension cultures yielded two lignans: the new cubebin dimethyl ether (1) and urinatetralin (2), a new lignan from P. niruri, but reported earlier from P. urinaria. This is the first report of

  2. Flavonoid Production, Growth and Differentiation of Stelechocarpus burahol (Bl.) Hook. F. and Th. Cell Suspension Culture.

    Aini Habibah, Noor; Moeljopawiro, Sukarti; Dewi, Kumala; Indrianto, Ari

    2017-01-01

    Stelechocarpus burahol is a plant containing flavonoid compounds that have the potential for use as an antihyperuricemic for gout medication. This study was performed to assess flavonoid production, growth and cell differentiation of S. burahol in cell suspension culture. Mesocarp was planted in Murashige and Skoog (MS) medium supplemented with 7.5 mg L-1 picloram for the induction of callus. Non-embryonic callus obtained was used in the formation of cell suspension cultures. Growth of cells was determined by fresh and dry weights. During the culturing, the fresh weight, dry weight and flavonoid content were determined as a result of culture status. The growth of the S. burahol cell suspension was slow, the stationary phase occurred at 30 days. The production of flavonoids was not in line with the growth of cells and the maximum production occurred on the 15th day of the log phase. The globular-shaped cells dominated the cell suspension culture at all ages. Fluorescein diacetate (FDA) staining of cells derived from cell cultures aged for 36 days showed that some cells were still viable. The results show that flavonoid production, growth and cell differentiation of a S. burahol cell suspension culture differed according to the culture age.

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

    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

  4. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

    Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

    2014-05-01

    Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Suspension culture combined with chemotherapeutic agents for sorting of breast cancer stem cells

    Li, Hai-zhi; Yi, Tong-bo; Wu, Zheng-yan

    2008-01-01

    Cancer stem cell (CSC) hypothesis has not been well demonstrated by the lack of the most convincing evidence concerning a single cell capable of giving rise to a tumor. The scarcity in quantity and improper approaches for isolation and purification of CSCs have become the major obstacles for great development in CSCs. Here we adopted suspension culture combined with anticancer regimens as a strategy for screening breast cancer stem cells (BrCSCs). BrCSCs could survive and be highly enriched in non-adherent suspension culture while chemotherapeutic agents could destroy most rapidly dividing cancer cells and spare relatively quiescent BrCSCs. TM40D murine breast cancer cells were cultured in serum-free medium. The expression of CD44 + CD24 - was measured by flow cytometry. Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The rate of apoptosis was examined by flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Selected cells in different amounts were injected subcutaneously into BALB/C mice to observe tumor formation. Cells of passage 10 in suspension culture had the highest percentage of CD44 + CD24 - (about 77 percent). A single tumor cell in 0.35 PPC could generate tumors in 3 of 20 BALB/C mice. Suspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs

  6. An efficient method for the establishment of cell suspension cultures in potato (Solanum tuberosum L.)

    Sajid, Z.A.

    2016-01-01

    Cell suspension cultures offers an In vitro system that can be used as a tool for various studies involving mutant selection, mass propagation, protoplast isolation, gene transfer and selection of cell-lines which are resistant to various biotic or abiotic stresses. Research work on the development of cell suspension cultures was carried out to establish the most efficient method in Potato (cv. Desiree). Healthy, well-proliferating tissues from different types of callus cultures (compact, friable, embryogenic or non-embryogenic) were inoculated on various media combinations, i.e., MS, MS2 or AA liquid medium containing 18.09 micro M 2, 4-D. A fixed quantity (0.5-1.0 g) of callus tissue from 60-day-old callus cultures was transferred to 10-25 ml of liquid medium in 100 ml Erlenmeyer flask. Cultures were placed on an orbital shaker and agitated at different speeds (75, 100 or 125 rpm) under 16-h photoperiod at 25 ± 2 degree C. Medium was changed after every 3 days and fractionated tissue was filtered after every 6 days through sterile mesh (100-800 micro m) to develop a cell-line by transferring resulting suspension to fresh medium under the same conditions. Results indicated that eight-week-old translucent, friable, off-white callus cultures were an excellent starting material for the initiation of homogeneous cell suspension cultures as compared to other tested sources. Of the three tested media (MS, MS2 or AA medium containing 18.09 micro M 2, 4-D), MS2 was found to be a better medium for the initiation of cell suspension cultures. Cell suspension cultures, placed in 16-h photoperiod at 25 ± 2 degree C and agitated at 120 rpm using a gyratory shaker showed excellent results. Several other factors influencing quick establishment of cell suspension cultures in this cultivar are also discussed in this communication. (author)

  7. Relations between fatty acid synthesis, pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

    Beynen, A.C.; Geelen, M.J.H.

    1984-01-01

    1. 1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis. 2. 2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off

  8. The Importance of REST for Development and Function of Beta Cells

    Martin, David; Grapin-Botton, Anne

    2017-01-01

    that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST......Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among...... these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes...

  9. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

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

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

    1980-01-01

    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

  11. The generation of resting membrane potentials in an inner ear hair cell system.

    Bracho, H; Budelli, R

    1978-01-01

    1. The macula sacculi in the mudpuppy is an inner ear sensory area accessible for intracellular recordings in vitro and in vivo. 2. The resting potentials recorded in vitro can be explained by the electrodiffusion theory assuming a uniform ionic selective in the membranes of the neuroepithelial cells. 3. The resting potentials recorded in vivo are significantly larger than predicted by the electrodiffusion theory, probably because of an electrogenic metabolic process present in the neuroepithelial cells. 4. An equivalent circuit is proposed to explain the resting electrogenesis in the neuroepithelial cells present in the sensory area. Images Plate 1 PMID:702400

  12. Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2014-01-01

    An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

  13. Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

    Chee, P P; Tricoli, D M

    1988-06-01

    A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14-17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.

  14. Enhanced Mulberroside A Production from Cell Suspension and Root Cultures of Morus alba Using Elicitation.

    Komaikul, Jukrapun; Kitisripanya, Tharita; Tanaka, Hiroyuki; Sritularak, Boonchoo; Putalun, Waraporn

    2015-07-01

    Morus alba L. has been used in Asian traditional medicine as an anti-inflammatory, anti-asthmatic, anthelmintic and as a whitening agent in cosmetic products. Mulberroside A is the major active compound from M. alba root bark. In this study, cell suspension and root cultures of M. alba were established, and the effect of the elicitors on the enhancement of mulberroside A production in M. alba was investigated. The cell suspension and root cultures of M. alba were exposed to elicitors and then mulberroside A contents were determined by an indirect competitive ELISA method. High levels of mulberroside A were obtained by addition of 100 and 200 μM salicylic acid with 24 h exposure time in cell suspension cultures (37.9 ± 1.5 and 34.0 ± 4.7 mg/g dry wt., respectively). Furthermore, addition of yeast extract at 2 mg/mL with 24 h exposure time can significantly increase mulberroside A contents from both cell suspension (3.2-fold) and root cultures (6.6-fold). Mulberroside A contents from both cell suspension and root cultures after treatment with elicitors are similar or higher than those found in the intact root and root bark of several years old M. alba. These results indicate that mulberry tissue cultures using the elicitation method are interesting alternative sources for mulberroside A production.

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

    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

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

    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

  17. REST controls self-renewal and tumorigenic competence of human glioblastoma cells.

    Luciano Conti

    Full Text Available The Repressor Element 1 Silencing Transcription factor (REST/NRSF is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

  18. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

    Kaushali Thakore-Shah

    Full Text Available REST (RE1 silencing transcription factor, also known as NRSF (neuron-restrictive silencer factor, is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.

  19. REST-mediated recruitment of polycomb repressor complexes in mammalian cells

    Dietrich, Nikolaj; Lerdrup, Mads; Landt, Eskild

    2012-01-01

    Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1......, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest¿/¿ and Eed¿/¿ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently...... of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest¿/¿ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context...

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

    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.

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

    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

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

    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...

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

    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...

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

    Begum, Parvin; Fugetsu, Bunshi

    2013-01-01

    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

  5. Src-family kinases negatively regulate NFAT signaling in resting human T cells.

    Alan Baer

    Full Text Available T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.

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

    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

  7. Poisson-Boltzmann theory of charged colloids: limits of the cell model for salty suspensions

    Denton, A R

    2010-01-01

    Thermodynamic properties of charge-stabilized colloidal suspensions and polyelectrolyte solutions are commonly modelled by implementing the mean-field Poisson-Boltzmann (PB) theory within a cell model. This approach models a bulk system by a single macroion, together with counterions and salt ions, confined to a symmetrically shaped, electroneutral cell. While easing numerical solution of the nonlinear PB equation, the cell model neglects microion-induced interactions and correlations between macroions, precluding modelling of macroion ordering phenomena. An alternative approach, which avoids the artificial constraints of cell geometry, exploits the mapping of a macroion-microion mixture onto a one-component model of pseudo-macroions governed by effective interparticle interactions. In practice, effective-interaction models are usually based on linear-screening approximations, which can accurately describe strong nonlinear screening only by incorporating an effective (renormalized) macroion charge. Combining charge renormalization and linearized PB theories, in both the cell model and an effective-interaction (cell-free) model, we compute osmotic pressures of highly charged colloids and monovalent microions, in Donnan equilibrium with a salt reservoir, over a range of concentrations. By comparing predictions with primitive model simulation data for salt-free suspensions, and with predictions from nonlinear PB theory for salty suspensions, we chart the limits of both the cell model and linear-screening approximations in modelling bulk thermodynamic properties. Up to moderately strong electrostatic couplings, the cell model proves accurate for predicting osmotic pressures of deionized (counterion-dominated) suspensions. With increasing salt concentration, however, the relative contribution of macroion interactions to the osmotic pressure grows, leading predictions from the cell and effective-interaction models to deviate. No evidence is found for a liquid

  8. Cell suspension culture and mutants selection for resistance to PEG induced water stress in alfalfa (Medicago sativa L.)

    Zhang Xiaodong; Lin Tingan

    1994-01-01

    Elements affecting suspension cell culture in alfalfa (Medicago sativa L.) were studied and a method of rapid establishment of embryogenic suspension cell lines was introduced. Effects of γ ray irradiation on the growth of suspension cells were studied, and the optimum dose of irradiation for inducing mutants from suspension cells was about 20 ∼ 60 Gy. Effects of PEG and NaCl induced water stress on the growth of suspension cells were also investigated, and the results showed that the congregants of preliminary suspension culture were more susceptible than the established suspension cell lines. With 20 Gy of γ ray irradiation on suspension cell line (JL416), six clones were obtained with 70 days of selection on medium of 15% PEG (about-11 bar). A number of regenerated plants were obtained from these clones. One clone was also gained from medium containing 20% PEG (about-15 bar). The selected mutant cell lines (JP15 and JP20) has strong resistances to high concentration of PEG and NaCl induced water stress

  9. Ultrasound Characterization of Microbead and Cell Suspensions by Speed of Sound Measurements of Neutrally Buoyant Samples

    Cushing, Kevin W.; Garofalo, Fabio; Magnusson, Cecilia

    2017-01-01

    . The density of the microparticles is determined by using a neutrally buoyant selection process that involves centrifuging of microparticles suspended in different density solutions, CsCl for microbeads and Percoll for cells. The speed of sound at 3 MHz in the neutrally buoyant suspensions is measured...... and fixed cells, such as red blood cells, white blood cells, DU-145 prostate cancer cells, MCF-7 breast cancer cells, and LU-HNSCC-25 head and-neck squamous carcinoma cells in phosphate buffered saline. The results show agreement with published data obtained by other methods....

  10. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture

    Ruth Olmer

    2018-05-01

    Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture

  11. Determinants of resting cerebral blood flow in sickle cell disease

    Bush, Adam M.; Borzage, Matthew T.; Choi, Soyoung; Václavů, Lena; Tamrazi, Benita; Nederveen, Aart J.; Coates, Thomas D.; Wood, John C.

    2016-01-01

    Stroke is common in children with sickle cell disease and results from an imbalance in oxygen supply and demand. Cerebral blood flow (CBF) is increased in patients with sickle cell disease to compensate for their anemia, but adequacy of their oxygen delivery has not been systematically demonstrated.

  12. Programmatic access to logical models in the Cell Collective modeling environment via a REST API.

    Kowal, Bryan M; Schreier, Travis R; Dauer, Joseph T; Helikar, Tomáš

    2016-01-01

    Cell Collective (www.cellcollective.org) is a web-based interactive environment for constructing, simulating and analyzing logical models of biological systems. Herein, we present a Web service to access models, annotations, and simulation data in the Cell Collective platform through the Representational State Transfer (REST) Application Programming Interface (API). The REST API provides a convenient method for obtaining Cell Collective data through almost any programming language. To ensure easy processing of the retrieved data, the request output from the API is available in a standard JSON format. The Cell Collective REST API is freely available at http://thecellcollective.org/tccapi. All public models in Cell Collective are available through the REST API. For users interested in creating and accessing their own models through the REST API first need to create an account in Cell Collective (http://thecellcollective.org). thelikar2@unl.edu. Technical user documentation: https://goo.gl/U52GWo. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Madala, NE

    2012-07-01

    Full Text Available Nicotiana tabacum cell suspensions, 2g wet wt/ml, rapidly took up 1 mM isonitrosoacetophenone (INAP), a plant-derived stress metabolite with anti-oxidative and anti-fungal properties, producing 40-hexopyranosyloxy-30-methoxyisonitrosoacetophenone...

  14. Studies on Rapidly Frozen Suspensions of Yeast Cells by Differential Thermal Analysis and Conductometry

    Mazur, Peter

    1963-01-01

    Few, if any, yeast cells survived rapid cooling to -196°C and subsequent slow warming. After rapid freezing, the suspensions absorbed latent heat of fusion between -15° and 0°C during warming, and the relation between the amount of heat absorbed and the concentration of cells was the same as that in equivalent KCl solutions, indicating that frozen suspensions behave thermally like frozen solutions. The amount of heat absorbed was such that more than 80 per cent of the intracellular solution had to be frozen. The conductometric behavior of frozen suspensions showed that cell solutes were still inside the cells and surrounded by an intact cell membrane at the time heat was being absorbed. Two models are consistent with these findings. The first assumes that intracellular freezing has taken place; the second that all freezable water has left the cells and frozen externally. The latter model is ruled out because rapidly cooled cells do not shrink by an amount equal to the volume of water that would have to be withdrawn to prevent internal freezing. PMID:13934216

  15. Distinct migration and contact dynamics of resting and IL-2-activated human natural killer cells

    Per Erik Olofsson

    2014-03-01

    Full Text Available Natural killer (NK cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor–ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e. the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and IL-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney (HEK 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ motile scanning of the target cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior and contact dynamics.

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

    Jalil, Mahanom; Annuar, Mohamad Suffian Mohamad; Tan, Boon Chin; Khalid, Norzulaani

    2015-01-01

    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.

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

    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.

  18. NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

    Sullivan, S.G.; Rosenthal, J.S.; Winston, A.; Stern, A.

    1988-01-01

    NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red blood cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells. 9 refs.; 3 figs.; 1 table

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

    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.

  20. Resting blood lactate in individuals with sickle cell disease

    Jefferson Petto

    2011-02-01

    Full Text Available BACKGROUND: The most common hereditary hemoglobin disorder, affecting 20 million individuals worldwide, is sickle cell disease. The vascular obstruction resulting from the sickling of cells in this disease can produce local hypoxemia, pain crises and infarction in several tissues, including the bones, spleen, kidneys and lungs. METHODS: The present study is characterized as a case control study, with the aim of identifying the baseline blood lactate concentration in individuals with hemoglobin SS and SC diseases. One-way ANOVA with the Tukey post-test was used to analyze the results and a p-value < 0.05 was considered significant. Calculations were made using the INSTAT statistical program. The graphs were generated using the ORING program. The study sample was composed of 31 men and women residing in the city of Santo Antônio de Jesus, Bahia, Brazil. The individuals were divided into two groups: Group GC of 16 subjects who did not present with any type of structural hemoglobinopathy; and Group GE composed of 15 individuals with ages between 2 and 35 years old, who had the SS and SC genotypes. Sample analyses were performed with 3 mL of blood during fasting. RESULTS: The baseline blood lactate concentration of the SS and SC individuals was higher than that of the control group (p<0.001 with means of 4.86 ± 0.95; 3.30 ± 0.33; 1.31 ± 0.08 IU/L for SS, SC and controls, respectively. This corroborates the initial research hypothesis. CONCLUSION: The baseline blood lactate of SS and SC individuals is 3 to 4 times higher than that of healthy subjects, probably due to the fact that these patients have a metabolic deviation to the anaerobic pathway.

  1. Resting blood lactate in individuals with sickle cell disease

    Petto, Jefferson; de Jesus, Jaqueline Brito; Vasques, Leila Monique Reis; Pinheiro, Renata Leão Silva; Oliveira, Aila Mascarenhas; Spinola, Kelly Aparecida Borges; Silva, Wellington dos Santos

    2011-01-01

    Background The most common hereditary hemoglobin disorder, affecting 20 million individuals worldwide, is sickle cell disease. The vascular obstruction resulting from the sickling of cells in this disease can produce local hypoxemia, pain crises and infarction in several tissues, including the bones, spleen, kidneys and lungs. Objective To determine red blood group genes in a Brazilian populations. Methods The present study is characterized as a case control study, with the aim of identifying the baseline blood lactate concentration in individuals with hemoglobin SS and SC diseases. One-way ANOVA with the Tukey post-test was used to analyze the results and a p-value < 0.05 was considered significant. Calculations were made using the INSTAT statistical program. The graphs were generated using the ORING program. The study sample was composed of 31 men and women residing in the city of Santo Antônio de Jesus, Bahia, Brazil. The individuals were divided into two groups: Group GC of 16 subjects who did not present with any type of structural hemoglobinopathy; and Group GE composed of 15 individuals with ages between 2 and 35 years old, who had the SS and SC genotypes. Sample analyses were performed with 3 mL of blood during fasting. Results The baseline blood lactate concentration of the SS and SC individuals was higher than that of the control group (p<0.001) with means of 4.86 ± 0.95; 3.30 ± 0.33; 1.31 ± 0.08 IU/L for SS, SC and controls, respectively. This corroborates the initial research hypothesis. Conclusion The baseline blood lactate of SS and SC individuals is 3 to 4 times higher than that of healthy subjects, probably due to the fact that these patients have a metabolic deviation to the anaerobic pathway. PMID:23284239

  2. Characterization of technetium(vII) reduction by cell suspensions of thermophilic bacteria and archaea.

    Chernyh, Nikolay A; Gavrilov, Sergei N; Sorokin, Vladimir V; German, Konstantin E; Sergeant, Claire; Simonoff, Monique; Robb, Frank; Slobodkin, Alexander I

    2007-08-01

    Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [(99)Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota.

  3. Enhancement of Shikonin Production in Suspension Cultures of Lithospermum erythrorhizon Cells by Gamma-irradiation

    Baek, Myung Hwa; Chung, Byung Yeoup; Kim, Jae Sung; An, Beyoung Chul; Lee, Young Bok

    2005-01-01

    The shikonin and several derivatives produced by the roots of Boraginacae family plants are purple compounds that have been used in several parts of the World as antimicrobial and antitumor agents in human pharmaceuticals. Shikonin has been reported as the most successful specimen of the mass production of plant secondary metabolites by cell suspension culture. Numerous studies have elucidated the regulation of production of these compounds in cell suspension cultures. It has known that ultrasonic and gamma irradiation can enhance the production of secondary metabolites. Thus, in present study, we investigate the effects of gamma-irradiation on the shikonin production and the key enzymes in the shikonin biosynthetic pathway of L. erythrorhizon cells

  4. Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

    Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

    2017-10-01

    Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

    Cicala, Claudia; Arthos, James; Censoplano, Nina; Cruz, Catherine; Chung, Eva; Martinelli, Elena; Lempicki, Richard A.; Natarajan, Ven; VanRyk, Donald; Daucher, Marybeth; Fauci, Anthony S.

    2006-01-01

    The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs

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

    Research Centre for Plant Growth and Development, School of Agricultural Sciences and Agribusiness, University of .... source of regulatory molecules that modulate cell division .... nucellar tissue from embryo callus derived from seed of.

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

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

    2017-09-01

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

  8. Experiment of amnion epithelial cell suspension liquid used for acute rabbit corneal alkali burn

    Yan-Yan Zhang

    2017-10-01

    Full Text Available AIM: To investigate the effects of amnion epithelial cell(AECsuspension liquid on the biological behavior of the rabbit's corneal epithelium, combined with the in vitro and in vivo experiments. METHODS: The rabbit's corneal epithelium were cultured in the lower chamber of transwell, and AEC suspension liquid was dropwised in the upper chamber. There was only culture medium in the upper chamber of the control group. The proliferation of rabbit's corneal epithelium was observed with CCK-8 automated colorimetry and the expression of PCNA was detected by immunocytochemistry. We used the scratch wound assay to detect the migration of corneal epithelial cell(CEC. The in vivo models were established by placing a 10mm diameter corneal trephine in the center of the cornea, within 1mol/L NaOH for 1min. We divided those into three groups: treatment group of AEC suspension liquid eye drop, AEC suspension liquid subconjunctival injection and the control group without any treatment. Using the slit-lamp biomicroscope and fluorescence staining to observe the cornea per week. After 28d we took the eyeballs with the HE staining. The expression of VEGF was detected by immunohistochemistry. RESULTS: The activity of CEC with AEC treatment was much higher than the control group(PPIn vivo, the inflammation of the corneal and the CNV of the AEC group were all significantly reduced compared with the control group(PPCONCLUSION: AEC suspension liquid can promote the proliferation and migration of the rabbit's corneal epithelium. The potential of AEC suspension liquid as a therapy for acute corneal alkali burn.

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

    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

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

    Turek, Ilona; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.; Marondedze, Claudius

    2015-01-01

    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.

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

    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.

  12. Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions.

    Medina, M I; Botella, M A; Quesada, M A; Valpuesta, V

    1997-05-05

    A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

  13. Predominance of membrane damage in yeast cells in suspension with monochromatic 163-nm vacuum ultraviolet light

    Ito, T.; Ito, A.

    1980-01-01

    Effects of monochromatic 163-nm ultraviolet light on aqueous suspensions of yeast cells were studied under N 2 and O 2 bubbling conditions. This is a continuation of previous attempts at using a bromine resonance lamp immersed in cell suspension as a means of treating cells with water radicals (163-nm photons decompose water molecules into H atoms and OH' radicals). We found that inactivation occurred only under O 2 bubbling. Genetic changes were induced, but this was attributed to the effects of far-uv components which contaminate the emission. A characteristic feature of the vacuum uv inactivation was a decrease in survival when cells were held in liquid after irradiation. The presence of p-nitrosodimethylaniline (a known OH' scavenger) during irradiation prevented the O 2 -dependent enhancement of inactivation. Cells irradiated under N 2 bubbling showed no such enhancement. Thus, the fast access of oxygen is a necessary condition for fixing initial damage. Initial damage of this type seems to be amplified during subsequent incubation, causing further killing. Cells irradiated under N 2 bubbling were not, however, free of damage, since dye permeability across the cell membrane of irradiated samples increased markedly with both N 2 and O 2 as tested by photodynamic induction of genetic changes using normally unpenetrable dye as a sensitizer. Spectrophotometric evidence for the presence of toluidine blue in the irradiated cells are also presented

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

    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.

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

    Smith, Zachary J; Chu, Kaiqin; Wachsmann-Hogiu, Sebastian

    2012-01-01

    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.

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

    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.

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

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

    2003-01-01

    Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  18. Rotational magnetic pulses enhance the magnetofection efficiency in vitro in adherent and suspension cells

    Dahmani, Ch.; Mykhaylyk, O.; Helling, Fl.; Götz, St.; Weyh, Th.; Herzog, H.-G.; Plank, Ch.

    2013-01-01

    The association of magnetic nanoparticles with gene delivery vectors in combination with the use of gradient magnetic fields (magnetofection) enables improved and synchronised gene delivery to cells. In this paper, we report a system comprising rotating permanent magnets to generate defined magnetic field pulses with frequencies from 2.66 to 133 Hz and a field amplitude of 190 or 310 mT at the location of the cells. Low-frequency pulses of 2.66–10 Hz with a magnetic flux density of 190 mT were applied to the examined cells for 30–120 s after magnetofection. These pulses resulted in a 1.5–1.9-fold enhancement in the transfection efficiency compared with magnetofection with only a static magnetic field in both adherent and suspension cells. The magnetic field amplitudes of 190 and 310 mT had similar effects on the transfection efficacy. No increase in the percentage of transgene-expressing suspension cells and no cytotoxic effects (based on the results of the MTT assay) were observed after applying alternating magnetic fields. - Highlights: ► We developed a magnetic system capable of generating defined magnetic pulses based on permanent magnets. ► The main advantage of the system is the lack of heat-induced fluctuations in the working parameters. ► Our system succeeded in enhancing the transfection of adherent human lung epithelial cells and human suspension cells. ► The enhancement in the transfection efficiency compared with static magnetic field is due to the magnetic field pulses. ► The approach could be used as a complementary method for drug targeting

  19. Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

    Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

    2016-01-01

    Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

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

    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.

  1. Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

    Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

    2016-05-15

    Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death.

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2015-11-01

    Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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

    2012-01-01

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

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

    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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    Kim, Jae Sung; Lee, Young Keun; Chung, Byung Yeoup; Lee, Young Bok; Hwang Hye Yeon

    2004-10-01

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark, and was increased by adding 1 μM Cu 2+ and 100 μM methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of γ radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS (β-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs

  6. Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

    Hagendoorn, MJM.; Wagner, A. M.; Segers, G.; Van Der Plas, LHW.; Oostdam, A.; Van Walraven, H. S.

    1994-10-01

    In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

  7. Rapid preparation of a noncultured skin cell suspension that promotes wound healing.

    Yoon, Cheonjae; Lee, Jungsuk; Jeong, Hyosun; Lee, Sungjun; Sohn, Taesik; Chung, Sungphil

    2017-06-01

    Autologous skin cell suspensions have been used for wound healing in patients with burns and against normal pigmentation in vitiligo. To separate cells and the extracellular matrix from skin tissue, most researchers use enzymatic digestion. Therefore, this process is difficult to perform during a routine surgical procedure. We aimed to prepare a suspension of noncultured autologous skin cells (NCSCs) using a tissue homogenizer as a new method instead of harsh biochemical reagents. The potential clinical applicability of NCSCs was analyzed using a nude-rat model of burn healing. After optimization of the homogenizer settings, cell viability ranged from 52 to 89%. Scanning electron microscopy showed evidence of keratinocyte-like cell morphology, and several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the NCSCs. The rat model revealed that NCSCs accelerated skin regeneration. NCSCs could be generated using a tissue homogenizer for enhancement of wound healing in vivo. In the NCSC group of wounds, on day 7 of epithelialization, granulation was observed, whereas on day 14, there was a significant increase in skin adnexa regeneration as compared to the control group (PBS treatment; p study suggests that the proposed process is rapid and does not require the use of biochemical agents. Thus, we recommend a combination of surgical treatment with the new therapy for a burn as an effective method.

  8. A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

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

    2012-02-01

    The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance. Copyright © 2011 Wiley Periodicals, Inc.

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

    Sara KHOSRAVINIA

    2012-11-01

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

  10. Suspension Matrices for Improved Schwann-Cell Survival after Implantation into the Injured Rat Spinal Cord

    Patel, Vivek; Joseph, Gravil; Patel, Amit; Patel, Samik; Bustin, Devin; Mawson, David; Tuesta, Luis M.; Puentes, Rocio; Ghosh, Mousumi

    2010-01-01

    Abstract Trauma to the spinal cord produces endogenously irreversible tissue and functional loss, requiring the application of therapeutic approaches to achieve meaningful restoration. Cellular strategies, in particular Schwann-cell implantation, have shown promise in overcoming many of the obstacles facing successful repair of the injured spinal cord. Here, we show that the implantation of Schwann cells as cell suspensions with in-situ gelling laminin:collagen matrices after spinal-cord contusion significantly enhances long-term cell survival but not proliferation, as well as improves graft vascularization and the degree of axonal in-growth over the standard implantation vehicle, minimal media. The use of a matrix to suspend cells prior to implantation should be an important consideration for achieving improved survival and effectiveness of cellular therapies for future clinical application. PMID:20144012

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

    Ashihara, Hiroshi; Sagishima, Kyoko; Kubota, Kaoru

    1989-01-01

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

  12. KINETIC STUDY OF PALMITIC ACID ESTERIFICATION CATALYZED BY Rhizopus oryzae RESTING CELLS

    JONH J MÉNDEZ

    2009-01-01

    Full Text Available ABSTRACT In the present study, a kinetic model for the biocatalytic synthesis of esters using Rhizopus oryzae resting cells is proposed. The kinetic study has been made in a range of 30-50 °C and atmospheric pressure. The Influence of operating variables, water content, pH, amount of mycelium was studied. Different values of temperature, initial mycelium concentration and acid/alcohol molar ratio were tested. Initial rates were estimated from the slope of the concentration of palmitic acid, or their corresponding ester at conversions of less than 10%, versus time and reported as mmol l-1 min -1. The values of kinetic constants were computed using the freeware program SIMFIT (http:\\\\www.simfit.man.ac.uk. Key words: bound lipase, esterification, fungal resting cells, Rhizopus oryzae, palmitic acid, propanol. RESUMEN En el presente estudio, un modelo cinético para la síntesis de esteres usando Rhizopus oryzae resting cells es propuesto. El estudio cinético fue realizado en un rango de temperatura de 30-50 ºC a presión atmosférica reducida. La influencia de las variables de operación tales como temperatura, pH y contenido de agua fueron estudiadas. Diferentes valores de concentración de micelio y relación molar de ácido/alcohol son ensayadas, Las velocidades iníciales se estimaron de la curva de concentración de acido palmítico, y su correspondiente conversión a ester en menos del 10%, frente a tiempo y reportadas en mmol I-1 min -1. Los valores de las constantes cinéticas fueron calculados usando el programa freeware SIMFIT (http:\\\\www.simfit.man.ac.uk. Palabras clave: Lipasas, esterificación, resting cells, Rhizopus oryzae, acido palmítico, propanol.

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

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

    2015-12-01

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

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

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

    2016-01-01

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

  15. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    Guida, Natascia [IRCSS SDN, Naples 80131 (Italy); Laudati, Giusy [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Anzilotti, Serenella [IRCSS SDN, Naples 80131 (Italy); Secondo, Agnese [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Montuori, Paolo [Department of Public Health, ‘Federico II’ University of Naples, Naples (Italy); Di Renzo, Gianfranco [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Canzoniero, Lorella M.T. [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy); Formisano, Luigi, E-mail: cformisa@unisannio.it [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy)

    2015-11-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  16. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2015-01-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  17. Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells.

    Rao, Xiaolan; Shen, Hui; Pattathil, Sivakumar; Hahn, Michael G; Gelineo-Albersheim, Ivana; Mohnen, Debra; Pu, Yunqiao; Ragauskas, Arthur J; Chen, Xin; Chen, Fang; Dixon, Richard A

    2017-01-01

    Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass ( Panicum virgatum ) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation. Together, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.

  18. Potent Inhibition of HIV-1 Replication in Resting CD4 T Cells by Resveratrol and Pterostilbene.

    Chan, Chi N; Trinité, Benjamin; Levy, David N

    2017-09-01

    HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health-promoting benefits. Resveratrol has been shown to inhibit the replication of several viruses, including herpes simplex viruses 1 and 2, papillomaviruses, severe acute respiratory syndrome virus, and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at a low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, an HIV-1 and simian immunodeficiency virus protein that increases deoxynucleoside triphosphate (dNTP) levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene, or related compounds as adjuvants in anti-HIV preexposure prophylaxis (PrEP) formulations. Copyright © 2017 American Society for Microbiology.

  19. Electrochemical performance of solid oxide fuel cells having electrolytes made by suspension and solution precursor plasma spraying

    Marr, M.; Kuhn, J.; Metcalfe, C.; Harris, J.; Kesler, O.

    2014-01-01

    Yttria-stabilized zirconia (YSZ) electrolytes were deposited by suspension plasma spraying (SPS) and solution precursor plasma spraying (SPPS). The electrolytes were evaluated for permeability, microstructure, and electrochemical performance. With SPS, three different suspensions were tested to explore the influence of powder size distribution and liquid properties. Electrolytes made from suspensions of a powder with d50 = 2.6 μm were more gas-tight than those made from suspensions of a powder with d50 = 0.6 μm. A peak open circuit voltage of 1.00 V was measured at 750 °C with a cell with an electrolyte made from a suspension of d50 = 2.6 μm powder. The use of a flammable suspension liquid was beneficial for improving electrolyte conductivity when using lower energy plasmas, but the choice of liquid was less important when using higher energy plasmas. With SPPS, peak electrolyte conductivities were comparable to the peak conductivities of the SPS electrolytes. However, leak rates through the SPPS electrolytes were higher than those through the electrolytes made from suspensions of d50 = 2.6 μm powder. The electrochemical test data on SPPS electrolytes are the first reported in the literature.

  20. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

    Rebersek, Matej; Kanduser, Masa; Miklavcic, Damijan

    2011-01-01

    Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer. A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment. Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected. We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation

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

    Muthu Thiruvengadam

    2016-11-01

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

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

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

    2016-01-01

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

  3. Regulation of intracellular calcium in resting and stimulated rat basophilic leukemia cells

    Mohr, F.C.

    1988-01-01

    Intracellular calcium regulation was studied in a cell line of mast cells, the rat basophilic leukemia (RBL) cells with the purpose of determining (1) The properties of the plasma membrane calcium permeability pathway and (2) The role of intracellular calcium stores. The first set of experiments showed that depolarization did not induce calcium entry or secretion in resting cells and did inhibit antigen-stimulated calcium uptake and secretion. In the second set of experiments the ionic basis of antigen-induced depolarization was studied using the fluorescent potential-sensitive probe bis-oxonol. The properties of the calcium entry pathway were more consistent with a calcium channel than a calcium transport mechanism such as Na:Ca exchange. The third set of experiments examined the effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on RBL cells. CCCP inhibited antigen-stimulated 45 Ca uptake and secretion by depolarizing the plasma membrane

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

    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.

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

    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.

  6. Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

    Chappell, J.; Nable, R.

    1987-01-01

    Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [ 14 C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity, an enzyme of general isoprenoid metabolism, paralleled the changes in [ 14 C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [ 14 C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [ 3 H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures

  7. Establishment of Aquilaria malaccensis Callus, cell suspension and adventitious root systems

    Norazlina Noordin; Rusli Ibrahim

    2010-01-01

    Aquilaria malaccensis is a tropical forest tree from the family Thymelaeaceae, an endangered forest species and was listed in CITES since 1995. Locally known as Pokok Karas, this tree produces agar wood or gaharu, a highly valuable, resinous and fragrant forest product. Karas has been highly recognized for its vast medicinal values and gaharu has been widely use for perfumery, incense and religious purposes. The phyto chemical studies of agar wood showed that Sesqui terpenoid and Phenyl ethy chromone derivatives are the principal compounds that have anti allergic and anti microbe activities. Cell and organ culture systems provide large scale production of biomass and offers feasibilities for the production of secondary metabolites. This paper describes the work done for establishing reproducible systems for callus initiation and production of cell suspension cultures as well as production of adventitious roots that will later be amenable for the production of secondary metabolites of A. malaccensis. Hence, further manipulation with Methyl Jasmonate, a chemical elicitor could be done to induce secondary metabolites using callus, cell suspension and adventitious roots systems. (author)

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

    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

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

    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.

  10. Production of Monascus pigments as extracellular crystals by cell suspension culture.

    Lu, Fengling; Liu, Lujie; Huang, Yaolin; Zhang, Xuehong; Wang, Zhilong

    2018-01-01

    It is generally accepted that Monascus pigments are predominantly cell-bound, including both intracellular and surface-bound pigments. This long-term misconception was corrected in the present work. Production of extracellular crystal pigments by submerged culture of Monascus sp. was confirmed by microscopic observation and collection of Monascus pigments from extracellular broth by direct membrane filtration. Following up the new fact, the bioactivity of mycelia as whole-cell biocatalyst for biosynthesis and biodegradation of Monascus pigments had been detailedly examined in both an aqueous solution and a nonionic surfactant micelle aqueous solution. Based on those experimental results, cell suspension culture in an aqueous medium was developed as a novel strategy for accumulation of high concentration of Monascus pigments. Thus, glucose feeding during submerged culture in the aqueous medium was carried out successfully and high orange Monascus pigments concentration of near 4 g/L was achieved.

  11. Biosynthesis of NAD from nicotinic acid and nicotinamide by resting cells of Arthrobacter globiformis

    Kuwahara, Masaaki

    1978-01-01

    Isotopically labeled nicotinic acid and nicotinamide were incorporated into the metabolites of nicotinic acid-dependent pathway (Preiss-Handler pathway) of the NAD biosynthesis by resting cells of Arthrobacter globiformis. Azaserine and adenosine markedly stimulated the accumulation of NAD in the cells. Radioactive nicotinic acid and nicotinamide were also incorporated into an unknown compound when the cells were incubated in the presence of azaserine. Cell-free extract of the organism showed the NAD synthetase activity, which required ammonium ion and ATP for the amidation of deamido-NAD. Adenosine inhibited the enzyme activity. The organism possessed nicotinamidase, suggesting deamidation is the first step in the biosynthesis of NAD from nicotinamide. The activity was inhibited by NAD, NADP and NMN. (auth.)

  12. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  13. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  14. Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

    Szechynska-Hebda, M.; Wedzony, M.; Dubas, E.; Kieft, H.; Lammeren, van A.A.M.

    2006-01-01

    Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation,

  15. Bottlenecks in the generation and maintenance of morphogenic banana cell suspensions and plant regeneration via somatic embryogenesis therefrom

    Schoofs, H.; Panis, B.; Strosse, H.; Mosqueda, A. M.; Torres, J. L.; Roux, N.; Doležel, Jaroslav; Swennen, R.

    2001-01-01

    Roč. 8, č. 2 (2001), s. 3-7 ISSN 0989-8972 R&D Projects: GA MŠk ME 376 Institutional research plan: CEZ:AV0Z5038910 Keywords : banana cell suspensions * plant regeneration Subject RIV: EA - Cell Biology

  16. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

    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 po...

  17. Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

    Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

    1982-01-01

    We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

  18. In situ bioremediation of trichloroethylene-contaminated water by a resting-cell methanotrophic microbial filter

    Taylor, R.T.; Duba, A.G.; Durham, W.B.; Hanna, M.L.; Jackson, K.J.; Jovanovich, M.C.; Knapp, R.B.; Knezovich, J.P.; Shah, N.N.; Shonnard, D.R.; Wijesinghe, A.M.

    1992-10-01

    The Lawrence Livermore National Laboratory is testing and developing an in situ microbial filter technology for remediating migrating subsurface plumes contaminated with low concentrations of trichloroethylene (TCE). Their current focus is the establishment of a replenishable bioactive zone (catalytic filter) along expanding plume boundaries by the Injection of a representative methanotrophic bacterium, Methylosinus trichosporium OB3b. We have successfully demonstrated this microbial filter strategy using emplaced, attached resting cells (no methane additions) in a 1.1-m flow-through test bed loaded with water-saturated sand. Two separate 24 h pulses of TCE (109 ppb and 85 ppb), one week apart, were pumped through the system at a flow velocity of 1.5 cm/h; no TCE (<0.5 ppb) was detected on the downstream side of the microbial filter. Subsequent excavation of the wet sand confirmed the existence of a TCE-bioactive zone 19 days after it had been created. An enhanced longevity of the cellular, soluble-form methane monooxygenase produced by this methanotroph Is a result of our laboratory bioreactor culturing conditions. Additional experiments with cells in sealed vials and emplaced in the 1.1-m test bed yielded a high resting-cell finite TCE biotransformation capacity of ∼ 0.25 mg per mg of bacteria; this is suitable for a planned sand-filled trench field demonstration at a Lawrence Livermore National Laboratory site

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

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

    2016-01-01

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

  20. Characterization of Ni-YSZ anodes for solid oxide fuel cells fabricated by suspension plasma spraying with axial feedstock injection

    Metcalfe, Craig; Kuhn, Joel; Kesler, Olivera

    2013-12-01

    Composite Ni-Y0.15Zr0.85O1.925 anodes were fabricated by axial-injection suspension plasma spraying in open atmosphere conditions. The composition of the anode is controllable by adjustment of the plasma gas composition, stand-off distance, and suspension feed rate. The total porosity is controllable through the addition of carbon black to the suspension as a sacrificial pore-forming material as well as by adjustment of the suspension feed rate. The size of the NiO particles in suspension affects both the composition and total porosity, with larger NiO particles leading to increased Ni content and porosity in the deposited coatings. The surface roughness increases with a decrease of the in-flight droplet momentum, which results from both smaller NiO particles in suspension and the addition of low density pore-forming materials. A solid oxide fuel cell was fabricated with both electrodes and electrolyte fabricated by axial-injection plasma spraying. Peak power densities of 0.718 W cm-2 and 1.13 W cm-2 at 750 °C and 850 °C, respectively, were achieved.

  1. High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

    Kaldis, Angelo; Ahmad, Adil; Reid, Alexandra; McGarvey, Brian; Brandle, Jim; Ma, Shengwu; Jevnikar, Anthony; Kohalmi, Susanne E; Menassa, Rima

    2013-01-01

    The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. PMID:23297698

  2. Experimental and analytical study of oxygen depletion in stirred cell suspensions

    Whillans, D.W.; Rauth, A.M.

    1980-01-01

    The determination and maintenance of constant low but non-zero levels of oxygen is critical in the study of the radiation chemical interactions of nitroimidazoles in mammalian cells in vitro. As well, many of these chemicals have increased toxicity toward hypoxic compared to aerobic cells, although absolute hypoxia probably is not required. Both of these phenomena must be investigated in systems where significant consumption of oxygen takes place, either through radiation depletion or by cellular metabolism. In this paper an analysis has been made of the form of oxygen depletion in stirred cell suspensions with overlying gas phase, and it has been found to conform to the relationship (C[t] - C/sub infinity/) = (C[0] - C/sub infinity/) exp(-k 1 t), where C/sub infinity/ = C/sub g/ - R/k 1 . Here C[t] is the oxygen tension throughout the solution; C/sub g/, the equivalent level in the overlying gas phase; R (concentration units per sec), the depletion rate; k 1 (sec/sup -1/), a physical constant independent of oxygen concentration and depletion rate; and C/sub infinity/, the oxygen level in solution approached at long times. This relationship has been confirmed in detail using a Clark-type oxygen sensor and a high-stability amplifier design due to Koch. Since oxygen levels down to a few hundred parts per million can be determined with accuracy, it has been possible to measure precisely the oxygen levels present in our experimental systems. Implications of these results for the interpretation of data obtained in stirred cell suspension with overlying gas phase under conditions of consumption are discussed

  3. Chitosan mediated enhancement of hydrolysable tannin in Phyllanthus debilis Klein ex Willd via plant cell suspension culture.

    V, Malayaman; N, Sisubalan; R P, Senthilkumar; S, Sheik Mohamed; R, Ranjithkumar; M, Ghouse Basha

    2017-11-01

    Phyllanthus debilis Klein ex Willd. is wild medicinal plant used in the traditional system of medicine. This plant has been actively used for hepatoprotection and to cure many diseases including jaundice and so on; which leads to complete extinction of this particular species. Therefore, the chitosan mediated cost effective cell suspension method has been developed for the production of hydrolysable tannin. The hydrolysable tannins are the main therapeutically active constituents with antioxidant, anticancer, and antimicrobial properties. An in vitro cell suspension culture was optimized by adding chitosan for production of hydrolysable tannin. According to the growth kinetics, a maximum biomass of 4.46±0.06g fresh cell weight and 1.33±0.04g dry cell weight were obtained from the optimal suspension medium consisted of MS medium+0.5mgL -1 BAP+1.5mgL -1 NAA. Chitosan was treated at the stationary phase which leads to the highest accumulation of hydrolysable tannin compared to the untreated control. Hydrolysable tannin was observed and compared using HPLC at the Rt of 4.91 in both chitosan treated and untreated cells. This is the first ever report where use of chitosan has been done to enhance the production of the hydrolysable tannin in P. debilis using cell suspension culture technique. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. [Formation of protodioscin and deltoside isomers in suspension cultures of Nepal yam (Dioscorea deltoidea Wall.) cells].

    Khandy, M T; Titova, M V; Konstantinova, S V; Kochkin, D V; Ivanov, I M; Nosov, A M

    2016-01-01

    Changes in the content of the furostanol glycosides protodioscin and deltoside, particularly that of the (25S)-isomers of the glycosides, during suspension cultivation of different lines of Nepal yam (Dioscorea deltoidea Wall.) cells of the strain IFR-DM-0.5 has been investigated. The composition of furostanol glycosides has been characterized, and the dynamics of the accumulation of individual glycosides during lengthy subcultivation of cells maintained in flasks or in a barbotage bioreactor has been analyzed. A positive correlation between the growth and accumulation of substances that belonged to the class of furostanol glycosides has been demonstrated for cultured dioscorea cells, whereas the content of some of the individual glycosides varied considerably between the lines of the strain, cultures maintained under different conditions, and even between cells in different phases of the growth cycle. The increased content of (25R)-forms of the glycosides (protodioscin and deltoside) was correlated with a decrease in the cellular growth rate, whereas an increase in culture growth intensity occurred concomitantly to an increase of the amount of (25S)-isomers. This may be indicative of the specific stimulatory effect of (25S)-glycosides, but not the (25R)-forms, on cell proliferation in vitro. Thus, the concentration of (25S)-forms may increase due to the autoselection of cells capable of intensive division during prolonged cultivation.

  5. Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron

    Doolittle, R.L.; Richter, G.W.

    1981-01-01

    Cultures of Kupffer cells and of hepatocytes, prepared from single rat livers, synthesized ferritin protein equally efficiently. In culture but not in suspension, both sorts of cells responded significantly to stimulation with iron by increased ferritin synthesis. As determined by isoelectric focusing, the isoferritin profiles of newly synthesized 14 -labeled Kupffer cell and hepatocyte ferritin were identical, each having three bands. However, unlabeled ferritin, extracted from nonparenchymal liver cells (mainly Kupffer and endothelial cells) of iron-loaded rats, contained an acidic isoferritin that was not present in hepatocyte ferritin. Investigation of ferritin synthesis in cultured peritoneal and alveolar macrophages yielded similar results. The isofocusing profile of newly synthesized peritoneal macrophage ferritin was indistinguishable from the profile of fresh Kupffer cell or hepatocyte ferritin. Thus, the three isoferritins common to Kupffer cells, hepatocytes, and extrahepatic macrophages are neither cell- nor tissue-specific. However, modifications on intracellular storage may affect the isofocusing properties. The findings, although consistent with the LnH24-n subunit model of ferritin protein, indicate identical restrictive genomic control of the H:L ratios in these sorts of cells. Further, they make it probable that Kupffer cell ferritin iron, originating by endogenous synthesis, is the principal source of Kupffer cell hemosiderin iron

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

    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.

  7. Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae).

    Yang, Li; Zhu, Jianhua; Song, Liyan; Shi, Xiaojian; Li, Xingyi; Yu, Rongmin

    2012-01-01

    A new sesquiterpene glycoside, artemisinic acid 3-β-O-β-D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3-β-hydroxyartemisinic acid (2, 36.69%) and 3-β-hydroxyartemisinic acid β-D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC₅₀ value of 0.56 µmol mL⁻¹.

  8. Bioconversion of isoeugenol to vanillin and vanillic acid using the resting cells of Trichosporon asahii.

    Ashengroph, Morahem; Amini, Jahanshir

    2017-12-01

    40 isoeugenol-tolerant yeasts were isolated from the rhizosphere soil samples which in turn were collected from aromatic plants in different regions of Iran, and further tested for their ability to grow on a minimal medium containing isoeugenol as the sole carbon and energy source. Nine isolates which were able to grow on isoeugenol were examined for their ability to convert isoeugenol into vanillin under growing cell experiments. Of the tested yeasts, the highest conversion efficiency was observed in isolate MP24. The isolate was identified as Trichosporon asahii based on morphological, biochemical and molecular (ITS region) characters and tested to effectively convert isoeugenol into vanillin under resting cell system. A comparative analysis of thin layer chromatography (TLC), UV-Vis spectrometry, and high-performance liquid chromatography (HPLC) verified that vanillin and vanillic acid are accumulated as two major metabolites using T. asahii strain MP24 resting cells. In the presence of 7.5 g/l of wet weight cells of the strain MP24 pre-grown on isoeugenol and harvested at the end of the exponential growth phase, the optimal concentration of vanillin reached 2.4 g/l with a molar conversion of 52.5% in the potassium phosphate buffer (100 mM, pH 5.8) supplemented with 5 g/l of isoeugenol and 2% (v/v) N , N -dimethylformamide (DMF). The total concentration of vanillin and vanillic acid obtained from the bioconversion process was 4.2 g/l (total molar yield of 88.3%). Until now, no data has been published on the conversion of isoeugenol into vanillin by the strains of the genus Trichosporon .

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

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

    2014-01-01

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

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

    Sujanya, S; Devi, B Poornasri; Sai, Isha

    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).

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

    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 (

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

    Woerdenbag, H.J.; Van Uden, W.; Frijlink, H.W.; Lerk, C.F.; Pras, N.; Malingre, T.M.

    1990-01-01

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

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

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

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

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

    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.

  15. Electrical pulse – mediated enhanced delivery of silver nanoparticles into living suspension cells for surface enhanced Raman spectroscopy

    Lin, J; Li, B; Feng, S; Chen, G; Li, Y; Huang, Z; Chen, R; Yu, Y; Huang, H; Lin, S; Li, C; Su, Y; Zeng, H

    2012-01-01

    Electrical pulse-mediated enhanced silver nanoparticles delivery is a much better method for intracellular surface-enhanced Raman spectroscopy (SERS) measurements of suspension cells. Robust and high-quality SERS spectra of living suspension cells were obtained based on an electroporation-SERS method, which can overcomes the shortcoming of non-uniform distribution of silver nanoparticles localized in the cell cytoplasm after electroporation and reduces the amount variance of silver nanoparticles delivered into different cells. The electroporation parameters include three 150 V (375 V/cm) electric pulses of 1, 5, and 5 ms durations respectively. Our results indicate that considerable amount of silver nanoparticles can be rapidly delivered into the human promyelocytic leukemia HL60 cells, and the satisfied SERS spectra were obtained while the viability of the treated cells was highly maintained (91.7%). The electroporation-SERS method offers great potential approach in delivering silver nanoparticles into living suspension cells, which is useful for widely biomedical applications including the real-time intracellular SERS analysis of living cells

  16. Xyloglucan biosynthesis by Golgi membranes from suspension-cultured sycamore (Acer pseudoplatanus) cells

    White, A.R.; Xin, Yi

    1990-01-01

    Xyloglucan is a major hemicellulose polysaccharide in plant cell walls. Biosynthesis of such cell wall polysaccharides is closely linked to the process of plant cell growth and development. Xyloglucan polysaccharides consist of a β-1,4 glucan backbone synthesized by xyloglucan synthase and sidechains of xylose, galactose, and fucose added by other transferase enzymes. Most plant Golgi and plasma membranes also contain glucan synthases I ampersand II, which make β-1,4 and β-1,3 glucans, respectively. All of these enzymes have very similar activities. Cell walls on suspension-cultured cells from Acer pseudoplatanus (sycamore maple) were enzymatically softened prior to cell disruption by passing through a 30 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on top-loaded or flotation sucrose density gradients. Samples were collected by gradient fractionation and assayed for membrane markers and xyloglucan and glucan synthase activities. Standard marker assays (cyt. c reductase for eR, IDPase ampersand UDPase for Golgi, and eosin 5'-malelmide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of 14 C-labeled sugars from UDP-glucose and UDP-xylose was used to detect xyloglucan synthase, glucan synthases I ampersand II, and xylosyl transferase in Golgi membrane fractions. These activities overlapped, although distinct peaks of xyloglucan synthase and xylosyl transferase were found. Ca ++ had a stimulatory effect on glucan synthases I ampersand II, while Mn ++ had an inhibitory effect on glucan synthase I in the presence of Ca ++ . The similarity of these various synthase activities demonstrates the need for careful structural characterization of newly synthesized polysaccharides

  17. Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

    Lescure, A M; Massenet, O; Briat, J F

    1990-11-15

    Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.

  18. Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

    Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

    2013-09-01

    The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

  19. Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

    Baldauf, Hanna-Mari; Stegmann, Lena; Schwarz, Sarah-Marie; Ambiel, Ina; Trotard, Maud; Martin, Margarethe; Burggraf, Manja; Lenzi, Gina M; Lejk, Helena; Pan, Xiaoyu; Fregoso, Oliver I; Lim, Efrem S; Abraham, Libin; Nguyen, Laura A; Rutsch, Frank; König, Renate; Kim, Baek; Emerman, Michael; Fackler, Oliver T; Keppler, Oliver T

    2017-03-07

    Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

  20. Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues.

    Xiong, Jimin; Gronthos, Stan; Bartold, P Mark

    2013-10-01

    Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth-supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal-regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem-cell populations that demonstrate similar properties to mesenchymal stromal

  1. CD4+ and CD8+ T cell activation are associated with HIV DNA in resting CD4+ T cells.

    Leslie R Cockerham

    Full Text Available The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1 in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.

  2. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

  4. Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

    Weld, R; Heinemann, J; Eady, C

    2001-03-01

    The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

  5. Rosmarinic acid and antioxidant enzyme activities in Lavandula vera MM cell suspension culture: a comparative study.

    Georgiev, Milen; Abrashev, Radoslav; Krumova, Ekaterina; Demirevska, Klimentina; Ilieva, Mladenka; Angelova, Maria

    2009-11-01

    The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members-non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

  6. Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures.

    Kwon, Jun-Young; Lee, Kyoung-Hoon; Cheon, Su-Hwan; Ryu, Hyun-Nam; Kim, Sun Jin; Kim, Dong-Il

    2012-03-01

    Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L(-1) in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.

  7. Alternative REST Splicing Underappreciated

    Chen, Guo-Lin; Miller, Gregory

    2017-01-01

    As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription, an N-terminal REST4 isoform caused by alternative splicing - inclusion of an extra exon (N3c) which introduces a pre-mature stop codon - has been implicated in...

  8. Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. 13C NMR and enzymatic assay

    Mesnard, F.; Marty, D.; Monti, J.P.; Gillet-Manceau, F.; Fliniaux, M.A.

    1999-01-01

    The metabolism of labelled pyruvate followed by 13 C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloid synthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

  9. Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara).

    Guiderdoni, E; Chaïr, H

    1992-11-01

    More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.

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

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

    1998-01-01

    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...... of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... 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...

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

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

    2016-01-01

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

  12. Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

    Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

    2006-01-01

    Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

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

    Bhattacharyya Dipto

    2012-05-01

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

  14. Metabolism of [14C] bicarbonate by Streptococcus lactis: the synthesis, uptake and excretion of aspartate by resting cells

    Hillier, A.J.; Rice, G.H.; Jago, G.R.

    1978-01-01

    Resting cells of Streptococcus lactis C10 were able to synthesize aspartic acid de novo but could not actively transport aspartic acid into the cell. Intracellular aspartate was excreted from the cell in the presence of glucose but did not exchange with any extracellular amino acids. The results indicate that Str. lactis C10 obtains the aspartic acid it requires for growth by bicarbonate fixation instead of by the utilization of extracellular aspartic acid. (author)

  15. Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing.

    Lin, Louis M; Huang, George T-J; Rosenberg, Paul A

    2007-08-01

    There is continuing controversy regarding the potential for inflammatory apical cysts to heal after nonsurgical endodontic therapy. Molecular cell biology may provide answers to a series of related questions. How are the epithelial cell rests of Malassez stimulated to proliferate? How are the apical cysts formed? How does the lining epithelium of apical cysts regress after endodontic therapy? Epithelial cell rests are induced to divide and proliferate by inflammatory mediators, proinflammatory cytokines, and growth factors released from host cells during periradicular inflammation. Quiescent epithelial cell rests can behave like restricted-potential stem cells if stimulated to proliferate. Formation of apical cysts is most likely caused by the merging of proliferating epithelial strands from all directions to form a three-dimensional ball mass. After endodontic therapy, epithelial cells in epithelial strands of periapical granulomas and the lining epithelium of apical cysts may stop proliferating because of a reduction in inflammatory mediators, proinflammatory cytokines, and growth factors. Epithelial cells will also regress because of activation of apoptosis or programmed cell death through deprivation of survival factors or by receiving death signals during periapical wound healing.

  16. Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, María Angeles; Sabater-Jara, Ana Belén

    2018-04-21

    cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

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

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

    1969-07-15

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

  18. Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.).

    Chaïr, H; Legavre, T; Guiderdoni, E

    1996-06-01

    We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 2∶1 or 1∶1 ratio, led to 0.8 × 10(-5) and 1.6 × 10(-5) resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and β-glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.

  19. Agrobacterium tumefaciens-mediated genetic transformation of embryogenesis cell suspensions of banana cultivar Grande naine (AAA

    Idalmis Bermúdez-Caraballoso

    2004-01-01

    Full Text Available The black Sigatoka (Mycosphaerella fijiensis Morelet has become in the last years, the most destructive disease that affects the production of banana and plantains world-wide. The present work was made with the objective to obtain transgenic plants of banana cultivar Grand naine (AAA resistant to this disease with the use of genetic transformation. Embryogenenic cell suspensions obtained from somatic embryos formed from immature male flowers, were used for the transformation by Agrobacterium tumefaciens. The bacterial strain EHA-105 was used with the binary plasmids pHCA-58, pHCG-59 and pHGA-91, which contain different combinations of genes that encode for the antifungal chitinase, glucanase enzymes and the AP-24 osmotin. The commercial herbicide BASTA® was used as selective agent. One hundred ten putative transformed lines of the three constructions were obtained, after three selection months in the culture medium. The transgenic events were verified by means of Polymerase Chain Reaction analysis. Key words: AP-24, chitinase, glucanase, Musa, Mycosphaerella fijiensis

  20. Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures.

    Bartholomeusz, Trixie Ann; Molinié, Roland; Roscher, Albrecht; Felpin, François-Xavier; Gillet, Françoise; Lebreton, Jacques; Mesnard, François; Robins, Richard J

    2005-08-01

    The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

  1. Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved.

    Bartholomeusz, Trixie Ann; Bhogal, Ramneek K; Molinié, Roland; Felpin, François-Xavier; Mathé-Allainmat, Monique; Meier, Anna-Carolin; Dräger, Birgit; Lebreton, Jacques; Roscher, Albrecht; Robins, Richard J; Mesnard, François

    2005-10-01

    Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [(13)C,(2)H(3)-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-(15)N]nornicotine resulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation.

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

    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.

  3. Characterization of metal-supported axial injection plasma sprayed solid oxide fuel cells with aqueous suspension plasma sprayed electrolyte layers

    Waldbillig, D.; Kesler, O.

    A method for manufacturing metal-supported SOFCs with atmospheric plasma spraying (APS) is presented, making use of aqueous suspension feedstock for the electrolyte layer and dry powder feedstock for the anode and cathode layers. The cathode layer was deposited first directly onto a metal support, in order to minimize contact resistance, and to allow the introduction of added porosity. The electrolyte layers produced by suspension plasma spraying (SPS) were characterized in terms of thickness, permeability, and microstructure, and the impact of substrate morphology on electrolyte properties was investigated. Fuel cells produced by APS were electrochemically tested at temperatures ranging from 650 to 750 °C. The substrate morphology had little effect on open circuit voltage, but substrates with finer porosity resulted in lower kinetic losses in the fuel cell polarization.

  4. Characterization of metal-supported axial injection plasma sprayed solid oxide fuel cells with aqueous suspension plasma sprayed electrolyte layers

    Waldbillig, D. [University of British Columbia, Department of Materials Engineering, 309-6350 Stores Road, Vancouver, BC (Canada); Kesler, O. [University of Toronto, Department of Mechanical and Industrial Engineering, 5 King' s College Road, Toronto, Ontario (Canada)

    2009-06-15

    A method for manufacturing metal-supported SOFCs with atmospheric plasma spraying (APS) is presented, making use of aqueous suspension feedstock for the electrolyte layer and dry powder feedstock for the anode and cathode layers. The cathode layer was deposited first directly onto a metal support, in order to minimize contact resistance, and to allow the introduction of added porosity. The electrolyte layers produced by suspension plasma spraying (SPS) were characterized in terms of thickness, permeability, and microstructure, and the impact of substrate morphology on electrolyte properties was investigated. Fuel cells produced by APS were electrochemically tested at temperatures ranging from 650 to 750 C. The substrate morphology had little effect on open circuit voltage, but substrates with finer porosity resulted in lower kinetic losses in the fuel cell polarization. (author)

  5. Use of embryogenic cell suspension and meristem-tip cultures for mutation breeding of apomictic Musa species

    Novak, F.J.; Afza, R.; Duren, M. van

    1990-01-01

    Full text: Breeding by crossing is difficult for banana and plantain. The plants are heterozygous, therefore mutagenic treatment may uncover a recessive allele by mutating or deleting a corresponding dominant allele. Meristem tips were excised from in vitro growing shoots and used for mutation experiments. Induction was carried out by irradiating shoot tips with γ rays and/or by treatment of explants with ethylmethanesulfonate (EMS). Cell suspension was initiated from corm and leaf tissue excised from in vitro grown plantlets. Mutagenised cell suspensions were derived from leaf and corm tissues irradiated with 60 Co γ rays - (10 to 60 Gy, 8 Gy/min). Musa clones exhibited differences in radiosensitivity and post-irradiation recovery. Doses of 20 to 40 Gy seem suitable for mutation induction. The EMS concentration of 25 mM for 4 hours was found effective for isolated shoot tips. Considerable phenotypic variation was observed among plants regenerated from in vitro shoot tips after mutagenic treatment. Leaf and corm explants kept their morphogenic ability in embryogenic cell suspensions after irradiation up to 25 Gy. (author)

  6. Evaluation of treatment response to autologous transplantation of noncultured melanocyte/keratinocyte cell suspension in patients with stable vitiligo.

    Ramos, Mariana Gontijo; Ramos, Daniel Gontijo; Ramos, Camila Gontijo

    2017-01-01

    Vitiligo is a chronic disease characterized by the appearance of achromic macules caused by melanocyte destruction. Surgical treatments with melanocyte transplantation can be used for stable vitiligo cases. To evaluate treatment response to the autologous transplantation of noncultured epidermal cell suspension in patients with stable vitiligo. Case series study in patients with stable vitiligo submitted to noncultured epidermal cell suspension transplantation and evaluated at least once, between 3 and 6 months after the procedure, to observe repigmentation and possible adverse effects. The maximum follow-up period for some patients was 24 months. Of the 20 patients who underwent 24 procedures, 25% showed an excellent rate of repigmentation, 50% good repigmentation, 15% regular, and 10% poor response. The best results were observed in face and neck lesions, while the worst in extremity lesions (88% and 33% of satisfactory responses, respectively). Patients with segmental vitiligo had a better response (84%) compared to non-segmental ones (63%). As side effects were observed hyperpigmentation of the treated area and the appearance of Koebner phenomenon in the donor area. Some limitations of the study included the small number of patients, a subjective evaluation, and the lack of long-term follow-up on the results. CONCLUSION: Noncultured epidermal cell suspension transplantation is efficient and well tolerated for stable vitiligo treatment, especially for segmental vitiligo on the face and neck.

  7. Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy.

    Tu-Anh Tran

    Full Text Available BACKGROUND: In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4+CD25+ regulatory T cells (Tregs are a CD4+ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART. METHODOLOGY/PRINCIPAL FINDINGS: We found evidence of infection of resting Tregs (HLADR(-CD69(-CD25(hiFoxP3+CD4+ T cells purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir. CONCLUSIONS: Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.

  8. Synergistic reduction of toluylene blue induced by acetaldehyde and menadione in yeast cell suspension: Application to determination of yeast cell activity

    Shiro Yamashoji

    2017-03-01

    Full Text Available Membrane permeant acetaldehyde and menadione induced the synergistic reduction of toluylene blue (TB acting as non-membrane permeant redox indicator in yeast cell suspension. NADH and acetaldehyde also induced the synergistic TB reduction in permeabilized yeast cells and phosphate buffer, but menadione had no ability to promote TB reduction. The pre-incubation of acetaldehyde inhibited the above synergistic reduction of TB in intact and permeabilized yeast cell suspension. The pre-incubation of acetaldehyde might promote NADH oxidation by alcohol dehydrogenase, because acetaldehyde decreased the intracellular NAD(PH concentration. The above facts indicate that the synergistic reduction of TB is controlled by the order of addition of menadione and acetaldehyde. The synergistic reduction of TB by menadione and acetaldehyde was proportional to viable yeast cell number from 104 to 2×106 cells/ml, and this assay was applicable to cytotoxicity test. The time required for the above assay was only 2 min.

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

    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.

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

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

    2017-08-01

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

  11. B Cells Negatively Regulate the Establishment of CD49b(+)T-bet(+) Resting Memory T Helper Cells in the Bone Marrow.

    Hojyo, Shintaro; Sarkander, Jana; Männe, Christian; Mursell, Mathias; Hanazawa, Asami; Zimmel, David; Zhu, Jinfang; Paul, William E; Fillatreau, Simon; Löhning, Max; Radbruch, Andreas; Tokoyoda, Koji

    2016-01-01

    During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs) to the bone marrow (BM) in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b(+)T-bet(+) antigen-experienced CD4 T cells in the spleen. CD49b(+)T-bet(+) antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b(+)T-bet(+) precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

  12. B cells negatively regulate the establishment of CD49b+T-bet+ resting memory T helper cells in the bone marrow

    Shintaro eHojyo

    2016-02-01

    Full Text Available During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs to the bone marrow (BM in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b+T-bet+ antigen-experienced CD4 T cells in the spleen. CD49b+T-bet+ antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b+T-bet+ precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

  13. Effect of substrate and cathode parameters on the properties of suspension plasma sprayed solid oxide fuel cell electrolytes

    Waldbillig, D.; Tang, Z.; Burgess, A. [British Columbia Univ., Vancouver, BC (Canada); Kesler, O. [Toronto Univ., ON (Canada)

    2008-07-01

    An axial injection suspension plasma spray system has been used to produce layers of fully stabilized yttriastabilized zirconia (YSZ) that could be used as solid oxide fuel cell (SOFC) electrolytes. Suspension plasma spraying is a promising technique for the rapid production of coatings with fine microstructures and controlled porosity without requiring a post-deposition heat treatment. This new manufacturing technique to produce SOFC active layers requires the build up of a number of different plasma sprayed SOFC functional layers (cathode, electrolyte and anode) sequentially on top of each other. To understand the influence of the substrate and previouslydeposited coating layers on subsequent coating layer properties, YSZ layers were deposited on top of plasma sprayed composite lanthanum strontium manganite (LSM)/YSZ cathode layers that were first deposited on porous ferritic stainless steel substrates. Three layer half cells consisting of the porous steel substrate, composite cathode, and suspension plasma sprayed electrolyte layer were then characterized. A systematic study was performed in order to investigate the effect of parameters such as substrate and cathode layer roughness, substrate surface pore size, and cathode microstructure and thickness on electrolyte deposition efficiency, cathode and electrolyte permeability, and layer microstructure. (orig.)

  14. Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell.

    Qi, Xiang-Hui; Zhu, Jing-Fei; Yun, Jun-Hua; Lin, Jing; Qi, Yi-Lin; Guo, Qi; Xu, Hong

    2016-09-01

    Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G. oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80 g/L xylitol by mixed culture from 30 g/L d-xylulose in 28 h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10 g/L xylitol was produced by the mixed culture from 30 g/L d-arabitol in 30 h with a yield of 0.837 g/g, and the max volumetric productivity of 0.990 g/L h was obtained at 22 h. These contrast to the fact that wild-type strain G. oxydans only produced 8.10 g/L xylitol in 30 h with a yield of 0.270 g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

    Abdel-Mohsen, Mohamed; Kuri-Cervantes, Leticia; Grau-Exposito, Judith; Spivak, Adam M; Nell, Racheal A; Tomescu, Costin; Vadrevu, Surya Kumari; Giron, Leila B; Serra-Peinado, Carla; Genescà, Meritxell; Castellví, Josep; Wu, Guoxin; Del Rio Estrada, Perla M; González-Navarro, Mauricio; Lynn, Kenneth; King, Colin T; Vemula, Sai; Cox, Kara; Wan, Yanmin; Li, Qingsheng; Mounzer, Karam; Kostman, Jay; Frank, Ian; Paiardini, Mirko; Hazuda, Daria; Reyes-Terán, Gustavo; Richman, Douglas; Howell, Bonnie; Tebas, Pablo; Martinez-Picado, Javier; Planelles, Vicente; Buzon, Maria J; Betts, Michael R; Montaner, Luis J

    2018-04-18

    The persistence of HIV reservoirs, including latently infected, resting CD4 + T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 + T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 + T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV + or SIV + subjects, we found that most of the circulating memory CD32 + CD4 + T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a T H 2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4 + T cells in peripheral blood or lymphoid tissue; isolated CD32 + resting CD4 + T cells accounted for less than 3% of the total HIV DNA in CD4 + T cells. Cell-associated HIV DNA and RNA loads in CD4 + T cells positively correlated with the frequency of CD32 + CD69 + CD4 + T cells but not with CD32 expression on resting CD4 + T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4 + T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4 + T cells enriched for transcriptionally active HIV after long-term ART. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  16. Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

    Lattanzio, Vincenzo; Caretto, Sofia; Linsalata, Vito; Colella, Giovanni; Mita, Giovanni

    2018-06-01

    Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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

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

    2017-01-01

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

  18. Red blood cell (RBC) suspensions in confined microflows: Pressure-flow relationship.

    Stauber, Hagit; Waisman, Dan; Korin, Netanel; Sznitman, Josué

    2017-10-01

    Microfluidic-based assays have become increasingly popular to explore microcirculation in vitro. In these experiments, blood is resuspended to a desired haematocrit level in a buffer solution, where frequent choices for preparing RBC suspensions comprise notably Dextran and physiological buffer. Yet, the rational for selecting one buffer versus another is often ill-defined and lacks detailed quantification, including ensuing changes in RBC flow characteristics. Here, we revisit RBC suspensions in microflows and attempt to quantify systematically some of the differences emanating between buffers. We measure bulk flow rate (Q) of RBC suspensions, using PBS- and Dextran-40, as a function of the applied pressure drop (ΔP) for two hematocrits (∼0% and 23%). Two distinct microfluidic designs of varying dimensions are employed: a straight channel larger than and a network array similar to the size of individual RBCs. Using the resulting pressure-flow curves, we extract the equivalent hydrodynamic resistances and estimate the relative viscosities. These efforts are a first step in rigorously quantifying the influence of the 'background' buffer on RBC flows within microfluidic devices and thereby underline the importance of purposefully selecting buffer suspensions for microfluidic in vitro assays. Copyright © 2017. Published by Elsevier Ltd.

  19. Effects of exogenous growth regulators on cell suspension culture of yin-hong grape (vitis vinifera l.) and establishment of the optimum medium

    Chao, Y.; Feng, J.C.; Yan, W.Y.; Xiao, Y.; Jun, Y.Y

    2015-01-01

    Callus induced by stem of Yin-hong grape (Vitis vinifera L.) was used as materials and B5 medium as basic medium. The major growth parameters of cell suspension cultures with various levels of 1-Naphthaleneacetic acid (NAA) and 6-Benzyl aminopurine (6-BA) were investigated to provide a basis for the optimum medium of suspension cell cultures of Yin-hong grape regarding cell number, packed cell volume (PCV), dry cell weight (DCW), cell viability, and morphology. All data were analysed by of two-way analysis of variance (ANOVA). Results showed that the treatment of 6-BA and NAA would effect the cell growth dynamics, probably causing logarithmic phase in advance at higher levels of 6-BA. Different concentration of 6-BA and NAA had significant effects on cells number, PCV, DCW and viability (p<0.05), while no-significant effect was observed on the cells morphology. The optimum medium for suspension cell cultures of Yin-hong grape was identified as B5+1.5 mg/L6-BA+1.5 mg/LNAA+ 250 mg/L casein hydrolysate + 30 g/L sucrose. With the optimum medium, the maximum number of suspension cells after the logarithmic growth phase was 34.78 * 108 / mL, the highest cell viability reached 86.45%.; DCW reached 3.84 g/L and PCV reached 0.092 mL/mL after eight days cultivating. (author)

  20. Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

    2011-01-01

    Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

  1. Enhanced production of phenolic acids in cell suspension culture of Salvia leriifolia Benth. using growth regulators and sucrose.

    Modarres, Masoomeh; Esmaeilzadeh Bahabadi, Sedigheh; Taghavizadeh Yazdi, Mohammad Ehsan

    2018-04-01

    Salvia leriifolia Benth. (Lamiaceae) is an endangered medicinal plant with hypoglycemic, anti-inflammatory and analgesic properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. In the present study, an efficient procedure has been developed for establishment of cell suspension culture of S. leriifolia as a strategy to obtain an in vitro phenolic acids producing cell line for the first time. The effect of growth regulators and various concentrations of sucrose have been analyzed, to optimize biomass growth and phenolic acids production. The callus used for this purpose was obtained from leaves of 15-day-old in vitro seedlings, on Murashige and Skoog (MS) basal medium supplemented with different hormone balances including benzylaminopurine (BAP) and indole butyric acid (IBA); 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN); naphthaleneacetic acid (NAA) and BAP. Modified MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA was the optimal condition for callus formation with the highest induction rate (100%), the best callus growth and the highest phenolic acids content. No callus induction was observed in combinations of IBA and BAP. Cell suspension cultures were established by transferring 0.5 g of callus to 30 mL liquid MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA. Dynamics of phenolic acids production has been investigated during the growth cycle of the suspension cultures. The maximum content of caffeic acid and salvianolic acid B were observed on the 15th day of the cultivation cycle while the highest amount of rosmarinic acid was observed on the first day. In response to various sucrose concentrations, cell cultures with 40 g/L sucrose not only produced the highest dry biomass but also the highest induction of caffeic acid and salvianolic acid B. The highest amount of rosmarinic acid was observed in media containing 50 g/L sucrose. These prepared cell suspension cultures provided a useful

  2. Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions.

    Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng

    2016-05-06

    Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn(2+)-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions*

    Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng

    2016-01-01

    Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn2+-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. PMID:26994136

  4. Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture.

    Brownleader, M D; Dey, P M

    1993-01-01

    Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of beta-L-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240-300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif--Ser (Hyp)4--within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.

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

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

    2016-04-15

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

  6. Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

    Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

    2017-10-01

    This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

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

    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.

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

    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.

    2012-01-01

    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 O 2 •− /HO 2 • , HO • , and H 2 O 2 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 1 O 2 was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO 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 1 O 2 upon X-ray irradiation opens novel approaches in the design of therapy strategies.

  9. RESTful NET

    Flanders, Jon

    2008-01-01

    RESTful .NET is the first book that teaches Windows developers to build RESTful web services using the latest Microsoft tools. Written by Windows Communication Foundation (WFC) expert Jon Flanders, this hands-on tutorial demonstrates how you can use WCF and other components of the .NET 3.5 Framework to build, deploy and use REST-based web services in a variety of application scenarios. RESTful architecture offers a simpler approach to building web services than SOAP, SOA, and the cumbersome WS- stack. And WCF has proven to be a flexible technology for building distributed systems not necessa

  10. Ion channel regulation of the dynamical instability of the resting membrane potential in saccular hair cells of the green frog (Rana esculenta)

    Jorgensen, F; Kroese, ABA

    2005-01-01

    Aims: We investigated the ion channel regulation of the resting membrane potential of hair cells with the aim to determine if the resting membrane potential is poised close to instability and thereby a potential cause of the spontaneous afferent spike activity. Methods: The ionic mechanism and the

  11. Laboratory treatability studies preparatory to field testing a resting-cell in situ microbial filter bioremediation strategy

    Taylor, R.T.; Hanna, M.L.

    1995-04-01

    Prior to a down-hole-column treatability test of a Methylosinus trichosporium OB3b attached-resting-cell in situ biofilter strategy, a set of three sequential laboratory experiments were carried out to define several key operational parameters and to evaluate the likely degree of success at a NASA Kennedy Space Center site. They involved the cell attachment to site-specific sediments, the intrinsic resting-cell biotransformation capacities for the contaminants of interest plus their time-dependent extents of biodegradative removal at the concentrations of concern, and a scaled in situ mini-flow-through-column system that closely mimics the subsurface conditions during a field-treatability or pilot test of an emplaced resting-cell filter. These experiments established the conditions required for the complete metabolic removal of a vinyl chloride (VC), cis-dichlororthylene (cis-DCE) and trichloroethylene (TCE) mixture. However, the gas chromatographic (GC) procedures that we utilized and the mini-flow-through column data demonstrated that, at most, only about 50--70% of the site-water VC, cis-DCE, and TCE would be biodegraded. This occurred because of a limiting level of dissolved oxygen, which was exacerbated by the simultaneous presence of several additional previously unrecognized groundwater components, especially methane, that are also competing substrates for the whole-cell soluble methane monooxygenase (sMMO) enzyme complex. Irrespective, collectively the simplicity of the methods that we have developed and the results obtainable with them appear to provide relevant laboratory-based test-criteria before taking our microbial filter strategy to an in situ field treatability or pilot demonstration stage at other sites in the future

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

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

    2015-01-01

    © 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.

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

    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.

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

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

    1998-01-01

    of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... 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......Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7...

  15. NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments.

    Eriksson, Stefanie; Elbing, Karin; Söderman, Olle; Lindkvist-Petersson, Karin; Topgaard, Daniel; Lasič, Samo

    2017-01-01

    Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.

  16. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    Owens, T

    1988-01-01

    on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

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

    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

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

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

    2014-01-01

    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 + -K cells) was established. Induction of Neurogenin3 expression in Nkx6.1 + -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 + -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 reaggregation

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

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

    1987-01-01

    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. ( 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 ( 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

  20. An "escape clock" for estimating the turnover of SIV DNA in resting CD4⁺ T cells.

    Jeanette Reece

    Full Text Available Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT SIV DNA present in early infection by CTL escape mutant (EM strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.

  1. Biosynthesis of phenolic compounds inVitis vinifera cell suspension cultures: Study on hydroxycinnamoyl CoA:ligase.

    Lotfy, S; Lofty, S; Fleuriet, A; Ramos, T; Macheix, J J

    1989-02-01

    In cell suspensions cultures from grape berry pulp (Vitis vinifera cv. Gamay fréaux)hydroxycinnamoyl CoA ligase (CoAL) displayed maximum activity (100 %) forp-coumaric acid and then, in decreasing order, for ferulic acid (81.3 %) and caffeic acid (60.4 %). No activity was detected with sinapic and cinnamic acids. The changes in CoAL activity during the growth cycle of the culture displayed two peaks : the highest (6 h after subculturing) was linked with a strong increase in protein caused by dilution ; the second was weaker and occurred on the 7th day of culture.Grape cell suspension accumulated mainly peonidin (Pn) and cyanidin (Cy) glucosides (Pn 3-glucoside, Cy 3-glucoside, Pn 3-acetylglucoside, Pn 3-caffeylglucoside, Pn 3-p-coumarylglucoside, and Cy 3-p-coumarylglucoside). Maximum accumulation of anthocyanins was associated with the exponential growth phase of the culture and might be the result of the substantial increase in CoAL activity resulting from the effect of dilution. The second enzyme activity peak was probably oriented towards the acylation of anthocyanins since the percentage of acylated forms increased with time after subculturing.

  2. Cell suspension method to improve green spot in in-vitro culture of jarak pagar (Jatropha curcas L ) mutant lines

    Ita Dwimahyani

    2007-01-01

    Jatropha curcas has a high potential as an alternative energy source, since it can produce natural oil which could be processed into fuel replacing fossil energy. Increasing demand of biodiesel has resulted in increasing demand for high quality of Jatropha germplasm. Cell suspension method is expected to assure the production of a homogeneous germplasm of Jatropha. A laboratory experiment was conducted to evaluate the effectiveness cell suspension method in of Jatropha curcas cotyledon. The explant used in this experiment was Jatropha curcas seed mutant line (JH-38) which has superiority in plant height, early maturity and unseasonable fruiting. Two kinds of in-vitro medium were used for callus induction, i.e. medium A (MS + 2,4-D 2.0 mg/l + BAP 0.5 mg/l + malt extract 0.1 g + agar 8.0 g/l) and medium B (MS + 2,4-D 3.0 mg/l + BAP 0,5 mg/l + malt extract 0,1 g + agar 8.0 g/l). The same medium composition without agar was used for cell generating, and medium ECS (MS + glutamine 0.5 g + casein hydrolysate 0.5 g + IAA 0.5 mg/l + BAP 3.0 mg/l + agar 8.0 g/l for cell growth. Results of the experiment showed that the optimum growth of calli was obtained by explant JH-38/3 in medium A. The growth level of embryonic cell ranged from 0 to 130 %. The optimum percentage green spot is shown by JH-38/1 explant in medium A. (author)

  3. A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

    Avettand-Fenoël, Véronique; Nembot, Georges; Mélard, Adeline; Blanc, Catherine; Lascoux-Combe, Caroline; Slama, Laurence; Allegre, Thierry; Allavena, Clotilde; Yazdanpanah, Yazdan; Duvivier, Claudine; Katlama, Christine; Goujard, Cécile; Seksik, Bao Chau Phung; Leplatois, Anne; Molina, Jean-Michel; Meyer, Laurence; Autran, Brigitte; Rouzioux, Christine

    2013-01-01

    Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI). We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM]) and short-lived (transitional-memory [TTM] and effector-memory cells [TEM]) resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells), although 10-fold less (p = 0.0005) than in equally infected TCM (4.5), TTM (4.7) and TEM (4.6) cells. CD3−CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells), unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells). The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility, suggesting that

  4. A single HIV-1 cluster and a skewed immune homeostasis drive the early spread of HIV among resting CD4+ cell subsets within one month post-infection.

    Charline Bacchus

    Full Text Available Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI. We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM] and short-lived (transitional-memory [TTM] and effector-memory cells [TEM] resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells, although 10-fold less (p = 0.0005 than in equally infected TCM (4.5, TTM (4.7 and TEM (4.6 cells. CD3-CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells, unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells. The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility

  5. Load-cell based characterization system for a “Violin-Mode” shadow-sensor in advanced LIGO suspensions

    Lockerbie, N. A.; Tokmakov, K. V.

    2016-01-01

    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.

  6. Load-cell based characterization system for a "Violin-Mode" shadow-sensor in advanced LIGO suspensions

    Lockerbie, N. A.; Tokmakov, K. V.

    2016-07-01

    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.

  7. Load-cell based characterization system for a “Violin-Mode” shadow-sensor in advanced LIGO suspensions

    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.

  8. Antigen presentation by resting B cells. Radiosensitivity of the antigen-presentation function and two distinct pathways of T cell activation

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1984-01-01

    In this report we have examined the ability of small resting B cells to act as antigen-presenting cells (APC) to antigen-specific MHC-restricted T cells as assessed by either T cell proliferation or T cell-dependent B cell stimulation. We found that 10 of 14 in vitro antigen-specific MHC-restricted T cell clones and lines and three of four T cell hybridomas could be induced to either proliferate or secrete IL-2 in the presence of lightly irradiated (1,000 rads) purified B cells and the appropriate foreign antigen. All T cell lines and hybridomas were stimulated to proliferate or make IL-2 by macrophage- and dendritic cell-enriched populations and all T cells tested except one hybridoma caused B cell activation when stimulated with B cells as APC. Furthermore, lightly irradiated, highly purified syngeneic B cells were as potent a source of APC for inducing B cell activation as were low density dendritic and macrophage-enriched cells. Lymph node T cells freshly taken from antigen-primed animals were also found to proliferate when cultured with purified B cells and the appropriate antigen. This APC function was easily measured when the cells were irradiated with 1,000 rads, but was greatly diminished or absent when they were irradiated with 3,300 rads. In addition, this radiosensitivity allowed us to easily distinguish B cell antigen presentation from presentation by the dendritic cell and macrophage, as the latter was resistant to 3,300 rads. Finally, one T cell clone that failed to proliferate when B cells were used as APC was able to recruit allogeneic B cells to proliferate in the presence of syngeneic B cells and the appropriate antigen. This result suggests that there are at least two distinct pathways of activation in T cells, one that leads to T cell proliferation and one that leads to the secretion of B cell recruitment factor(s)

  9. The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension

    Zhang Hong-Yu

    2010-11-01

    Full Text Available Abstract Background The ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. It has been shown that chronic hindlimb unloading downregulates the satellite cell activity. This study investigated the role of low-frequency electrical stimulation on satellite cell activity during a 28 d hindlimb suspension in rats. Results Mechanical unloading resulted in a 44% reduction in the myofiber cross-sectional area as well as a 29% and 34% reduction in the number of myonuclei and myonuclear domains, respectively, in the soleus muscles (P vs the weight-bearing control. The number of quiescent (M-cadherin+, proliferating (BrdU+ and myoD+, and differentiated (myogenin+ satellite cells was also reduced by 48-57% compared to the weight-bearing animals (P P Conclusion This study shows that electrical stimulation partially attenuated the decrease in muscle size and satellite cells during hindlimb unloading. The causal relationship between satellite cell activation and electrical stimulation remain to be established.

  10. Analysis of survival of C-18 cells after irradiation in suspension with chelated and ionic bismuth-212 using microdosimetry

    Stinchcomb, T.G.; Roeske, J.C.

    1994-01-01

    A previous analysis of non-stochastic dose based on data obtained during irradiations of C-18 cells in suspension by α particles emitted from two forms (chelated and ionic) of 212 Bi was made using survival curves. No appreciable difference in slope (1/D o ) was found between the two forms. Such non-stochastic analyses do not account for the large differences in specific energies deposited in the individual cell nuclei. This microdosimetric (1/z o ) of the individual C-18 cells using the distribution of specific energies deposited in the individual cell nuclei. The resulting sensitivity is greater for the α particles emitted from the chelated 212 Bi than from the ionic 212 Bi. An attempt to account for this greater sensitivity in terms of greater LET of α particles passing through the cell nuclei from the chelated 212 Bi is unsuccessful. Instead the greater sensitivity disappears if the microdosimetric analysis uses average values for the radii of the cell and of its nucleus rather than the values (from the peak in the cell size distribution) used by the non-stochastic dose analysis. 13 refs., 7 figs

  11. Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells.

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

    2010-09-01

    A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

  12. Production of metabolites from chloro biphenyls by resting cells of Pseudomonas strain LB400 after growth on different carbon sources

    Billingsley, K.A.; Ward, O.P.

    1999-01-01

    The transformation of polychlorinated biphenyl (PCB), when exposed to microorganisms, into chlorobenzoic acid metabolites was studied. PCBs are widely used in electrical transformers and have become widely dispersed in the environment. A proposed system for clean up of PCBs involves the combined use of anaerobic reductive dehalogenation of highly chlorinated congeners followed by aerobic degradation of moderately chlorinated PCBs, mediated by oxidative ring attack. Much of this work focused on biphenyl-grown cells, in particular Pseudomonas strain LB400 grown on biphenyl, which contains a multicomponent enzyme system. It was shown that resting cells of Pseudomonas strain LB400, grown on glycerol or glucose, could also transform purified PCB congeners and mixtures of PCBs in Aroclors, however, transformation rates were less than those observed with biphenyl-grown cells. 29 refs., 2 tabs., 2 figs

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

    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.

  14. Anti-Cancer Activity of Resveratrol and Derivatives Produced by Grapevine Cell Suspensions in a 14 L Stirred Bioreactor

    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.

  15. Computational fluid dynamics (CFD) analysis of airlift bioreactor: effect of draft tube configurations on hydrodynamics, cell suspension, and shear rate.

    Pawar, Sanjay B

    2018-01-01

    The biomass productivity of microalgae cells mainly depends on the hydrodynamics of airlift bioreactor (ABR). Thus, the hydrodynamics of concentric tube ABR was initially studied using two-phase three-dimensional CFD simulations with the Eulerian-Lagrangian approach. The performance of ABR (17 L) was examined for different configurations of the draft tube using various drag models such as Grace, Ishii-Zuber, and Schiller-Naumann. The gas holdups in the riser and the downcomer were well predicted using E-L approach. This work was further extended to study the dispersion of microalgae cells in the ABR using three-phase CFD simulations. In this model (combined E-E and E-L), the solid phase (microalgae cells) was dispersed into the continuous liquid phase (water), while the gas phase (air bubbles) was modeled as a particle transport fluid. The effect of non-drag forces such as virtual mass and lift forces was also considered. Flow regimes were explained on the basis of the relative gas holdup distribution in the riser and the downcomer. The microalgae cells were found in suspension for the superficial gas velocities of 0.02-0.04 m s -1 experiencing an average shear of 23.52-44.56 s -1 which is far below the critical limit of cell damage.

  16. Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae

    Koay Suan See

    2011-06-01

    Full Text Available Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of variousailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production.The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time oncell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of differentconcentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50mg/L had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45g/L sucrose without MeJA showed the highest pigment content (0.69±0.22Cv/g-FCM. The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40Cv/g-FCM. This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures. Rev. Biol. Trop. 59 (2: 597-606. Epub 2011 June 01.

  17. Metabolism of pentachlorophenol in cell suspension cultures of wheat (Triticum aestivum L.). Tetrachlorocatechol as a primary metabolite

    Schaefer, W.; Sandermann, H. Jr.

    1988-01-01

    Wheat cell suspension cultures were incubated with [U- 14 C] pentachlorophenol (PCP; 1 ppm, 48 h, 27 0 C). Soluble metabolites were formed in ∼ 50% yield, another ∼ 31% of the applied radioactivity being incorporated into the insoluble residue. The soluble metabolite fraction, and its β-D-glucoside conjugate components, the total insoluble residue, and its lignin components, were all found to contain a novel polar PCP derivative besides smaller amounts of tetrachlorohydroquinone and PCP. The novel derivative also predominated in intact wheat plants and was identified as tetrachlorocatechol by TLC, HPLC, GC, and EI as well as CI mass spectroscopy. Tetrachlorocatechol is a potential mutagen, so that the soluble and insoluble conjugates formed in wheat from PCP may present a toxicological hazard

  18. Biotransformation of artemisinin using cell suspension cultures of Catharanthus roseus (L.) G.Don and Lavandula officinalis L.

    Patel, Suman; Gaur, Rashmi; Verma, Priyanka; Bhakuni, Rajendra S; Mathur, Archana

    2010-08-01

    Artemisinin, an antimalarial compound, at 5 mg/40 ml, was transformed by cell suspension cultures of Catharanthus roseus (L.) G.Don and Lavandula officinalis L. into deoxyartemisinin with yields >78% (3.93 mg deoxyartemisinin from 5 mg artemisinin). Maximum conversion (78.6 and 78%) occurred after 6 and 7 days of adding artemisinin to 20 and 9 days old cultures of C. roseus and L. officinalis, respectively. The procedure was scaled up by and 500 mg artemisinin was transformed into 390 mg deoxyartemisinin. Addition of artemisinin at the beginning of the culture cycle resulted in >50% reduction in dry biomass production with no bioconversion. Conversion of artemisinin occurred intracellularly followed by leaching of the product into the medium.

  19. A fundamental research of growth, metabolism and product formation of tobacco suspension cells at different scales

    Ullisch, David

    2012-01-01

    For over two decades, plant cell cultures have been promising hosts for the expression of recombinant proteins such as hormones, growth factors, full-size antibodies and antigens. So far, over 700 different plant cell cultures are stored in the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig. Among these plant cell cultures, the tobacco cell line Nicotiana tabacum Bright Yellow 2 (BY-2) was chosen as a good host cell line for the production of recombinant proteins...

  20. Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

    Eric eRuelland

    2014-11-01

    Full Text Available Basal phosphoinositide-dependent phospholipase C (PI-PLC activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently.

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

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

    2017-10-01

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

  2. Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg.) cell suspension cultures under cadmium and zinc stress

    P. Thangavel; Stephanie Long; Rakesh Minocha

    2007-01-01

    Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC2 and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12...

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

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

    2016-09-01

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

  4. Structural characterisation of xyloglucan secreted by suspension-cultured cells of Nicotiana plumbaginifolia.

    Sims, I M; Munro, S L; Currie, G; Craik, D; Bacic, A

    1996-10-31

    Linkage analysis of a xyloglucan from the extracellular medium of suspension cultures of Nicotiana plumbaginifolia showed mostly 4-Glcp and 4,6-Glcp, terminal Xylp and 2-Xylp, and terminal Araf, along with approximately 10% (w/w) O-acetyl groups, equivalent to approximately 0.28 mol acetyl per mol of glycosyl residue. Methylation with methyl trifluoromethanesulfonate under neutral conditions, followed by re-methylation with CD3I under basic conditions, and conversion into partially methylated alditol acetates showed that O-acetyl groups were primarily attached to C-6 of approximately 44% of the 4-Glcp backbone not substituted with Xylp residues and to C-5 of approximately 15% of the terminal Araf residues. These positions of the O-acetyl groups were confirmed by 1H-NMR. Oligosaccharides generated by digestion of native xyloglucan with endo-(1-->4)-beta-glucanase were separated by a combination of gel-filtration chromatography and anion-exchange HPLC, and analysed by glycosyl linkage analysis and by electrospray ionisation-mass spectrometry (ESI-MS). The major oligosaccharide subunits were Glc4Xyl2 and Glc5Xyl2, of which 50-60% are substituted with one terminal Araf residue attached to O-2 of a Xylp residue, and a further 20-25% are substituted with two terminal Araf residues attached to O-2 of the Xylp residues. ESI-MS showed that many of the oligosaccharide subunits carried one, two, and, occasionally three O-acetyl groups.

  5. Correlated accumulation of anthocyanins and rosmarinic acid in mechanically stressed red cell suspensions of basil (Ocimum basilicum).

    Strazzer, Pamela; Guzzo, Flavia; Levi, Marisa

    2011-02-15

    A red basil cell line (T2b) rich in rosmarinic acid (RA) was selected for the stable production of anthocyanins (ACs) in the dark. Cell suspension cultures were subjected to mechanical stress through increased agitation (switch from 90 to 150 rpm) to determine the relationship between AC and RA accumulation. Cell extracts were analyzed by HPLC and LC-MS, and the resulting data were processed with multivariate statistical analysis. MS and MS/MS spectra facilitated the putative annotation of several complex cyanidin-based ACs, which were esterified with coumaric acid and, in some cases, also with malonic acid. It was also possible to identify various RA-related molecules, some caffeic and coumaric acid derivatives and some flavanones. Mechanical stress increased the total AC and RA contents, but reduced biomass accumulation. Many metabolites were induced by mechanical stress, including RA and some of its derivatives, most ACs, hydroxycinnamic acids and flavonoids, whereas the abundance of some RA dimers was reduced. Although AC and RA share a common early biosynthetic pathway (from phenylalanine to 4-coumaroyl-CoA) and could have similar or overlapping functions providing antioxidant activity against stress-generated reactive oxygen species, there appeared to be no competition between their individual pathways. Copyright © 2010 Elsevier GmbH. All rights reserved.

  6. Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

    Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

    2014-10-16

    The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

  7. Induction of lethal and genetic damage by vacuum-ultraviolet (163 nm) irradiation of aqueous suspensions of yeast cells

    Ito, T.; Kobayashi, K.

    1976-01-01

    Yeast cells suspended in distilled water were irradiated with monochromatic 163 nm photons by immersing a specially designed discharge tube into the suspension. This was thought to be a useful means of investigating in vivo effects of radiation-induced water radicals on well cells in the complete absence of ionic species, since 163 nm photons can dissociate water only via excitation. These experiments showed that the water radicals (excluding e/sub aq/ - ) exerted both lethal and genetic (gene-conversion) effects quite potently, and the characteristic protection against these effects was observable when 2-mercaptoethanol or, in particular, p-aminobenzoic acid, a specific scavenger for OH radicals, was added to the medium prior to irradiation. Nearly complete protection from both lethal and genetic effects was observed in some cases with p-aminobenzoic acid. These results establish unequivocally that the OH radical, and not the hydrogen atom (H radical), possesses the damaging potency in the cell. Comparisons with γ-ray experiments revealed several differences between 163 nm photons and γ rays in the protective actions of radical scavengers, which may be attributable to reactive species other than OH radicals produced by the γ rays

  8. Use of Resting Cells of Native Screened Rhodotorula sp. CW03 in Biotransformation of Caffeine to Theophylline and Paraxanthine

    M. Ashengroph

    2015-07-01

    Full Text Available Introduction & Objective: In recent years, microorganisms have been applied as biocatalysts for making pharmaceutically natural products. Microbial biotransformation of caffeine suggests a dual approach for biodegradation of toxic caffeine from polluted environments and a method for the production of medically and pharmaceutically valuable dimethylxanthines. The present work describes the identification of native yeasts capable of biotransformation of caffeine into theophylline and paraxanthine. Materials & Methods: In this experimental study fourteen yeast strains which were able to de-grade caffeine isolated based on their morphology were selected as biocatalysts for biotrans-formation of caffeine as a low-cost substrate to high value added dimethylxanthines such as theophylline and theobromine. The selected strains were characterized based on phenotypic and genetic tests. Screening was performed by Thin Layer Chromatography (TLC and High Performance Liquid Chromatography (HPLC analyses. Results: The results obtained using TLC and HPLC analyses suggest formation of two main metabolites of theophylline and paraxanthine from biotransformation of caffeine under resting cells of Rhodotorula sp. CW03 (GenBank accession number KF414531. The results showed that under resting cell conditions a maximum concentration of theophylline 380 mg/l (molar yield of 16.4% and paraxanthine 880 mg/l (molar yield of 37.9% were obtained after 72 h and 120 h of conversion time, respectively. Conclusion: In the current investigation, done for the first time in Iran, we describe the isola-tion and identification of yeast strains with caffeine degradation ability which can be proposed as safe and cost-effective biocatalysts in production of value added dimethylxanthines from caffeine as a low-cost substrate.(Sci J Hamadan Univ Med Sci 2015; 22 (2: 83-92

  9. Detection of anthraquinones and identification of 1,4-naphthohydroquinone in cell suspension cultures of Rudgea jasminoides (Cham.) Müll. Arg. (Rubiaceae)

    Oliveira, Marisa de Cacia; Negri, Giusepina; Salatino, Antônio; Braga, Márcia Regina

    2007-01-01

    In Rubiaceae, anthraquinones and naphthoquinones are secondary metabolites characteristic of the subfamily Rubioideae, in which Rudgea jasminoides is included. Thin-layer chromatography using specific solvent systems and spray reagents indicated the presence of anthraquinones constitutively produced by cell suspension cultures of R. jasminoides. GC/MS analysis detected 1,4-naphthohydroquinone as a product of biosynthesis only after elicitation of the cells with yeast extract (Saccharomyces ce...

  10. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells

    Zhang, Z.; Schuler, T.; Zupancic, M.; Wietgrefe, S.; Staskus, K. A.; Reimann, K. A.; Reinhart, T. A.; Rogan, M.; Cavert, W.; Miller, C. J.; Veazey, R. S.; Notermans, D.; Little, S.; Danner, S. A.; Richman, D. D.; Havlir, D.; Wong, J.; Jordan, H. L.; Schacker, T. W.; Racz, P.; Tenner-Racz, K.; Letvin, N. L.; Wolinsky, S.; Haase, A. T.

    1999-01-01

    In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in

  11. Membranes replace irradiated blast cells as growth requirement for leukemic blast progenitors in suspension culture

    Nara, N.; McCulloch, E.A.

    1985-01-01

    The blast cells of acute myeloblastic leukemia (AML) may be considered as a renewal population, maintained by blast stem cells capable of both self-renewal and the generation of progeny with reduced or absent proliferative potential. This growth requires that two conditions be met: first, the cultures must contain growth factors in media conditioned either by phytohemagglutinin (PHA)-stimulated mononuclear leukocytes (PHA-LCM), or by cells of the continuous bladder carcinoma line HTB9 (HTB9-CM). Second, the cell density must be maintained at 10(6) blasts/ml; this may be achieved by adding irradiated cells to smaller numbers of intact blasts. The authors are concerned with the mechanism of the feeding function. They present evidence that (a) cell-cell contact is required. (b) Blasts are heterogeneous in respect to their capacity to support growth. (c) Fractions containing membranes from blast cells will substitute for intact cells in promoting the generation of new blast progenitors in culture. (d) This membrane function may be specific for AML blasts, since membranes from blasts of lymphoblastic leukemia or normal marrow cells were inactive

  12. Biosynthesis of 14C-phytoene from tomato cell suspension cultures (Lycopersicon esculentum) for utilization in prostate cancer cell culture studies.

    Campbell, Jessica K; Rogers, Randy B; Lila, Mary Ann; Erdman, John W

    2006-02-08

    This work describes the development and utilization of a plant cell culture production approach to biosynthesize and radiolabel phytoene and phytofluene for prostate cancer cell culture studies. The herbicide norflurazon was added to established cell suspension cultures of tomato (Lycopersicon esculentum cv. VFNT cherry), to induce the biosynthesis and accumulation of the lycopene precursors, phytoene and phytofluene, in their natural isomeric forms (15-cis-phytoene and two cis-phytofluene isomers). Norflurazon concentrations, solvent carrier type and concentration, and duration of culture exposure to norflurazon were screened to optimize phytoene and phytofluene synthesis. Maximum yields of both phytoene and phytofluene were achieved after 7 days of treatment with 0.03 mg norflurazon/40 mL fresh medium, provided in 0.07% solvent carrier. Introduction of 14C-sucrose to the tomato cell culture medium enabled the production of 14C-labeled phytoene for subsequent prostate tumor cell uptake studies. In DU 145 prostate tumor cells, it was determined that 15-cis-phytoene and an oxidized product of phytoene were taken up and partially metabolized by the cells. The ability to biosynthesize, radiolabel, and isolate these carotenoids from tomato cell cultures is a novel, valuable methodology for further in vitro and in vivo investigations into the roles of phytoene and phytofluene in cancer chemoprevention.

  13. CellBase, a comprehensive collection of RESTful web services for retrieving relevant biological information from heterogeneous sources.

    Bleda, Marta; Tarraga, Joaquin; de Maria, Alejandro; Salavert, Francisco; Garcia-Alonso, Luz; Celma, Matilde; Martin, Ainoha; Dopazo, Joaquin; Medina, Ignacio

    2012-07-01

    During the past years, the advances in high-throughput technologies have produced an unprecedented growth in the number and size of repositories and databases storing relevant biological data. Today, there is more biological information than ever but, unfortunately, the current status of many of these repositories is far from being optimal. Some of the most common problems are that the information is spread out in many small databases; frequently there are different standards among repositories and some databases are no longer supported or they contain too specific and unconnected information. In addition, data size is increasingly becoming an obstacle when accessing or storing biological data. All these issues make very difficult to extract and integrate information from different sources, to analyze experiments or to access and query this information in a programmatic way. CellBase provides a solution to the growing necessity of integration by easing the access to biological data. CellBase implements a set of RESTful web services that query a centralized database containing the most relevant biological data sources. The database is hosted in our servers and is regularly updated. CellBase documentation can be found at http://docs.bioinfo.cipf.es/projects/cellbase.

  14. One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum.

    Khaskheli, Abid Ali; Talpur, Farah Naz; Cebeci Aydin, Aysun; Jawaid, Sana; Surhio, Muhammad Ali; Afridi, Hassan Imran

    2017-10-01

    Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.

  15. Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells.

    Voitsekhovskaja, Olga V; Schiermeyer, Andreas; Reumann, Sigrun

    2014-01-01

    Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.

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

    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.

  17. A modified suspension spray combined with particle gradation method for preparation of protonic ceramic membrane fuel cells

    Xie, Kui; Wang, Songlin; Chen, Xiaorui; Jiang, Tao; Lin, Bin; Wei, Ming; Liu, Xingqin; Meng, Guangyao [Department of Materials Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026 (China); Yan, Ruiqiang [Department of Materials Engineering, Taizhou University, Linhai, Zhejiang 317000 (China); Dong, Dehua [Department of Chemical Engineering, Monash University, Clayton, VIC 3800 (Australia)

    2008-05-01

    In order to prepare a dense proton-conductive Ba(Zr{sub 0.1}Ce{sub 0.7})Y{sub 0.2}O{sub 3-{delta}} (BZCY7) electrolyte membrane, a proper anode composition with 65% Ni{sub 2}O{sub 3} in weight ratio was determined after investigating the effects of anode compositions on anode shrinkages for co-sintering. The thermal expansion margins between sintered anodes and electrolytes, which were less than 1% below 750 C, also showed good thermal expansion compatibility. A suspension spray combined with particle gradation method had been introduced to prepare dense electrolyte membrane on porous anode support. After a heat treatment at 1400 C for 5 h, a cell with La{sub 0.5}Sr{sub 0.5}CoO{sub 3-{delta}} (LSCO) cathode was assembled and tested with hydrogen and ammonia as fuels. The outputs reached as high as 330 mW cm{sup -2} in hydrogen and 300 mW cm{sup -2} in ammonia at 700 C, respectively. Comparing with the interface of another cell prepared by dry-pressing method, this one also showed a good interface contact between electrodes and electrolyte. To sum up, this combined technique can be considered as commercial fabrication technology candidate. (author)

  18. First insights in the Eu(III) speciation in plant cell suspension cultures

    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.

  19. First insights in the Eu(III) speciation in plant cell suspension cultures

    Moll, Henry; Sachs, Susanne

    2017-01-01

    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 "7F_2 transition and prolonged luminescence lifetimes.

  20. Production of L-DOPA by suspension grown cells of Mucuna pruriens.

    Wichers, H.J.

    1985-01-01

    In vitro cultured plant cells can be utilized for the production of valuable metabolites. Many biochemical and physiological characteristics of ln vitro cultured plant cells depend on various environmental parameters, such as the composition of the growth medium in which many parameters are

  1. Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway.

    Yin, Mengmeng; Yuan, Yin; Cui, Yurong; Hong, Xian; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jurgen; Xi, Jiaoya

    2015-01-01

    Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms. RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal. Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal. Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

  2. Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

    Pech-Kú, Roberto; Muñoz-Sánchez, J Armando; Monforte-González, Miriam; Vázquez-Flota, Felipe; Rodas-Junco, Beatriz A; González-Mendoza, Víctor M; Hernández-Sotomayor, S M Teresa

    2018-04-01

    Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl 3 -treatment (500μM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Limits on replenishment of the resting CD4+ T cell reservoir for HIV in patients on HAART.

    Ahmad R Sedaghat

    2007-08-01

    Full Text Available Whereas cells productively infected with human immunodeficiency virus type 1 (HIV-1 decay rapidly in the setting of highly active antiretroviral therapy (HAART, latently infected resting CD4(+ T cells decay very slowly, persisting for the lifetime of the patient and thus forming a stable reservoir for HIV-1. It has been suggested that the stability of the latent reservoir is due to low-level viral replication that continuously replenishes the reservoir despite HAART. Here, we offer the first quantitative study to our knowledge of inflow of newly infected cells into the latent reservoir due to viral replication in the setting of HAART. We make use of a previous observation that in some patients on HAART, the residual viremia is dominated by a predominant plasma clone (PPC of HIV-1 not found in the latent reservoir. The unique sequence of the PPC serves as a functional label for new entries into the reservoir. We employ a simple mathematical model for the dynamics of the latent reservoir to constrain the inflow rate to between 0 and as few as 70 cells per day. The magnitude of the maximum daily inflow rate is small compared to the size of the latent reservoir, and therefore any inflow that occurs in patients on HAART is unlikely to significantly influence the decay rate of the reservoir. These results suggest that the stability of the latent reservoir is unlikely to arise from ongoing replication during HAART. Thus, intensification of standard HAART regimens should have minimal effects on the decay of the latent reservoir.

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

    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.

  5. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions

    Akenhead, Michael L.; Horrall, Nolan M.; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y.

    2015-01-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s−1 shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia). PMID:26065495

  6. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

    Akenhead, Michael L; Horrall, Nolan M; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y

    2015-09-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

  7. Heterotrophic Bioleaching of Sulfur, Iron, and Silicon Impurities from Coal by Fusarium oxysporum FE and Exophiala spinifera FM with Growing and Resting Cells.

    Etemadzadeh, Shekoofeh Sadat; Emtiazi, Giti; Etemadifar, Zahra

    2016-06-01

    Coal is the most abundant fossil fuel containing sulfur and other elements which promote environmental pollution after burning. Also the silicon impurities make the transportation of coal expensive. In this research, two isolated fungi from oil contaminated soil with accessory number KF554100 (Fusarium oxysporum FE) and KC925672 (Exophiala spinifera FM) were used for heterotrophic biological leaching of coal. The leaching were detected by FTIR, CHNS, XRF analyzer and compared with iron and sulfate released in the supernatant. The results showed that E. spinifera FM produced more acidic metabolites in growing cells, promoting the iron and sulfate ions removal while resting cells of F. oxysporum FE enhanced the removal of aromatic sulfur. XRF analysis showed that the resting cells of E. spinifera FM proceeded maximum leaching for iron and silicon (48.8, 43.2 %, respectively). CHNS analysis demonstrated that 34.21 % of sulfur leaching was due to the activities of resting cells of F. oxysporum FE. Also F. oxysporum FE removed organic sulfur more than E. spinifera FM in both growing and resting cells. FTIR data showed that both fungi had the ability to remove pyrite and quartz from coal. These data indicated that inoculations of these fungi to the coal are cheap and impurity removals were faster than autotrophic bacteria. Also due to the removal of dibenzothiophene, pyrite, and quartz, we speculated that they are excellent candidates for bioleaching of coal, oil, and gas.

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

    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.

  9. Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

    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.

  10. Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

    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.

  11. Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.

    Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S

    2016-12-01

    Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P skin wounds. Biotechnol. Bioeng. 2016;113: 2725-2738. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Coupling limonene formation and oxyfunctionalization by mixed-culture resting cell fermentation.

    Willrodt, Christian; Hoschek, Anna; Bühler, Bruno; Schmid, Andreas; Julsing, Mattijs K

    2015-09-01

    Metabolic engineering strategies mark a milestone for the fermentative production of bulk and fine chemicals. Yet, toxic products and volatile reaction intermediates with low solubilities remain challenging. Prominent examples are artificial multistep pathways like the production of perillyl acetate (POHAc) from glucose via limonene. For POHAc, these limitations can be overcome by mixed-culture fermentations. A limonene biosynthesis pathway and cytochrome P450 153A6 (CYP153A6) as regioselective hydroxylase are used in two distinct recombinant E. coli. POHAc formation from glucose in one recombinant cell was hindered by ineffective coupling of limonene synthesis and low rates of oxyfunctionalization. The optimization of P450 gene expression led to the formation of 6.20 ± 0.06 mg gcdw (-1) POHAc in a biphasic batch cultivation with glucose as sole carbon and energy source. Increasing the spatial proximity between limonene synthase and CYP153A6 by a genetic fusion of both enzymes changed the molar limonene/POHAc ratio from 3.2 to 1.6. Spatial separation of limonene biosynthesis from its oxyfunctionalization improved POHAc concentration 3.3-fold to 21.7 mg L(-1) as compared to a biphasic fermentation. Mixed-cultures of E. coli BL21 (DE3) containing the limonene biosynthesis pathway and E. coli MG1655 harboring either CYP153A6, or alternatively a cymene monooxygenase, showed POHAc formation rates of 0.06 or 0.11 U gcdw (-1) , respectively. This concept provides a novel framework for fermentative syntheses involving toxic, volatile, or barely soluble compounds or pathway intermediates. © 2015 Wiley Periodicals, Inc.

  13. Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

    McCue, Kent F.; Conn, Eric E.

    1990-01-01

    Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

  14. Establishing in vitro Zinnia elegans cell suspension culture with high tracheary elements differentiation

    Twumasi, P.; Schel, J.H.N.; Ieperen, van W.; Woltering, E.J.; Emons, A.M.C.

    2009-01-01

    The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and

  15. Electrical Impedance Spectroscopy for Detection of Cells in Suspensions Using Microfluidic Device with Integrated Microneedles

    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.

  16. Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry.

    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

  17. Biochemical and morphological responses to abiotc elicitor chitin in suspension-cultured sugarcane cells

    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.

  18. Evaluation of ferromagnetic fluids and suspensions for the site-specific radiofrequency-induced hyperthermia of MX11 sarcoma cells in vitro

    Brusentsov, Nikolai A.; Gogosov, V.V.; Brusentsova, T.N.; Sergeev, A.V.; Jurchenko, N.Y.; Kuznetsov, Anatoly A.; Kuznetsov, Oleg A. E-mail: oleg@louisiana.edu; Shumakov, L.I

    2001-07-01

    Seventeen different ferromagnetic fluids and suspensions were prepared and evaluated for application in radiofrequency-induced hyperthermia. Specific power absorption rates were measured at 0.88 MHz to range from 0 to 240 W per gram of iron for different preparations. Survival of MX11 cells mixed with ferrofluids and subjected to radiofrequency was much lower than with RF without ferrofluid or ferrofluid alone.

  19. Evaluation of ferromagnetic fluids and suspensions for the site-specific radiofrequency-induced hyperthermia of MX11 sarcoma cells in vitro

    Brusentsov, Nikolai A.; Gogosov, V.V.; Brusentsova, T.N.; Sergeev, A.V.; Jurchenko, N.Y.; Kuznetsov, Anatoly A.; Kuznetsov, Oleg A.; Shumakov, L.I.

    2001-01-01

    Seventeen different ferromagnetic fluids and suspensions were prepared and evaluated for application in radiofrequency-induced hyperthermia. Specific power absorption rates were measured at 0.88 MHz to range from 0 to 240 W per gram of iron for different preparations. Survival of MX11 cells mixed with ferrofluids and subjected to radiofrequency was much lower than with RF without ferrofluid or ferrofluid alone

  20. Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats.

    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.

  1. Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system

    Maršík, Petr; Šíša, Miroslav; Lacina, O.; Moťková, Kateřina; Langhansová, Lenka; Rezek, Jan; Vaněk, Tomáš

    2017-01-01

    Roč. 220, JAN (2017), s. 383-392 ISSN 0269-7491 R&D Projects: GA ČR(CZ) GA14-22593S Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * Ibuprofen * Metabolism * Plant cells * Sequestration Subject RIV: CE - Biochemistry OBOR OECD: Plant sciences, botany Impact factor: 5.099, year: 2016

  2. Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

    Cavalcanti, João Henrique F; Quinhones, Carla G S; Schertl, Peter; Brito, Danielle S; Eubel, Holger; Hildebrandt, Tatjana; Nunes-Nesi, Adriano; Braun, Hans-Peter; Araújo, Wagner L

    2017-12-01

    Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation. © 2017 Scandinavian Plant Physiology Society.

  3. Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis.

    Gas-Pascual, Elisabet; Simonovik, Biljana; Heintz, Dimitri; Bergdoll, Marc; Schaller, Hubert; Bach, Thomas J

    2015-08-01

    The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

  4. High production of succinyl isoflavone glycosides by Bacillus licheniformis ZSP01 resting cells in aqueous miscible organic medium.

    Zhang, Sen; Chen, Guoguang; Chu, Jianlin; Wu, Bin; He, Bingfang

    2015-01-01

    To achieve efficient production of succinyldaidzin and succinylgenistin, resting cells of a solvent-stable strain Bacillus licheniformis ZSP01 were used to react with pure isoflavones or soybean flour extract in a reaction medium with 10% dimethyl sulfoxide. Strikingly, 0.8 mM daidzein, 0.8 mM genistein, 2.0 mM daidzin, and 2.0 mM genistin were transformed to succinyl isoflavone glycosides in 27 H (yield >90%). The soybean flour extract (6.1%, w/v) contained 0.32 mM daidzein, 0.84 mM daidzin, 0.38 mM genistein, and 1.04 mM genistin. Over 95% of total isoflavones (daidzein, daidzin, genistein, and genistin) in the soybean flour extract were converted to succinyl isoflavone glycosides after 27 H. Strain ZSP01 shows both high glycosylation and succinylation activities. These results suggest that B. licheniformis ZSP01 could be useful for the efficient production of succinyl soybean isoflavone glycosides. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  5. A fast platform for simulating semi-flexible fiber suspensions applied to cell mechanics

    Nazockdast, Ehssan, E-mail: ehssan@cims.nyu.edu [Courant Institute of Mathematical Sciences, New York University, New York, NY 10012 (United States); Center for Computational Biology, Simons Foundation, New York, NY 10010 (United States); Rahimian, Abtin, E-mail: arahimian@acm.org [Courant Institute of Mathematical Sciences, New York University, New York, NY 10012 (United States); Zorin, Denis, E-mail: dzorin@cs.nyu.edu [Courant Institute of Mathematical Sciences, New York University, New York, NY 10012 (United States); Shelley, Michael, E-mail: shelley@cims.nyu.edu [Courant Institute of Mathematical Sciences, New York University, New York, NY 10012 (United States); Center for Computational Biology, Simons Foundation, New York, NY 10010 (United States)

    2017-01-15

    We present a novel platform for the large-scale simulation of three-dimensional fibrous structures immersed in a Stokesian fluid and evolving under confinement or in free-space in three dimensions. One of the main motivations for this work is to study the dynamics of fiber assemblies within biological cells. For this, we also incorporate the key biophysical elements that determine the dynamics of these assemblies, which include the polymerization and depolymerization kinetics of fibers, their interactions with molecular motors and other objects, their flexibility, and hydrodynamic coupling. This work, to our knowledge, is the first technique to include many-body hydrodynamic interactions (HIs), and the resulting fluid flows, in cellular assemblies of flexible fibers. We use non-local slender body theory to compute the fluid–structure interactions of the fibers and a second-kind boundary integral formulation for other rigid bodies and the confining boundary. A kernel-independent implementation of the fast multipole method is utilized for efficient evaluation of HIs. The deformation of the fibers is described by nonlinear Euler–Bernoulli beam theory and their polymerization is modeled by the reparametrization of the dynamic equations in the appropriate non-Lagrangian frame. We use a pseudo-spectral representation of fiber positions and implicit time-stepping to resolve large fiber deformations, and to allow time-steps not excessively constrained by temporal stiffness or fiber–fiber interactions. The entire computational scheme is parallelized, which enables simulating assemblies of thousands of fibers. We use our method to investigate two important questions in the mechanics of cell division: (i) the effect of confinement on the hydrodynamic mobility of microtubule asters; and (ii) the dynamics of the positioning of mitotic spindle in complex cell geometries. Finally to demonstrate the general applicability of the method, we simulate the sedimentation of a

  6. A fast platform for simulating semi-flexible fiber suspensions applied to cell mechanics

    Nazockdast, Ehssan; Rahimian, Abtin; Zorin, Denis; Shelley, Michael

    2017-01-01

    We present a novel platform for the large-scale simulation of three-dimensional fibrous structures immersed in a Stokesian fluid and evolving under confinement or in free-space in three dimensions. One of the main motivations for this work is to study the dynamics of fiber assemblies within biological cells. For this, we also incorporate the key biophysical elements that determine the dynamics of these assemblies, which include the polymerization and depolymerization kinetics of fibers, their interactions with molecular motors and other objects, their flexibility, and hydrodynamic coupling. This work, to our knowledge, is the first technique to include many-body hydrodynamic interactions (HIs), and the resulting fluid flows, in cellular assemblies of flexible fibers. We use non-local slender body theory to compute the fluid–structure interactions of the fibers and a second-kind boundary integral formulation for other rigid bodies and the confining boundary. A kernel-independent implementation of the fast multipole method is utilized for efficient evaluation of HIs. The deformation of the fibers is described by nonlinear Euler–Bernoulli beam theory and their polymerization is modeled by the reparametrization of the dynamic equations in the appropriate non-Lagrangian frame. We use a pseudo-spectral representation of fiber positions and implicit time-stepping to resolve large fiber deformations, and to allow time-steps not excessively constrained by temporal stiffness or fiber–fiber interactions. The entire computational scheme is parallelized, which enables simulating assemblies of thousands of fibers. We use our method to investigate two important questions in the mechanics of cell division: (i) the effect of confinement on the hydrodynamic mobility of microtubule asters; and (ii) the dynamics of the positioning of mitotic spindle in complex cell geometries. Finally to demonstrate the general applicability of the method, we simulate the sedimentation of a

  7. A fast platform for simulating semi-flexible fiber suspensions applied to cell mechanics

    Nazockdast, Ehssan; Rahimian, Abtin; Zorin, Denis; Shelley, Michael

    2017-01-01

    We present a novel platform for the large-scale simulation of three-dimensional fibrous structures immersed in a Stokesian fluid and evolving under confinement or in free-space in three dimensions. One of the main motivations for this work is to study the dynamics of fiber assemblies within biological cells. For this, we also incorporate the key biophysical elements that determine the dynamics of these assemblies, which include the polymerization and depolymerization kinetics of fibers, their interactions with molecular motors and other objects, their flexibility, and hydrodynamic coupling. This work, to our knowledge, is the first technique to include many-body hydrodynamic interactions (HIs), and the resulting fluid flows, in cellular assemblies of flexible fibers. We use non-local slender body theory to compute the fluid-structure interactions of the fibers and a second-kind boundary integral formulation for other rigid bodies and the confining boundary. A kernel-independent implementation of the fast multipole method is utilized for efficient evaluation of HIs. The deformation of the fibers is described by nonlinear Euler-Bernoulli beam theory and their polymerization is modeled by the reparametrization of the dynamic equations in the appropriate non-Lagrangian frame. We use a pseudo-spectral representation of fiber positions and implicit time-stepping to resolve large fiber deformations, and to allow time-steps not excessively constrained by temporal stiffness or fiber-fiber interactions. The entire computational scheme is parallelized, which enables simulating assemblies of thousands of fibers. We use our method to investigate two important questions in the mechanics of cell division: (i) the effect of confinement on the hydrodynamic mobility of microtubule asters; and (ii) the dynamics of the positioning of mitotic spindle in complex cell geometries. Finally to demonstrate the general applicability of the method, we simulate the sedimentation of a cloud of

  8. Electrochemical testing of suspension plasma sprayed solid oxide fuel cell electrolytes

    Waldbillig, D.; Kesler, O.

    Electrochemical performance of metal-supported plasma sprayed (PS) solid oxide fuel cells (SOFCs) was tested for three nominal electrolyte thicknesses and three electrolyte fabrication conditions to determine the effects of electrolyte thickness and microstructure on open circuit voltage (OCV) and series resistance (R s). The measured OCV values were approximately 90% of the Nernst voltages, and electrolyte area specific resistances below 0.1 Ω cm 2 were obtained at 750 °C for electrolyte thicknesses below 20 μm. Least-squares fitting was used to estimate the contributions to R s of the YSZ bulk material, its microstructure, and the contact resistance between the current collectors and the cells. It was found that the 96% dense electrolyte layers produced from high plasma gas flow rate conditions had the lowest permeation rates, the highest OCV values, and the smallest electrolyte-related voltage losses. Optimal electrolyte thicknesses were determined for each electrolyte microstructure that would result in the lowest combination of OCV loss and voltage loss due to series resistance for operating voltages of 0.8 V and 0.7 V.

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

    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.

  10. Application of Pectin From Rauvolfia serpentina (L.) Benth to the Cryopreservation of Human Leucocyte Cell Suspensions.

    Zaitseva, O O; PoleZhaeva, T V; Khudvakov, A N; Solomina, O N; Golovchenko, V V

      BACKGROUND: Due to their valuable medicinal properties and high physiological activity, plant polysaccharides are currently being extensively studied. The present study aims to investigate rauwolfian (pectin for Rauvolfia serpentina) supplementation on human leukocytes cryopreservation. We determined the сharacteristics of leukocytes undergoing freezing with pectin at different temperatures. Donor leukocytes were frozen under the protection of comprehensive cryoprotectant solution and stored in electrical freezers (-20C, -40C, -80C). A regular decrease of all values starting from a higher temperature (-20С) through to the lower temperature (-80С) was identified. The study showed that pectin rauwolfian stimulated both the oxygen-independent and the oxygen-dependent killer response. We also found that the oxygen-dependent neutrophil killer effect was reduced as the storage temperature was lowered. It was determined that the LPO levels in the cells with added pectin-containing solutions remained the same before freezing, while their antioxidant activity positively increased, which is beneficial for neutrophils for their further freezing to -20C, -40C and -80C. The results of the study make it possible to assume that rauwolfian, a pectin extracted from Rauvolfia serpentina, has an exocellular protectant effect as part of cryopreservative solution on human white blood cells stored at different low temperatures. The versatility of the substance is probably due to the degree of the macromolecule branching, in particular, the structure of carbohydrate side chains, which contain a large number of hydroxyl groups.

  11. Cytotoxicity of Labruscol, a New Resveratrol Dimer Produced by Grapevine Cell Suspensions, on Human Skin Melanoma Cancer Cell Line HT-144

    Laetitia Nivelle

    2017-11-01

    Full Text Available A new resveratrol dimer (1 called labruscol, has been purified by centrifugal partition chromatography of a crude ethyl acetate stilbene extract obtained from elicited grapevine cell suspensions of Vitis labrusca L. cultured in a 14-liter stirred bioreactor. One dimensional (1D and two dimensional (2D nuclear magnetic resonance (NMR analyses including 1H, 13C, heteronuclear single-quantum correlation (HSQC, heteronuclear multiple bond correlation (HMBC, and correlation spectroscopy (COSY as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS were used to characterize this compound and to unambiguously identify it as a new stilbene dimer, though its relative stereochemistry remained unsolved. Labruscol was recovered as a pure compound (>93% in sufficient amounts (41 mg to allow assessment of its biological activity (cell viability, cell invasion and apoptotic activity on two different cell lines, including one human skin melanoma cancer cell line HT-144 and a healthy human dermal fibroblast (HDF line. This compound induced almost 100% of cell viability inhibition in the cancer line at a dose of 100 μM within 72 h of treatment. However, at all tested concentrations and treatment times, resveratrol displayed an inhibition of the cancer line viability higher than that of labruscol in the presence of fetal bovine serum. Both compounds also showed differential activities on healthy and cancer cell lines. Finally, labruscol at a concentration of 1.2 μM was shown to reduce cell invasion by 40%, although no similar activity was observed with resveratrol. The cytotoxic activity of this newly-identified dimer is discussed.

  12. Response of resting L5178Y-S and L5178Y-R cells with different radiosensitivity to radiation and hydroxyurea

    Afanas'ev, G.G.; Shumel', I.; Valitska, M.; Nekokojchitska, Eh.; Beer, Ya. Z.; Pelevina, I.I.

    1981-01-01

    A study was made of cloning capacity of radiosensitive L5178YS and radioresjstant L5J78YR cells of mouse leukaemia suspension culture after X-irradiation and postradiation treatment with hydroxyurea at 37 deg and 34 deg C. It was shown that both cell lines possess a good cloning capacity at the stationary phase of growth; they are more radiosensitive than cells at the logarithmic phase of growth and cannot repair potentially lethal damages either after decreasing the temperature or the administration of h droxyurea. The postradiation treatment with hydroxyurea enhances the effect of radiation by 2 times in the case of R cells, and by 2 and 1.3 times, depending on the incubation temperature, in the case of S cells

  13. Relations between fluxes and concentrations of Na in cell suspensions of Acer pseudoplatanus

    Pennarun, A.-M.

    1978-01-01

    Taking in account the data provided by preliminary compartmental analysis, the net influxes of 24 Na measured in Acer cells after a short loading period (45 minutes) followed by a short wash (1 minute) represent the influx across the plasmalemma (phi sub(0c)) and, after a long loading period (4 hours) followed by a long wash (2 hours) represent the quasi-steady influx from the external solution to the vacuole (phi sub(0v). At flux equilibrium and when the external Na concentration is high enough, the other unidirectional fluxes - phi sub(c0), phi sub(cv) and phi sub(vc) - can be determined from these measurements. This method was used to study the variation of Na flux in terms of the external concentrations and the resulting internal concentrations. The kinetics obtained confirm the active nature of the efflux phi sub(vc) across the tonoplast according to the conclusions given by the application of the USSING-TEORELL criterion to the results of compartmental analysis. On the contrary, they suggest a passive character for the efflux phi sub(c0) accross the plasmalemma which could be considered as active according to the USSING-TEORELL criterion. The contradiction could be eliminated by taking into consideration the important underestimation of the Na activity coefficient in the cytoplasm, due to the neglecting of water binding [fr

  14. Effects of 60Co γ-rays irradiation on cell growth and alkaloid accumulation of protocorm-like bodies in suspension cultures from Dendrobium huoshanense

    Hong Sali; Jin Qing; Huang Bei; Cai Yongping; Lin Yi

    2009-01-01

    Protocorm-like bodies (PLBs) in suspension cultures from Dendrobium huoshanense were irradiated by 60 Co γ-rays at doses of 5, 10, 20 and 30Gy, and alkaloid accumulation of PLBs was studied. The results showed that 60 Co γ-rays irradiation could improve the alkaloid content of PLBs, and the suitable dose was 10Gy. The fresh weight of 10Gy irradiated PLBs was 26.54g/flask, and the alkaloid content was 0.035% on the 36th day. The medium pH and electric conductivity of 10Gy irradiated PLBs changed slightly during the suspension culture period. The results suggested such cultural environment was suitable for PLBs growth continuely. Results also showed that 60 Co γ-rays irradiation could increase the activities of POD, SOD, CAT, PAL and decrease the activity of PPO, these were responsible for the improvement of cell growth and alkaloid accumulation in PLBs. (authors)

  15. Toxin- and cadmium-induced cell death events in tomato suspension cells resemble features of hypersensitive response

    Iakimova, E.T.; Woltering, E.J.; Yordanova, Z.P.

    2007-01-01

    Elicitors of different origin (fumonisin B1, fungal toxin), camptothecin (alkaloid from Camptotheca acuminata), mastoparan (wasp venom) and the heavy metal (cadmium) were tested for their ability to induce programmed cell death (PCD) in a model system of tomato cell culture, line MsK8. By employing

  16. Biosynthesis of schwertmannite by Acidithiobacillus ferrooxidans cell suspensions under different pH condition

    Liao Yuehua [Department of Environmental Engineering, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095 (China); Zhou Lixiang [Department of Environmental Engineering, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095 (China)], E-mail: lxzhou@njau.edu.cn; Liang Jianru; Xiong Huixin [Department of Environmental Engineering, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095 (China)

    2009-01-01

    Oxidation of FeSO{sub 4} solution with initial pH in the range of 1.40-3.51 by Acidithiobacillus ferrooxidans LX5 cell at 26 deg. C and subsequent precipitation of resulting Fe(III) were investigated in the present study. Results showed that the oxidation rate of Fe(II) was around 1.2-3.9 mmol l{sup -1} h{sup -1}. X-ray diffraction (XRD) indicated that the formed precipitates were composed of natrojarosite with schwertmannite when the initial pH was 3.51, while only schwertmannite was produced when initial pH was in the range of 1.60-3.44 and no precipitate occurred when initial pH {<=} 1.40. Scanning electron microscope (SEM) analyses showed that precipitates formed in solution with initial pH 3.51 were spherical particles of about 0.4 {mu}m in diameter and had a smooth surface, whereas precipitates in solution with initial pH {<=} 3.44 were spherical particles of approximately 1.0 {mu}m in diameter, having specific sea-urchin morphology. Specific surface area of the precipitates varied from 3.42 to 23.45 m{sup 2} g{sup -1}. X-ray fluorescence analyses revealed that schwertmannite formed in solution with initial pH in the range of 2.00-3.44 had similar elemental composition and could be expressed as Fe{sub 8}O{sub 8}(OH){sub 4.42}(SO{sub 4}){sub 1.79,} whereas Fe{sub 8}O{sub 8}(OH){sub 4.36}(SO{sub 4}){sub 1.82} and Fe{sub 8}O{sub 8}(OH){sub 4.29}(SO{sub 4}){sub 1.86} as its chemical formula when the initial pH was 1.80 and 1.60, respectively.

  17. A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

    Deryabin, Dmitry G; Efremova, Ludmila V; Vasilchenko, Alexey S; Saidakova, Evgeniya V; Sizova, Elena A; Troshin, Pavel A; Zhilenkov, Alexander V; Khakina, Ekaterina A; Khakina, Ekaterina E

    2015-08-08

    The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the

  18. Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

    Ying Hou

    Full Text Available Phenylpyruvic acid (PPA is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate reached 29.8 ± 2.1 g·L-1 (99.3% and 75.1 ± 2.5 g·L-1 (93.9% in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

  19. Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

    2016-01-01

    Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L-1 (99.3%) and 75.1 ± 2.5 g·L-1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

  20. Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Liu, Long; Du, Guocheng; Chen, Jian

    2016-01-01

    Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L−1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L−1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L−1 (99.3%) and 75.1 ± 2.5 g·L−1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation. PMID:27851793

  1. Application of Taguchi Design and Response Surface Methodology for Improving Conversion of Isoeugenol into Vanillin by Resting Cells of Psychrobacter sp. CSW4

    Ashengroph, Morahem; Nahvi, Iraj; Amini, Jahanshir

    2013-01-01

    For all industrial processes, modelling, optimisation and control are the keys to enhance productivity and ensure product quality. In the current study, the optimization of process parameters for improving the conversion of isoeugenol to vanillin by Psychrobacter sp. CSW4 was investigated by means of Taguchi approach and Box-Behnken statistical design under resting cell conditions. Taguchi design was employed for screening the significant variables in the bioconversion medium. Sequentially, B...

  2. Leydig Cell Tumor Associated with Testicular Adrenal Rest Tumors in a Patient with Congenital Adrenal Hyperplasia due to 11β-Hydroxylase Deficiency

    Charfi, Nadia; Kamoun, Mahdi; Feki Mnif, Mouna; Mseddi, Neila; Mnif, Fatma; Kallel, Nozha; Ben Naceur, Basma; Rekik, Nabila; Fourati, Hela; Daoud, Emna; Mnif, Zainab; Hadj Sliman, Mourad; Sellami-Boudawara, Tahia; Abid, Mohamed

    2012-01-01

    Congenital adrenal hyperplasia (CAH) describes a group of inherited autosomal recessive disorders characterized by enzyme defects in the steroidogenic pathways that lead to the biosynthesis of cortisol, aldosterone, and androgens. Chronic excessive adrenocorticotropic hormone (ACTH) stimulation may result in hyperplasia of ACTH-sensitive tissues in adrenal glands and other sites such as the testes, causing testicular masses known as testicular adrenal rest tumors (TARTs). Leydig cell tumors (...

  3. Effects of rest time after Li plating on safety behavior—ARC tests with commercial high-energy 18650 Li-ion cells

    Waldmann, Thomas; Wohlfahrt-Mehrens, Margret

    2017-01-01

    During charging at low temperatures, metallic Lithium can be deposited on the surface of graphite anodes of Li-ion cells. This Li plating does not only lead to fast capacity fade, it can also impair the safety behavior. The present study observes the effect of rest periods between Li plating and subsequent accelerated rate calorimetry (ARC) tests. As an example, commercial 3.25 Ah 18650-type cells with graphite anodes and NCA cathodes are cycled at 0 °C to provoke Li plating. It is found that the rest period at 25 °C between Li plating and the ARC tests has a significant influence on the onset temperature of exothermic reactions (T SH ), the onset temperature of thermal runaway (T TR ), the maximum temperature, the self-heating rate, and on damage patterns of 18650 cells. The results are discussed in terms of chemical intercalation of Li plating into adjacent graphite particles during the rest period. The exponential increase of capacity recovery and T SH as a function of time suggests a reaction of 1st order for the relaxation process.

  4. Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

    2014-08-01

    Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae

    Koay Suan See

    2011-06-01

    Full Text Available Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of variousailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production.The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time oncell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of differentconcentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50mg/L had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45g/L sucrose without MeJA showed the highest pigment content (0.69±0.22Cv/g-FCM. The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40Cv/g-FCM. This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures. Rev. Biol. Trop. 59 (2: 597-606. Epub 2011 June 01.elastoma malabathricum pertenece a la familia de las melastomáceas, es una planta medicinal importante ampliamente distribuida desde Madagascar hasta Australia, que se utiliza en remedios tradicionales para el tratamiento de diversas dolencias. Además de sus propiedades medicinales, se ha identificado como una fuente potencial de producción de antocianinas. En esta

  6. Chemical compatibility and properties of suspension plasma-sprayed SrTiO3-based anodes for intermediate-temperature solid oxide fuel cells

    Zhang, Shan-Lin; Li, Cheng-Xin; Li, Chang-Jiu

    2014-10-01

    La-doped strontium titanate (LST) is a promising, redox-stable perovskite material for direct hydrocarbon oxidation anodes in intermediate-temperature solid oxide fuel cells (IT-SOFCs). In this study, nano-sized LST and Sm-doped ceria (SDC) powders are produced by the sol-gel and glycine-nitrate processes, respectively. The chemical compatibility between LST and electrolyte materials is studied. A LST-SDC composite anode is prepared by suspension plasma spraying (SPS). The effects of annealing conditions on the phase structure, microstructure, and chemical stability of the LST-SDC composite anode are investigated. The results indicate that the suspension plasma-sprayed LST-SDC anode has the same phase structure as the original powders. LST exhibits a good chemical compatibility with SDC and Mg/Sr-doped lanthanum gallate (LSGM). The anode has a porosity of ∼40% with a finely porous structure that provides high gas permeability and a long three-phase boundary for the anode reaction. Single cells assembled with the LST-SDC anode, La0.8Sr0.2Ga0.8Mg0.2O3 electrolyte, and La0.8Sr0.2CoO3-SDC cathode show a good performance at 650-800 °C. The annealing reduces the impedances due to the enhancement in the bonding between the particles in the anode and interface of anode and LSGM electrolyte, thus improving the output performance of the cell.

  7. EDITORIAL: Colloidal suspensions Colloidal suspensions

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

    2011-05-01

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

  8. Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

    Bortesi, Luisa; Rademacher, Thomas; Schiermeyer, Andreas; Schuster, Flora; Pezzotti, Mario; Schillberg, Stefan

    2012-07-11

    Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5'-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.

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

    L. Koohi,

    2014-07-01

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

  10. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models

    Anders, Catherine B.; Chess, Jordan J.; Wingett, Denise G.; Punnoose, Alex

    2015-11-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  11. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models.

    Anders, Catherine B; Chess, Jordan J; Wingett, Denise G; Punnoose, Alex

    2015-12-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  12. Ultrastructural changes in the glial cells at neuromuscular synapses of Locusta migratoria occurring after nerve stimulation and subsequent rest: a morphometric analysis.

    Reinecke, M

    1979-10-01

    The glial processes ensheathing the motor nerve terminals on the retractor unguis muscle of Locusta migratoria are described. Ultrastructural changes observed after electrical nerve stimulation (20 Hz, 7 min) without or with subsequent rest (2 min, 1 h) are analysed morphometrically. Immediately after stimulation both the average terminal circumference (+ 23%) and its proportion covered by glial processes (+ 16%) are significantly increased. The mean number of Schwann cell processes per micron of terminal circumference (without stimulation: 0.86 +/- 0.04) is also affected: Immediately after stimulation it is increased by about 15% and after 2 min of rest even by 36%. The periaxonal cleft (without stimulation: 16.5 nm +/- 0.36) becomes wider immediately after stimulation by about 19%, an effect which is almost reversed after 1 h of rest. It is suggested that these changes are a consequence of the enlargement of the nerve terminal's surface upon massive exocytotic activity and that they are possibly mediated by mechanical attachment between glial and terminal plasma membranes.

  13. 21 Days head-down bed rest induces weakening of cell-mediated immunity - Some spaceflight findings confirmed in a ground-based analog.

    Kelsen, Jens; Bartels, Lars Erik; Dige, Anders; Hvas, Christian Lodberg; Frings-Meuthen, Petra; Boehme, Gisela; Thomsen, Marianne Kragh; Fenger-Grøn, Morten; Dahlerup, Jens Frederik

    2012-08-01

    Several studies indicate a weakening of cell-mediated immunity (CMI) and reactivation of latent herpes viruses during spaceflight. We tested the hypothesis that head-down bed rest (HDBR), a ground-based analog of spaceflight, mimics the impact of microgravity on human immunity. Seven healthy young males underwent two periods of 3 weeks HDBR in the test facility of the German Aerospace Center. As a nutritional countermeasure aimed against bone demineralisation, 90 mmol potassium bicarbonate (KHCO(3)) was administered daily in a crossover design. Blood samples were drawn on five occasions. Whole blood was stimulated with antigen i.e. Candida albicans, purified protein derivative (PPD) tuberculin, tetanus toxoid and Cytomegalovirus (CMV) (CMV-QuantiFERON). Flow cytometric analysis included CD4(+)CD25(+)CD127(-)FOXP3(+) regulatory T cells (Tregs), γδ T cells, B cells, NK cells and dendritic cells. In one of the two bed rest periods, we observed a significant decrease in production of interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following phytohemagglutinin (PHA) stimulation, with a rapid normalization being observed after HDBR. The cytokine levels showed a V-shaped pattern that led to a relativeTh2-shift in cytokine balance. Only three individuals responded to the specific T cell antigens without showing signs of an altered response during HDBR, nor did we observe reactivation of CMV or Epstein-Barr virus (EBV). Of unknown significance, dietary supplementation with KHCO(3) counteracted the decrease in IL-2 levels during HDBR, while there was no impact on other immunological parameters. We conclude that discrete alterations in CMI may be induced by HDBR in selected individuals. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque.

    Barboza-Silva, E; Castro, A C D; Marquis, R E

    2005-12-01

    Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.

  15. Establecimiento de un cultivo de celulas en suspensión de Eucalyptus cinérea Establishment of cell suspension culture of Eucalyptus cinerea

    Arias Zabala Mario

    2002-06-01

    Full Text Available Se desarrolla un protocolo para la obtención y establecimiento de suspensiones celulares de E. cinerea. La concentración de las hormonas 2,4 D Y BAP tienen un efecto significativo en la formación de callos friables de E. cinerea, obteniendo una respuesta periódica en la formación de callos friables con respecto a la concentración de las hormonas. Puede obtenerse hasta un 90% de formación de callos friables con varias combinaciones hormonales; primero, con concentraciones alrededor de 3,0 mg/L de 2,4 D Y 1,0 mg/L de BAp, y segundo, alrededor de 6,0 mg/L de 2,4 D Y1,0 mg/L de BAP. A partir de los callos anteriores, se obtienen suspensiones celulares con un activo crecimiento celular, con tiempos de duplicación entre cuatro y siete días, y alcanzando densidades celulares de 15 g células secas/L. Las suspensiones de E. cinerea ofrecen una herramienta importante para la propagación de esta especie vía embriogénesis somática, estudio de biorreactores, producción de metabolitos secundarios y procesos de biotransformación.A protocol is developed for obtaining and establishment of E. cinerea cell suspension. The concentration of hormones 2,4 D and BAP have a significant effect in the formation of friable callus of E. cinerea, obtaining a periodic answer in the formation of friable callus with regard to hormones concentration. It can be obtained until 90% of formation of friable callus in several hormone combinations, first with concentrations around 3,0 mg/L of 2,4 D and 1,0 mg/L of BAP; and second, around 6,0 mgIL of 2,4 D and 1,0 mg/L of BAP. Using the last callus, cell suspensions are obtained with an active cellular growth, with times of duplication between 4 and 7 days and obtaining cell densities of 15 g of dry cells/L. The suspensions of E. cinerea offer an important tool for the propagation of this specie by somatic embryogenesis, bioreactors study, production of secondary metabolites and biotransformation processes.

  16. Particle Suspension Mechanisms - Supplemental Material

    Dillon, M B

    2011-03-03

    This supplemental material provides a brief introduction to particle suspension mechanisms that cause exfoliated skin cells to become and remain airborne. The material presented here provides additional context to the primary manuscript and serves as background for designing possible future studies to assess the impact of skin cells as a source of infectious aerosols. This introduction is not intended to be comprehensive and interested readers are encouraged to consult the references cited.

  17. REST based mobile applications

    Rambow, Mark; Preuss, Thomas; Berdux, Jörg; Conrad, Marc

    2008-02-01

    Simplicity is the major advantage of REST based webservices. Whereas SOAP is widespread in complex, security sensitive business-to-business aplications, REST is widely used for mashups and end-user centric applicatons. In that context we give an overview of REST and compare it to SOAP. Furthermore we apply the GeoDrawing application as an example for REST based mobile applications and emphasize on pros and cons for the use of REST in mobile application scenarios.

  18. Cell division arrest by gamma-irradiation in photoautotrophic suspension culture of Euphorbia characias: maintenance of photosynthetic capacity and overaccumulation of sucrose

    Chagvardieff, P.; Dimon, B.; Carrier, P.; Triantaphylides, C.

    1989-01-01

    Gamma-irradiation (250 Gy) applied to photoautotrophic cell suspensions of Euphorbia characias L. in the exponential growth phase led to the arrest of cell division and to a subsequent overaccumulation of sucrose and dry matter. From the fourth day of culture, the chlorophyll content and gross photosynthesis were not depressed by gamma-treatment nor by sugar accumulation. In both cultures, no difference was observed between oxygen uptake in the light at CO 2 saturating concentration and in the dark, suggesting that no change in energy-dissipative reactions took place after irradiation. A slight increase in oxygen uptake in both light and dark was observed in irradiated cells during the first four days. However, in the absence of limiting factors, the photosynthetic capacities of the dividing and irradiated non-dividing photoautotrophic cells were identical but higher than that of the non-dividing cells in the stationary growth phase. This suggests that gamma-irradiation arrests cell division by a mechanism different to that occurring in stationary-phase cultures. This may be of value in investigating the metabolism of secondary products. (author)

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

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

    2017-12-01

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

  20. Estrogen response of MCF-7 cells grown on diverse substrates and in suspension culture: promotion of morphological heterogeneity, modulation of progestin receptor induction; cell-substrate interactions on collagen gels.

    Pourreau-Schneider, N; Berthois, Y; Mittre, H; Charpin, C; Jacquemier, J; Martin, P M

    1984-12-01

    In this study we observed the incidence of hormone sensitivity in the response of MCF-7 cells to estrogen stimulation when the cells were cultured in different contact environments (hydrophilic plastic, bovine corneal extracellular matrix, type I collagen and in suspension culture). The major purpose was to describe the influence of cell to cell and cell to substrate contacts on the morphological response to estrogen treatment. However, other parameters including growth and induction of progestin receptor were also explored, keeping in mind that the MCF-7 cell line, although representative of normal mammary epithelium in that it contains a similar hormone receptivity, was selected in vitro from a metastatic population in a pleural effusion. Although substrate conditions did not modify growth enhancement by estrogens, progestin receptor levels were significantly higher in three-dimensional spheroid cultures in which cell to cell contacts were optimal due to elimination of basal contact. A careful morphological survey of large surfaces lead to an objective opinion of the overall effect of the hormone treatment on the non-cloned cell line in which a marked heterogeneity in the response of individual cells was observed. In terms of morphofunctional differentiation, the edification of acini with dense microvillus coating was best in suspension culture. When sections were made perpendicular to the plane of cultures on collagen gel rafts two other phenomena were noted: decrease in intercellular junctions, resulting in reduced cell to cell cohesion, and accumulation biodegradation products in the collagen lattice. This suggested a hormone-mediated interaction between the metastatic cells and the fibrillar substrate, collagen I, one of the major constituents of tissue stroma. This estrogen response might be related to the metastatic phenotype and must be distinct from their hormone sensitivity in terms of growth and differentiation since hormone receptivity is generally

  1. Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. {sup 13}C NMR and enzymatic assay; Activite de la glutamine synthetase dans des suspensions cellulaires de solanacees productrices ou non d'alcaloides. RMN du {sup 13}C et dosage enzymatique

    Mesnard, F.; Marty, D.; Monti, J.P. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Biophysique, Groupe de Recherche des Biomolecules: micro-environnement et Metabolisme; Gillet-Manceau, F.; Fliniaux, M.A. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Phytotechnologie

    1999-09-01

    The metabolism of labelled pyruvate followed by {sup 13}C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloidsynthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

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

    Kwiatkowska, Aleksandra; Zebrowski, Jacek; Oklejewicz, Bernadetta; Czarnik, Justyna; Halibart-Puzio, Joanna; Wnuk, Maciej

    2014-01-01

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

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

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

    2014-05-02

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

  4. Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.) 1

    Henstrand, John M.; McCue, Kent F.; Brink, Kent; Handa, Avtar K.; Herrmann, Klaus M.; Conn, Eric E.

    1992-01-01

    Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated. ImagesFigure 1 PMID:16668708

  5. Leydig Cell Tumor Associated with Testicular Adrenal Rest Tumors in a Patient with Congenital Adrenal Hyperplasia due to 11β-Hydroxylase Deficiency

    Nadia Charfi

    2012-01-01

    Full Text Available Congenital adrenal hyperplasia (CAH describes a group of inherited autosomal recessive disorders characterized by enzyme defects in the steroidogenic pathways that lead to the biosynthesis of cortisol, aldosterone, and androgens. Chronic excessive adrenocorticotropic hormone (ACTH stimulation may result in hyperplasia of ACTH-sensitive tissues in adrenal glands and other sites such as the testes, causing testicular masses known as testicular adrenal rest tumors (TARTs. Leydig cell tumors (LCTs are make up a very small number of all testicular tumors and can be difficult to distinguish from TARTs. This distinction is interesting because LCTs and TARTs require different therapeutic approaches. Hereby, we present an unusual case of a 19-year-old patient with CAH due to 11β-hydroxylase deficiency, who presented with TARTs and an epididymal Leydig cell tumor.

  6. Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

    Toussaint, Frédéric; Pierman, Baptiste; Bertin, Aurélie; Lévy, Daniel; Boutry, Marc

    2017-05-04

    Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1  mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  7. Prontonic ceramic membrane fuel cells with layered GdBaCo{sub 2}O{sub 5+x} cathode prepared by gel-casting and suspension spray

    Lin, Bin; Zhang, Shangquan; Zhang, Linchao; Bi, Lei; Ding, Hanping; Liu, Xingqin; Gao, Jianfeng; Meng, Guangyao [Department of Materials Science and Engineering, University of Science and Technology of China (USTC), Hefei 230026 (China)

    2008-03-01

    In order to develop a simple and cost-effective route to fabricate protonic ceramic membrane fuel cells (PCMFCs) with layered GdBaCo{sub 2}O{sub 5+x} (GBCO) cathode, a dense BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (BZCY7) electrolyte was fabricated on a porous anode by gel-casting and suspension spray. The porous NiO-BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (NiO-BZCY7) anode was directly prepared from metal oxide (NiO, BaCO{sub 3}, ZrO{sub 2}, CeO{sub 2} and Y{sub 2}O{sub 3}) by a simple gel-casting process. A suspension of BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} powders synthesized by gel-casting was then employed to deposit BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (BZCY7) thin layer by pressurized spray process on NiO-BZCY7 anode. The bi-layer with 10 {mu}m dense BZCY7 electrolyte was obtained by co-sintering at 1400 C for 5 h. With layered GBCO cathode synthesized by gel-casting on the bi-layer, single cells were assembled and tested with H{sub 2} as fuel and the static air as oxidant. An open-circuit potential of 0.98 V, a maximum power density of 266 mW cm{sup -2}, and a low polarization resistance of the electrodes of 0.16 {omega} cm{sup 2} was achieved at 700 C. (author)

  8. Impact of noncovalent interactions between apple condensed tannins and cell walls on their transfer from fruit to juice: studies in model suspensions and application.

    Le Bourvellec, Carine; Le Quere, Jean-Michel; Renard, Catherine M G C

    2007-09-19

    The adsorption of procyanidins (condensed tannins) on cell-wall material was quantified by bringing into contact solutions of procyanidins and suspensions of cell-wall material. A model was developed on the basis of the Langmuir isotherm formulation and a factorial experimental design. The parameters that influenced the adsorption were the concentration and molecular weight of the procyanidins, the ionic strength of the solution, the temperature, and the apple cell-wall concentration. The model was applied to partitioning of procyanidins from apple between juice and mash. The parameters to be taken into account are the composition of the apples and, specifically, (i) the concentration and molecular weight of the procyanidins, (ii) their acidity and pH as a determinant of the ionic strength, and (iii) their cell-wall content and the temperature at pressing. To estimate the ability of the model to relate procyanidin concentrations in the juice to their concentration in the apple, apples of three varieties of widely different procyanidin compositions were pressed in conditions that prevent oxidation. In these conditions, yields in the juice were >80% for phenolic acids or catechin monomers but <50% for procyanidins, with the lowest rates obtained for the higher polymers in accordance with the model.

  9. Fracture in Kaolinite clay suspensions

    Kosgodagan Acharige, Sebastien; Jerolmack, Douglas J.; Arratia, Paulo E.

    2017-11-01

    Clay minerals are involved in many natural (landslides, river channels) and industrial processes (ceramics, cosmetics, oil recovery). They are plate shaped charged colloids and exhibit different flow properties than simpler colloids when suspended in a liquid such as thixotropy and shear-banding. kaolinite platelets are non-swelling, meaning that the stacks formed by the platelets do not have water layers, and thus the suspension does not have a sol-gel transition. However, it has been shown that kaolinite suspensions possesses a non-zero yield stress even at low concentrations, indicating that the particles arrange themselves in a structure through attractive interactions. Here, we experimentally investigate the sedimentation of kaolinite suspensions in a Hele-Shaw cell. The sedimentation of these dilute suspensions can display solid behavior like fracture, revealed in cross-polarized light, which is linked to the failure of the weakly-bonded structure (typical yield stress 10-2 Pa). By changing the interaction potential of the particles (by sonication or introducing salts), we show through these sedimentation experiments, how the fracture pattern can be avoided. Research was sponsored by the Army Research Laboratory and was accomplished under Grant Number 569074.

  10. The extracellular matrix deposited by asthmatic airway smooth muscle cells in a resting state reflects a healthy matrix

    Harkness, Louise; Ashton, Anthony; Burgess, Janette

    2015-01-01

    Introduction: The remodelled asthmatic airway features an altered extracellular matrix (ECM) & increased vasculature. Previous studies found asthmatic (A) airway smooth muscle cells (ASMCs) to deposit an ECM with enhanced bioactivity. These studies however investigated ECM deposited in the presence

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

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

    2016-12-01

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

  12. Determination of tenogenic differentiation in human mesenchymal stem cells by terahertz waves for measurement of the optical property of cellular suspensions

    Morita, Yasuyuki; Azuchi, Kosuke; Ju, Yang; Suzuki, Satoshi; Xu, Baiyao; Yamamoto, Shuhei

    2014-01-01

    Technology for identifying stem cell-to-tenocyte differentiation that is non-contact and non-destructive in vitro is essential in tissue engineering. It has been found that expression of various RNA and proteins produced by differentiated cells is elevated when human bone marrow mesenchymal stem cells (hBMSCs) differentiate into tenocytes. Also, such biomolecules have absorption bands in the terahertz range. Thus, we attempted to evaluate whether terahertz waves could be used to distinguish hBMSC-to-tenocyte differentiation. Terahertz time-domain spectroscopy (THz-TDS) using femtosecond laser pulses was used for terahertz measurements. HBMSCs differentiated into tenocytes with mechanical stimulation: 10% cyclical uniaxial stretching at 1 Hz for 24 or 48 h. Cellular suspensions before and after differentiation were measured with terahertz waves. Complex refractive index, consisting of a refractive index (real) and an extinction coefficient (imaginary) obtained from the transmitted terahertz signals, was evaluated before and after differentiation at 1.0 THz. As a result, the THz-TDS system enabled discrimination of hBMSC-to-tenocyte differentiation due to the marked contrast in optical parameter before and after differentiation. This is the first report of the potential of a THz-TDS system for the detection of tenogenic differentiation using a non-contact and non-destructive in vitro technique. (paper)

  13. Determination of tenogenic differentiation in human mesenchymal stem cells by terahertz waves for measurement of the optical property of cellular suspensions

    Morita, Yasuyuki; Azuchi, Kosuke; Ju, Yang; Suzuki, Satoshi; Xu, Baiyao; Yamamoto, Shuhei

    2014-06-01

    Technology for identifying stem cell-to-tenocyte differentiation that is non-contact and non-destructive in vitro is essential in tissue engineering. It has been found that expression of various RNA and proteins produced by differentiated cells is elevated when human bone marrow mesenchymal stem cells (hBMSCs) differentiate into tenocytes. Also, such biomolecules have absorption bands in the terahertz range. Thus, we attempted to evaluate whether terahertz waves could be used to distinguish hBMSC-to-tenocyte differentiation. Terahertz time-domain spectroscopy (THz-TDS) using femtosecond laser pulses was used for terahertz measurements. HBMSCs differentiated into tenocytes with mechanical stimulation: 10% cyclical uniaxial stretching at 1 Hz for 24 or 48 h. Cellular suspensions before and after differentiation were measured with terahertz waves. Complex refractive index, consisting of a refractive index (real) and an extinction coefficient (imaginary) obtained from the transmitted terahertz signals, was evaluated before and after differentiation at 1.0 THz. As a result, the THz-TDS system enabled discrimination of hBMSC-to-tenocyte differentiation due to the marked contrast in optical parameter before and after differentiation. This is the first report of the potential of a THz-TDS system for the detection of tenogenic differentiation using a non-contact and non-destructive in vitro technique.

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

    I. A. Siddiqui

    2013-12-01

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

  15. Effect of mercuric chloride on cellular morphology and acid phosphatase of tissue culture cells cultivated in suspension

    Li, M F; Traxler, G S

    1974-01-01

    Cells exposed to HgCl/sub 2/ (0.5 mg/liter) increased dramatically in size and stained poorly with May-Grunwald Giemsa stain and exhibited incompleteness in cell division. When the cell DNA was stained by the Feulgen technique, many multinucleated cells were apparent in the cultures treated with HgCl/sub 2/. Additionally, enlargement and alteration of the nucleoli were evident. Electron-micrographs of the experimental cells revealed that microvilli, ribosomes, mitochondria, and endoplasmic reticula were abundant in the control cells, but in contrast a scarcity of these organelles was observed together with notable cytoplasmic vacuolation in the HgCl/sub 2/-treated cells. In addition the nucleolini of the treated cells were enlarged and had begun to fuse, producing a mulberry appearance. Electronmicroscopic detection of acid phosphatase activity in the cells indicated that the periplasmic enzyme activity was present in control cells, but not in the cells exposed to HgCl/sub 2/. The possible reaction of Hg/sup + +/ with deoxyribonucleic acid and disulfides is discussed with respect to the observed cytopathic effect and impaired enzyme activity. 10 references, 5 figures.

  16. Application of Taguchi Design and Response Surface Methodology for Improving Conversion of Isoeugenol into Vanillin by Resting Cells of Psychrobacter sp. CSW4.

    Ashengroph, Morahem; Nahvi, Iraj; Amini, Jahanshir

    2013-01-01

    For all industrial processes, modelling, optimisation and control are the keys to enhance productivity and ensure product quality. In the current study, the optimization of process parameters for improving the conversion of isoeugenol to vanillin by Psychrobacter sp. CSW4 was investigated by means of Taguchi approach and Box-Behnken statistical design under resting cell conditions. Taguchi design was employed for screening the significant variables in the bioconversion medium. Sequentially, Box-Behnken design experiments under Response Surface Methodology (RSM) was used for further optimization. Four factors (isoeugenol, NaCl, biomass and tween 80 initial concentrations), which have significant effects on vanillin yield, were selected from ten variables by Taguchi experimental design. With the regression coefficient analysis in the Box-Behnken design, a relationship between vanillin production and four significant variables was obtained, and the optimum levels of the four variables were as follows: initial isoeugenol concentration 6.5 g/L, initial tween 80 concentration 0.89 g/L, initial NaCl concentration 113.2 g/L and initial biomass concentration 6.27 g/L. Under these optimized conditions, the maximum predicted concentration of vanillin was 2.25 g/L. These optimized values of the factors were validated in a triplicate shaking flask study and an average of 2.19 g/L for vanillin, which corresponded to a molar yield 36.3%, after a 24 h bioconversion was obtained. The present work is the first one reporting the application of Taguchi design and Response surface methodology for optimizing bioconversion of isoeugenol into vanillin under resting cell conditions.

  17. Suspension trauma; Le traumatisme de suspension

    Trudel, S. [Le Centre de sante et de services sociaux du rocher Perce, Chandler, PQ (Canada)

    2010-07-01

    This presentation discussed the precautions that should be taken to avoid falls from wind turbines or transmission towers. Suspension trauma was explained by a medical doctor in terms of physiology and the body's normal circulation and the elements that disturb normal physiology when in suspension. The trauma occurs following a fall, which carries the risk of 1or more disorders, such as massive hemorrhage, high cardiac pulse, and constriction of blood vessels. Nausea, vertigo, cardiac arrhythmia and sweating occur 15 to 20 minutes following the fall. The presentation emphasized the importance of having qualified personnel at the site and wearing proper harnesses and equipment that supports the neck. figs.

  18. Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

    Beauchamp, Nicholas J.; van Achterberg, Tanja A. E.; Engelse, Marten A.; Pannekoek, Hans; de Vries, Carlie J. M.

    2003-01-01

    Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of

  19. Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase.

    Becerra-Arteaga, Alejandro; Shuler, Michael L

    2007-08-15

    We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins. (c) 2007 Wiley Periodicals, Inc.

  20. Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

    Tobias Manigold

    2010-02-01

    Full Text Available In man, infection with South American Andes virus (ANDV causes hantavirus cardiopulmonary syndrome (HCPS. HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+CD8(+ T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473 years after the acute infection. Remarkably, Gn(465-473-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+CD27(-CD28(-CCR7(-CD127(- effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T

  1. Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

    Ana Clara Mignaqui

    Full Text Available Foot-and-mouth disease (FMD is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV. The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

  2. Recently activated naive CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2-type cytokines.

    Croft, M; Swain, S L

    1995-05-01

    Development of T cells during primary responses was investigated using pigeon cytochrome C-specific naive Th from TCR transgenic mice. Naive CD4 cells did not activate and help resting B cells. This failure was found to be primarily because the resting B cells were incapable of stimulating the naive Th. Provision of a costimulatory signal such as anti-CD28, or addition of APCs that express costimulatory molecules, such as dendritic cells, activated B cells, and B7+ and B7+ICAM(+)-expressing fibroblasts, induced naive Th activation and promoted T cell-dependent help for IgM secretion. T cell activation for as little as 24 h promoted helper activity, and Ig secretion required production of small amounts of IL-4 by the activated naive Th. On initial stimulation, naive Th secrete only IL-2. By mRNA analysis, activated naive Th were also shown to produce IL-4, however induction of IL-4 message only occurred 24 h after initial activation and required additional stimulation with Ag. A single exposure of naive CD4 to Ag/APC followed by 4 to 12 days in culture led to generation of effector Th which secreted IL-2 and some IFN-gamma, and no detectable IL-4 or IL-5, and which could only help B cells to IgM secretion. In contrast, similar cultures that received Ag/APC one or more times during this period generated effector cells capable of secreting easily detectable titers of IL-4 and IL-5, as well as IL-2 and IFN-gamma, and able to now promote IgG1 and IgE responses. Generation of these Th0-like effectors was accompanied by increasing amounts of IL-4 secreted during the culture period after each restimulation, and addition of anti-IL-4 in culture inhibited development of the capacity to produce Th2 cytokines. These studies reinforce the notion that naive CD4 must interact with a costimulatory professional APC, rather than a resting B cell, for initiation of the primary response, but show that such an interaction can result in rapid development of the ability to interact with

  3. Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension.

    Manela, Neta; Oliva, Moran; Ovadia, Rinat; Sikron-Persi, Noga; Ayenew, Biruk; Fait, Aaron; Galili, Gad; Perl, Avichai; Weiss, David; Oren-Shamir, Michal

    2015-01-01

    Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

  4. Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension

    Neta eManela

    2015-07-01

    Full Text Available Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG* of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as

  5. How Streptomyces anulatus Primes Grapevine Defenses to Cope with Gray Mold: A Study of the Early Responses of Cell Suspensions

    Parul Vatsa-Portugal

    2017-06-01

    Full Text Available Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of grapevine and is controlled with an intense application of fungicides. As alternatives to chemicals, beneficial microbes may promote plant health by stimulating the plant’s immune system. An actinomycete, Streptomyces anulatus S37, has been screened from the rhizosphere microbiome of healthy Vitis vinifera on the basis of its ability to promote grapevine growth and to induce resistance against various phytopathogens, including B. cinerea. However, molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain unknown. This study focuses on local defense events induced in grapevine cells during interactions with S. anulatus S37 before and after pathogen challenge. We demonstrated that S. anulatus S37 induced early responses including oxidative burst, extracellular alkalinization, activation of protein kinases, induction of defense gene expression and phytoalexin accumulation, but not the programmed cell death. Interestingly, upon challenge with the B. cinerea, the S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in cell death. In the presence of the EGTA, a calcium channel inhibitor, the induced oxidative burst, and the protein kinase activity were inhibited, but not the extracellular alkalinization, suggesting that Ca2+ may also contribute upstream to the induced defenses. Moreover, desensitization assays using extracellular pH showed that once increased by S. anulatus S37, cells became refractory to further stimulation by B. cinerea, suggesting that grapevine cells perceive distinctly beneficial and pathogenic microbes.

  6. Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids

    Kapur, S K; Wang, X; Shang, H; Yun, S; Li, X; Feng, G; Khurgel, M; Katz, A J

    2012-01-01

    Adipose-derived stromal/stem cells (ASCs) have been gaining recognition as an extremely versatile cell source for tissue engineering. The usefulness of ASCs in biofabrication is further enhanced by our demonstration of the unique properties of these cells when they are cultured as three-dimensional cellular aggregates or spheroids. As described herein, three-dimensional formulations, or self-assembling ASC spheroids develop their own extracellular matrix that serves to increase the robustness of the cells to mechanical stresses. The composition of the extracellular matrix can be altered based on the external environment of the spheroids and these constructs can be grown in a reproducible manner and to a consistent size. The spheroid formulation helps preserve the viability and developmental plasticity of ASCs even under defined, serum-free media conditions. For the first time, we show that multiple generations of adherent ASCs produced from these spheroids retain their ability to differentiate into multiple cell/tissue types. These demonstrated properties support the idea that culture-expanded ASCs are an excellent candidate cellular material for ‘organ printing’—the approach of developing complex tissue structures from a standardized cell ‘ink’ or cell formulation. (paper)

  7. Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells

    Rice Andrew P

    2006-10-01

    Full Text Available Abstract Background The latent reservoir of human immunodeficiency virus type 1 (HIV-1 in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART. Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9 that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. Results Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. Conclusion Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4+ T cells for a precise balance between

  8. Genetic Perturbations Suggest a Role of the Resting Potential in Regulating the Expression of the Ion Channels of the KCNA and HCN families in Octopus Cells of the Ventral Cochlear Nucleus

    Cao, Xiao-Jie; Oertel, Donata

    2017-01-01

    Low-voltage-activated K+ (gKL) and hyperpolarization-activated mixed cation conductances (gh) mediate currents, IKL and Ih, through channels of the Kv1 (KCNA) and HCN families respectively and give auditory neurons the temporal precision required for signaling information about the onset, fine structure, and time of arrival of sounds. Being partially activated at rest, gKL and gh contribute to the resting potential and shape responses to even small subthreshold synaptic currents. Resting gKL and gh also affect the coupling of somatic depolarization with the generation of action potentials. To learn how these important conductances are regulated we have investigated how genetic perturbations affect their expression in octopus cells of the ventral cochlear nucleus (VCN). We report five new findings: First, the magnitude of gh and gKL varied over more than two-fold between wild type strains of mice. Second, average resting potentials are not different in different strains of mice even in the face of large differences in average gKL and gh. Third, IKL has two components, one being α-dendrotoxin (α-DTX)-sensitive and partially inactivating and the other being α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, and non-inactivating. Fourth, the loss of Kv1.1 results in diminution of the α-DTX-sensitive IKL, and compensatory increased expression of an α-DTX-insensitive, tetraethylammonium (TEA)-sensitive IKL. Fifth, Ih and IKL are balanced at the resting potential in all wild type and mutant octopus cells even when resting potentials vary in individual cells over nearly 10 mV, indicating that the resting potential influences the expression of gh and gKL. The independence of resting potentials on gKL and gh shows that gKL and gh do not, over days or weeks, determine the resting potential but rather that the resting potential plays a role in regulating the magnitude of either or both gKL and gh. PMID:28065805

  9. Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

    Jow, Flora; Numann, Randy

    1998-01-01

    Cultured human capillary endothelial cells (HCEC) contain a large inward rectifier current, IK(IR), that can be abolished by removing external K+ or by adding 50 μm Ba2+.We show that IK(IR) is responsible for maintaining the hyperpolarized potential (−60.6 ± 0.5 mV, n = 83) of HCEC. Blocking IK(IR) with 50 μm Ba2+ shifts the zero current level and depolarizes HCEC by 36.5 ± 1.3 mV (n = 4).Increasing external Ca2+ concentration ([Ca2+]o) from 0.5 to 7 mm reduces the magnitude of IK(IR) by 36.5 ± 2.3 % (n = 5) and depolarizes the cells by 10.33 ± 2.4 mV (n = 3), whereas decreasing [Ca2+]o from 1.8 to 0.5 mm increases the amplitude of IK(IR) by 6.9 ± 1.9 % (n = 4). The relationship between [Ca2+]o and the percentage block of IK(IR) gives a Kd value of 5.4 ± 0.6 mm at −120 mV.IK(IR) is also blocked by other divalent ions, with Ba2+ >> Sr2+ > Mg2+ > Mn2+= Ca2+, and the block of peak current at −120 mV being 85.3 ± 3.2 % (n = 5) for 50 μm Ba2+, 62.9 ± 2.2 % (n = 5) for 5 mm Sr2+, 40.7 ± 2.5 % (n = 9) for 5 mm Mg2+, 33.4 ± 2.1 % (n = 5) for 5 mm Mn2+ and 32.9 ± 2.1 % (n = 5) for 5 mm Ca2+.The voltage dependence of Sr2+ block of peak IK(IR) occurred with a Kd value of 1.0 ± 0.09 mm for −140 mV, 1.9 ± 0.16 mm for −130 mV, 3.1 ± 0.28 mm for −120 mV, 4.6 ± 0.34 mm for −110 mV and 6.4 ± 0.5 mm for −100 mV (n = 5), with a calculated electrical distance (δ) of 0.44 from the outside. PMID:9729622

  10. Nano-hydroxyapatite colloid suspension coated on chemically modified porous silicon by cathodic bias: a suitable surface for cell culture

    Sanchez, Alejandra [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Electroquimica y Energia Quimica de la Universidad de Costa Rica (CELEQ), Universidad de Costa Rica, 2060 (Costa Rica); Gonzalez, Jerson [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Garcia-Pineres, Alfonso [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Investigacion en Biologia Celular y Molecular (CIBCM), Universidad de Costa Rica, 2060 (Costa Rica); Montero, Mavis L. [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Electroquimica y Energia Quimica de la Universidad de Costa Rica (CELEQ), Universidad de Costa Rica, 2060 (Costa Rica); Centro de Ciencia e Ingenieria en Materiales (CICIMA), Universidad de Costa Rica, 2060 (Costa Rica)

    2011-06-15

    The properties of porous silicon make it an interesting material for biological applications. However, porous silicon is not an appropriate surface for cell growth. Surface modification is an alternative that could afford a bioactive material. In this work, we report a method to yield materials by modification of the porous silicon surface with hydroxyapatite of nanometric dimensions, produced using an electrochemical process and coated on macroporous silicon substrates by cathodic bias. The chemical nature of the calcium phosphate deposited on the substrates after the experimental process and the amount of cell growth on these surfaces were characterized. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  11. Dissection of cis-regulatory element architecture of the rice oleosin gene promoters to assess abscisic acid responsiveness in suspension-cultured rice cells.

    Kim, Sol; Lee, Soo-Bin; Han, Chae-Seong; Lim, Mi-Na; Lee, Sung-Eun; Yoon, In Sun; Hwang, Yong-Sic

    2017-08-01

    Oleosins are the most abundant proteins in the monolipid layer surrounding neutral storage lipids that form oil bodies in plants. Several lines of evidence indicate that they are physiologically important for the maintenance of oil body structure and for mobilization of the lipids stored inside. Rice has six oleosin genes in its genome, the expression of all of which was found to be responsive to abscisic acid (ABA) in our examination of mature embryo and aleurone tissues. The 5'-flanking region of OsOle5 was initially characterized for its responsiveness to ABA through a transient expression assay system using the protoplasts from suspension-cultured rice cells. A series of successive deletions and site-directed mutations identified five regions critical for the hormonal induction of its promoter activity. A search for cis-acting elements in these regions deposited in a public database revealed that they contain various promoter elements previously reported to be involved in the ABA response of various genes. A gain-of-function experiment indicated that multiple copies of all five regions were sufficient to provide the minimal promoter with a distinct ABA responsiveness. Comparative sequence analysis of the short, but still ABA-responsive, promoters of OsOle genes revealed no common modular architecture shared by them, indicating that various distinct promoter elements and independent trans-acting factors are involved in the ABA responsiveness of rice oleosin multigenes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Photolabeling of tonoplast from sugar beet cell suspensions by [3H]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar Na+/H+ antiport

    Barkla, B.J.; Charuk, J.H.M.; Blumwald, E.; Cragoe, E.J. Jr.

    1990-01-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na + /H + antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na + /H + exchange in a competitive manner with a K i of 2.5 and 5.9 micromolar for ΔpH-dependent 22 Na + influx in tonoplast vesicles and Na + -dependent H + efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [ 3 H]MIA to tonoplast membranes revealed a high affinity binding component with a K d of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na + /H + antiport. Photolabeling of the tonoplast with [ 3 H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog

  13. Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

    Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

    1990-07-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

  14. Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

    Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

    1990-01-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

  15. Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures.

    Mesnard, F; Azaroual, N; Marty, D; Fliniaux, M A; Robins, R J; Vermeersch, G; Monti, J P

    2000-02-01

    Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.

  16. Is Rest Really Rest? Resting State Functional Connectivity during Rest and Motor Task Paradigms.

    Jurkiewicz, Michael T; Crawley, Adrian P; Mikulis, David J

    2018-04-18

    Numerous studies have identified the default mode network (DMN) within the brain of healthy individuals, which has been attributed to the ongoing mental activity of the brain during the wakeful resting-state. While engaged during specific resting-state fMRI paradigms, it remains unclear as to whether traditional block-design simple movement fMRI experiments significantly influence the default mode network or other areas. Using blood-oxygen level dependent (BOLD) fMRI we characterized the pattern of functional connectivity in healthy subjects during a resting-state paradigm and compared this to the same resting-state analysis performed on motor task data residual time courses after regressing out the task paradigm. Using seed-voxel analysis to define the DMN, the executive control network (ECN), and sensorimotor, auditory and visual networks, the resting-state analysis of the residual time courses demonstrated reduced functional connectivity in the motor network and reduced connectivity between the insula and the ECN compared to the standard resting-state datasets. Overall, performance of simple self-directed motor tasks does little to change the resting-state functional connectivity across the brain, especially in non-motor areas. This would suggest that previously acquired fMRI studies incorporating simple block-design motor tasks could be mined retrospectively for assessment of the resting-state connectivity.

  17. The necrotroph Botrytis cinerea induces a non-host type II resistance mechanism in Pinus pinaster suspension-cultured cells.

    Azevedo, Herlânder; Lino-Neto, Teresa; Tavares, Rui Manuel

    2008-03-01

    Models of non-host resistance have failed to account for the pathogenicity of necrotrophic agents. During the interaction of Pinus pinaster (maritime pine) with the non-host necrotrophic pathogen Botrytis cinerea, the generation and scavenging of reactive oxygen species (ROS) and the induction of the hypersensitive response (HR) were analyzed. Elicitation of maritime pine suspended cells with B. cinerea spores resulted in the biphasic induction of ROS. The phase I oxidative burst was dependent on calcium influx, while the phase II oxidative burst also depended on NADPH oxidase, protein kinase activity, and de novo transcription and protein synthesis. A decline was observed in catalase (CAT) and superoxide dismutase (SOD) activity, together with the down-regulation of Fe-Sod1, chlCu, Zn-Sod1 and csApx1, suggesting a coordinated response towards a decrease in the ROS-scavenging capacity of maritime pine cells during challenge. Following the second oxidative burst, programmed cell death events characteristic of the HR were observed. The results suggest the ROS-mediated and cell-breach-independent activation of Type II non-host resistance during the P. pinaster-B. cinerea interaction.

  18. Determination of specific growth stages of plant cell suspension cultures by monitoring conductivity changes in the medium.

    Hahlbrock, K; Ebel, J; Oaks, A; Auden, J; Liersch, M

    1974-03-01

    Conductivity changes in the medium of cultured soybean (Glycine max L.) cells were shown to be strictly correlated with nitrate uptake and growth of the cultures. A continuous record of the conductivity was used as a simple and reliable method of determining specific growth stages and concomitant peaks in the activities of nitrate reductase and phenylalanine ammonia-lyase.

  19. Rheology of organoclay suspension

    Hato, MJ

    2011-05-01

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

  20. Hydropneumatic suspension systems

    Bauer, Wolfgang

    2011-07-01

    Hydropneumatic suspensions systems combine the excellent properties of gas springs with the favourable damping properties of hydraulic fluids. The advantages of these systems are particularly appropriate for automotive applications, such as passenger cars, trucks and agricultural equipment. In this book, Dr. Bauer provides an extensive overview of hydropneumatic suspension systems. Starting with a comparison of different types of suspension systems, the author subsequently describes the theoretical background associated with spring and damping characteristics of hydropneumatic systems and furthermore explains the design of the most important system components. Additionally he gives an overview of level control systems and various special functions. Finally the technology is illustrated by design examples and the outlook for future hydropneumatic suspensions is discussed. (orig.)

  1. Suspension Trauma / Orthostatic Intolerance

    ... Suspension Trauma/Orthostatic Intolerance Safety and Health Information Bulletin SHIB 03-24-2004, updated 2011 This Safety ... the harness, the environmental conditions, and the worker's psychological state all may increase the onset and severity ...

  2. The Mystical Suspension

    Héctor Santiesteban Oliva

    2016-11-01

    Full Text Available Mistical suspension, silence, time, absolute, ontology, ineffability, aletheiaIn the mystical ecstasy there is a sensorial and intellectual suspension when contemplating the absolute, the ontological Being. Silence is not only significant: it is revealing. The greatest expression of experience inner silence . The word is insufficient when the ontological reality is revealed. Revelation or truth , the Greek concept of aletheia, takes on greater significance in that transcendental experience. It is also suspended phenomenological time and remains eternity open.

  3. Mesenchymal stromal cell secretomes are modulated by suspension time, delivery vehicle, passage through catheter, and exposure to adjuvants.

    Parsha, Kaushik; Mir, Osman; Satani, Nikunj; Yang, Bing; Guerrero, Waldo; Mei, Zhuyong; Cai, Chunyan; Chen, Peng R; Gee, Adrian; Hanley, Patrick J; Aronowski, Jaroslaw; Savitz, Sean I

    2017-01-01

    Extensive animal data indicate that mesenchymal stromal cells (MSCs) improve outcome in stroke models. Intra-arterial (IA) injection is a promising route of delivery for MSCs. Therapeutic effect of MSCs in stroke is likely based on the broad repertoire of secreted trophic and immunomodulatory cytokines produced by MSCs. We determined the differential effects of exposing MSCs to different types of clinically relevant vehicles, and/or different additives and passage through a catheter relevant to IA injections. MSCs derived from human bone marrow were tested in the following vehicles: 5% albumin (ALB), 6% Hextend (HEX) and 40% dextran (DEX). Each solution was tested (i) alone, (ii) with low-dose heparin, (iii) with 10% Omnipaque, or (iv) a combination of heparin and Omnipaque. Cells in vehicles were collected directly or passed through an IA catheter, and MSC viability and cytokine release profiles were assessed. Cell viability remained above 90% under all tested conditions with albumin being the highest at 97%. Viability was slightly reduced after catheter passage or exposure to heparin or Omnipaque. Catheter passage had little effect on MSC cytokine secretion. ALB led to increased release of angiogenic factors such as vascular endothelial growth factor compared with other vehicles, while HEX and DEX led to suppression of pro-inflammatory cytokines such as interleukin-6. However, when these three vehicles were subjected to catheter passage and/or exposure to additives, the cytokine release profile varied depending on the combination of conditions to which MSCs were exposed. Exposure of MSCs to certain types of vehicles or additives changes the profile of cytokine secretion. The activation phenotype of MSCs may therefore be affected by the vehicles used for these cells or the exposure to the adjuvants used in their administration. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Control of polyclonal immunoglobulin production from human lymphocytes by leukotrienes; leukotriene B4 induces an OKT8(+), radiosensitive suppressor cell from resting, human OKT8(-) T cells

    Atluru, D.; Goodwin, J.S.

    1984-01-01

    We report that leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of arachidonic acid, is a potent suppressor of polyclonal Ig production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood lymphocytes, while LTC4 and LTD4 have little activity in this system. Preincubation of T cells with LTB4 in nanomolar to picomolar concentrations rendered these cells suppressive of Ig production in subsequent PWM-stimulated cultures of fresh, autologous B + T cells. This LTB4-induced suppressor cell was radiosensitive, and its generation could be blocked by cyclohexamide but not by mitomycin C. The LTB4-induced suppressor cell was OKT8(+), while the precursor for the cell could be OKT8(-). The incubation of OKT8(-) T cells with LTB4 for 18 h resulted in the appearance of the OKT8(+) on 10-20% of the cells, and this could be blocked by cyclohexamide but not by mitomycin C. Thus, LTB4 in very low concentrations induces a radiosensitive OKT8(+) suppressor cell from OKT8(-) cells. In this regard, LTB4 is three to six orders of magnitude more potent than any endogenous hormonal inducer of suppressor cells previously described. Glucocorticosteroids, which block suppressor cell induction in many systems, may act by inhibiting endogenous production of LTB4

  5. REST represses a subset of the pancreatic endocrine differentiation program

    Martin, David; Kim, Yung-Hae; Sever, Dror

    2015-01-01

    in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic...... endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3...

  6. Evidence for the involvement of tetrahydrofolate in the demethylation of nicotine by Nicotiana plumbaginifolia cell-suspension cultures.

    Mesnard, François; Roscher, Albrecht; Garlick, Andrew P; Girard, Sandrine; Baguet, Evelyne; Arroo, Randolf R J; Lebreton, Jacques; Robins, Richard J; Ratcliffe, GeorgeR

    2002-04-01

    The conversion of nicotine to nornicotine by Nicotiana plumbaginifolia Viv. cells was investigated by analysing the redistribution of label during feeding experiments with (R,S)-[2H- methyl]nicotine, (R,S)-[13C- methyl]nicotine and (R,S)-[14C- methyl]nicotine, and the results show that the N-methyl group of nicotine can be recycled into primary metabolism. Nuclear magnetic resonance (NMR) analysis of ethanolic extracts of cells grown in the presence of (R,S)-[13C- methyl]nicotine, using 1H-13C correlation spectroscopy (HMQC, HMBC), revealed the presence of [3-13C]serine and [13C- methyl]methionine. Label was also identified in a cysteinyl derivative and in several methoxylated compounds, but no evidence was obtained with either NMR or ion-trap mass spectrometry for the presence of any intermediate between nicotine and nornicotine. However, experiments with (R,S)-[14C- methyl]nicotine indicated that 70-75% of the metabolised label was released as carbon dioxide. These results are consistent with a pathway in which the oxidative hydrolysis of the nicotine methyl produces an unstable intermediate, N'-hydroxymethylnornicotine, that breaks down spontaneously to nornicotine and formaldehyde, with the formaldehyde being metabolised either directly to formate and carbon dioxide, or through the tetrahydrofolate-mediated pathways of one-carbon metabolism. However since the key intermediate, N-hydroxymethylnornicotine, could not be detected, the possibility of a direct methyl group transfer to tetrahydrofolate cannot be excluded.

  7. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients.

    Celsa A Spina

    Full Text Available The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for "anti-latency" therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

  8. An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

    Spina, Celsa A.; Anderson, Jenny; Archin, Nancie M.; Bosque, Alberto; Chan, Jonathan; Famiglietti, Marylinda; Greene, Warner C.; Kashuba, Angela; Lewin, Sharon R.; Margolis, David M.; Mau, Matthew; Ruelas, Debbie; Saleh, Suha; Shirakawa, Kotaro; Siliciano, Robert F.; Singhania, Akul; Soto, Paula C.; Terry, Valeri H.; Verdin, Eric; Woelk, Christopher; Wooden, Stacey; Xing, Sifei; Planelles, Vicente

    2013-01-01

    The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not. PMID:24385908

  9. RESTful Web Services Cookbook

    Allamaraju, Subbu

    2010-01-01

    While the REST design philosophy has captured the imagination of web and enterprise developers alike, using this approach to develop real web services is no picnic. This cookbook includes more than 100 recipes to help you take advantage of REST, HTTP, and the infrastructure of the Web. You'll learn ways to design RESTful web services for client and server applications that meet performance, scalability, reliability, and security goals, no matter what programming language and development framework you use. Each recipe includes one or two problem statements, with easy-to-follow, step-by-step i

  10. D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

    Salonen, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

    2013-04-01

    Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

  11. Resting state electroencephalographic correlates with red cell long-chain fatty acids, memory performance and age in adolescent boys with attention deficit hyperactivity disorder.

    Sumich, Alexander; Matsudaira, Toshiko; Gow, Rachel V; Ibrahimovic, Almira; Ghebremeskel, Kebreab; Crawford, Michael; Taylor, Eric

    2009-12-01

    Abnormal fatty acid status has been implicated in the aetiology of attention deficit hyperactivity disorder (ADHD). Delayed maturation in ADHD may result in raised frontal low frequency (theta) electroencephalographic activity (EEG) and a reduction in posterior high frequency (beta, alpha) activity. The current study used sequential linear regression to investigate the association between age, resting-state EEG and levels of long-chain polyunsaturated omega-3 and omega-6 fatty acids in red blood cells in 46 adolescent boys with ADHD symptoms. Docosahexaenoic acid (DHA) levels were positively associated with fast frequency activity: alpha during eyes-open and beta during eyes-closed conditions. Frontal theta activity during both eyes-open and eyes-closed conditions was inversely associated with age and positively associated with eicosapentaenoic acid (EPA) levels. Alpha activity correlated positively with performance on fluency for categories (semantic memory). Theta activity correlated inversely with performance on delayed (25 min) verbal memory (recall + recognition/2). No associations were observed between long-chain omega-6 and EEG measures. Results support differential associations for DHA and EPA with fast and slow EEG activity respectively. Results support EEG activity as an objective biomarker of neural function associated with long-chain omega-3 fatty acids in ADHD.

  12. Phagocytosis in phosphate chromium (III) suspensions

    Cruz-Arencibia, Jorge; Fano Machín, Yoiz; Cruz-Morales, Ahmed; Tamayo Fuente, Radamés; Morín-Zorrilla, José

    2015-01-01

    Phagocytosis in vivo and in vitro of a suspension of chromic phosphate (III) labeled with 51 Cr and 32 P is studied. The radioactive particles dispersed in a media of 2 % gelatin in acetate buffer pH 4-4.5 have a predominant size of 0.8 μm and 5 μm. According with biodistribution experiments in rats after 30 minutes near the 80 % of radioactivity is registered in the liver, probably associated with phagocytosis of the particles by liver Kupffer cells. Is also showed that the suspension particles are phagocytized in vitro by mouse peritoneal macrophages. This facts indicate that the studied suspension have appropriate characteristics to be used in radiosynoviorthesis according to the principal action mechanism described for this procedure, particles phagocytosis by cells present in the inflamed synovium. (author)

  13. Hybrid superconducting magnetic suspensions

    Tixador, P.; Hiebel, P.; Brunet, Y.; Chaud, X.; Gautier-Picard, P.

    1996-01-01

    Superconductors, especially high T c ones, are the most attractive materials to design stable and fully passive magnetic suspensions which have to control five degrees of freedom. The hybrid superconducting magnetic suspensions present high performances and a simple cooling mode. They consist of a permanent magnet bearing, stabilized by a suitable magnet-superconductor structure. Several designs are given and compared in terms of forces and stiffnesses. The design of the magnet bearing plays an important part. The superconducting magnetic bearing participates less in levitation but must provide a high stabilizing stiffness. This is achieved by the magnet configuration, a good material in term of critical current density and field cooling. A hybrid superconducting suspension for a flywheel is presented. This system consists of a magnet thrust bearing stabilized by superconductors interacting with an alternating polarity magnet structure. First tests and results are reported. Superconducting materials are magnetically melt-textured YBaCuO

  14. Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

    Seif, R; Martin, R G

    1979-12-01

    Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

  15. Magnetic Suspension Technology Workshop

    Keckler, C.R.; Groom, N.J.; Britcher, C.P.

    1993-01-01

    In order to identify the state of magnetic suspension technology in such areas as rotating systems, pointing of experiments or subsystems, payload isolation, and superconducting materials, a workshop on Magnetic Suspension Technology was held at the Langley Research Center in Hampton, Virginia, on 2-4 Feb. 1988. The workshop included five technical sessions in which a total of 24 papers were presented. The technical sessions covered the areas of pointing, isolation, and measurement, rotating systems, modeling and control, and superconductors. A list of attendees is provided. Separate abstracts have been prepared for articles from this report

  16. Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

    Sierralta, W D; Jakob, F; Thole, H; Engel, P; Jungblut, P W

    1992-01-01

    Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.

  17. Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

    Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Anticoli, Simona; Manfredi, Francesco; Ferrantelli, Flavia; d'Ettorre, Gabriella; Schietroma, Ivan; Andreotti, Mauro; Federico, Maurizio

    2017-09-01

    Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4 + T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4 + T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4 + T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4 + T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4 + T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4 + T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

  18. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

    Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

    2008-12-01

    Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

  19. Compressible Fluid Suspension Performance Testing

    Hoogterp, Francis

    2003-01-01

    ... compressible fluid suspension system that was designed and installed on the vehicle by DTI. The purpose of the tests was to evaluate the possible performance benefits of the compressible fluid suspension system...

  20. Multiobjective suspension control problem

    Jager, de A.G.

    1995-01-01

    The paper describes a (controller) design problem in the field of suspension systems for transport vehicles. A ten degrees-of-freedom model for a tractor-semitrailer vehicle is presented, using parameters derived from a real vehicle, which should be used for design and verification purposes. Road

  1. REST based service composition

    Grönvall, Erik; Ingstrup, Mads; Pløger, Morten

    2011-01-01

    This paper presents an ongoing work developing and testing a Service Composition framework based upon the REST architecture named SECREST. A minimalistic approach have been favored instead of a creating a complete infrastructure. One focus has been on the system's interaction model. Indeed, an aim...

  2. EFECTO ANTIALIMENTARIO DE LOS EXTRACTOS DE SUSPENSIONES CELULARES DE Azadirachta indica SOBRE Spodoptera frugiperda J. E. Smith EN CONDICIONES DE LABORATORIO ANTIFEEDANT EFFECT OF CELL SUSPENSION EXTRACTS OF Azadirachta indica ON Spodoptera frugiperda J. E. Smith UNDER LABORATORY CONDITIONS

    Jacqueline Capataz Tafur

    2007-06-01

    Full Text Available Los extractos de neem (Azadirachta indica y sus ingredientes activos como la azadiractina, presentan acción antialimentaria e inhibición del desarrollo de muchos insectos. El objetivo de este estudio fue examinar el efecto antialimentario de los extractos de suspensiones celulares de neem elicitadas a diferentes condiciones de luz y temperatura. Los extractos fueron aplicados en discos de hojas de maíz y sometidas a bioensayos en larvas de segundo instar de Spodoptera frugiperda J. E. Smith (Lepidptera: Noctuidae. Los extractos intracelulares y del medio de cultivo de las suspensiones celulares de A. indica mostraron efecto biológico sobre larvas de S. frugiperda L2, con valores del 100 % de índice antialimentario para los extractos intracelulares de suspensiones de A. indica elicitadas a 15 °C y oscuridad, y del 39,3% para extractos extracelulares (medio de cultivo de suspensiones elicitadas a 35 °C y oscuridad.Extracts of neem (Azadirachta indica and their active ingredients as azadirachtin, present an action and inhibition of the development of many insects. In this study, the antifeedant effect of cell suspension extracts elicited to different conditions of light and temperature was examined. The extracts were applied in corn leaves disks and submitted to bioassays in larvae of second instar of Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae. The intracellular extracts and the culture media (supernatant of the cell suspensions of A. indica showed a biological effect on larvae of S. frugiperda L2, with values of the 100 % of antifeeding index for the intracellular extracts of cells elicited to 15 °C and darkness, and 39,3% of antifeeding index for extracellular extracts (medium of cultivation of elicited suspensions to 35°C , and darkness.

  3. On the concept of resting potential--pumping ratio of the Na⁺/K⁺ pump and concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell.

    Xu, Ning

    2013-01-01

    In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is -30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.

  4. Particle interactions in concentrated suspensions

    Mondy, L.A.; Graham, A.L.; Abbott, J.R.; Brenner, H.

    1993-01-01

    An overview is presented of research that focuses on slow flows of suspensions in which colloidal and inertial effects are negligibly small. The authors describe nuclear magnetic resonance imaging experiments to quantitatively measure particle migration occurring in concentrated suspensions undergoing a flow with a nonuniform shear rate. These experiments address the issue of how the flow field affects the microstructure of suspensions. In order to understand the local viscosity in a suspension with such a flow-induced, spatially varying concentration, one must know how the viscosity of a homogeneous suspension depends on such variables as solids concentration and particle orientation. The authors suggest the technique of falling ball viscometry, using small balls, as a method to determine the effective viscosity of a suspension without affecting the original microstructure significantly. They also describe data from experiments in which the detailed fluctuations of a falling ball's velocity indicate the noncontinuum nature of the suspension and may lead to more insights into the effects of suspension microstructure on macroscopic properties. Finally, they briefly describe other experiments that can be performed in quiescent suspensions (in contrast to the use of conventional shear rotational viscometers) in order to learn more about boundary effects in concentrated suspensions

  5. Administrative license suspension: Does length of suspension matter?

    Fell, James C; Scherer, Michael

    2017-08-18

    Administrative license revocation (ALR) laws, which provide that the license of a driver with a blood alcohol concentration at or over the illegal limit is subject to an immediate suspension by the state department of motor vehicles, are an example of a traffic law in which the sanction rapidly follows the offense. The power of ALR laws has been attributed to how swiftly the sanction is applied, but does the length of suspension matter? Our objectives were to (a) determine the relationship of the ALR suspension length to the prevalence of drinking drivers relative to sober drivers in fatal crashes and (b) estimate the extent to which the relationship is associated to the general deterrent effect compared to the specific deterrent effect of the law. Data comparing the impact of ALR law implementation and ALR law suspension periods were analyzed using structural equation modeling techniques on the ratio of drinking drivers to nondrinking drivers in fatal crashes from the Fatality Analysis Reporting System (FARS). States with an ALR law with a short suspension period (1-30 days) had a significantly lower drinking driver ratio than states with no ALR law. States with a suspension period of 91-180 days had significantly lower ratios than states with shorter suspension periods, while the three states with suspension lengths of 181 days or longer had significantly lower ratios than states with shorter suspension periods. The implementation of any ALR law was associated with a 13.1% decrease in the drinking/nondrinking driver fatal crash ratio but only a 1.8% decrease in the intoxicated/nonintoxicated fatal crash ratio. The ALR laws and suspension lengths had a significant general deterrent effect, but no specific deterrent effect. States might want to keep (or adopt) ALR laws for their general deterrent effects and pursue alternatives for specific deterrent effects. States with short ALR suspension periods should consider lengthening them to 91 days or longer.

  6. Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

    Ren, Huan [Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008 (China); Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078 (China); Gao, Zhangfeng [Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410008 (China); Wu, Nayiyuan; Zeng, Liu; Tang, Xinyue; Chen, Xiaoping; Liu, Zhaoqian; Zhang, Wei; Wang, Liansheng [Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008 (China); Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078 (China); Li, Zhi, E-mail: lizhi489@163.com [Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008 (China); Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078 (China)

    2015-08-07

    The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which including glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST.

  7. Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

    Ren, Huan; Gao, Zhangfeng; Wu, Nayiyuan; Zeng, Liu; Tang, Xinyue; Chen, Xiaoping; Liu, Zhaoqian; Zhang, Wei; Wang, Liansheng; Li, Zhi

    2015-01-01

    The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which including glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST

  8. Hydrodynamic dispersion of microswimmers in suspension

    Martin, Matthieu; Rafaï, Salima; Peyla, Philippe

    2014-11-01

    In our laboratory, we study hydrodynamics of suspensions of micro-swimmers. These micro-organisms are unicellular algae Chlamydomonas Rheinhardii which are able to swim by using their flagella. The swimming dynamics of these micro-swimmers can be seen as a random walk, in absence of any kind of interaction. In addition, these algae have the property of being phototactic, i.e. they swim towards the light. Combining this property with a hydrodynamic flow, we were able to reversibly separate algae from the rest of the fluid. But for sufficiently high volume fraction, these active particles interact with each other. We are now interested in how the coupling of hydrodynamic interactions between swimmers and phototaxis can modify the swimming dynamics at the scale of the suspension. To this aim, we conduct experiments in microfluidic devices to study the dispersion of the micro-organisms in a the liquid phase as a function of the volume fraction. We show that the dispersion of an assembly of puller type microswimmers is quantitatively affected by hydrodynamics interactions. Phd student.

  9. Dipoles at rest

    Griffiths, D.J.

    1992-01-01

    In a world populated by magnetic monopoles (as well as ordinary electric charges), there are two kinds of electric dipoles: those due to separated electric charges, and those due to current loops of magnetic charge. Similarly, there are two kinds of magnetic dipoles: those due to separated magnetic monopoles, and those due to electric current loops. This paper derives the potentials and fields of each of the four dipole species, and calculates the force, torque, energy, momentum, and angular momentum of each type, when placed (at rest) in a static external field (which may itself be produced by electric charges and currents, magnetic charges and currents, or all of these). Some implications and applications of the various results are discussed

  10. Magnetorheological suspension electromagnetic brake

    Bica, Ioan

    2004-01-01

    The magnetorheological suspension (MRS) brake is of the monoblock type. The main part of the electromagnetic brake is an electromagnet, between whose poles two MRS disks are placed. For distances between disks of 0.65x10 -3 m±10%, revolutions of the electric motor, coupled to the electromagnetic brake, ranging between 200 and 1600 rev/min and braking powers of up to 85 W, there are no differences in revolutions between the disks of the electromagnetic brake. For fixed revolutions of the electric motor, the revolution of the parallel disk can be modified continuously by means of the intensity of the magnetic field. In all cases, the quantity of MRS is of 0.35x10 -3 kg

  11. A simple route to prepare stable hydroxyapatite nanoparticles suspension

    Han Yingchao; Wang Xinyu; Li Shipu

    2009-01-01

    A simple ultrasound assisted precipitation method with addition of glycosaminoglycans (GAGs) is proposed to prepare stable hydroxyapatite (HAP) nanoparticles suspension from the mixture of Ca(H 2 PO 4 ) 2 solution and Ca(OH) 2 solution. The product was characterized by XRD, FT-IR, TEM, HRTEM and particle size, and zeta potential analyzer. TEM observation shows that the suspension is composed of 10-20 nm x 20-50 nm short rod-like and 10-30 nm similar spherical HAP nanoparticles. The number-averaged particle size of stable suspension is about 30 nm between 11.6 and 110.5 nm and the zeta potential is -60.9 mV. The increase of stability of HAP nanoparticles suspension mainly depends on the electrostatic effect and steric effect of GAGs. The HAP nanoparticles can be easily transported into the cancer cells and exhibit good potential as gene or drug carrier system.

  12. Innate B cells: oxymoron or validated concept? [v1; ref status: indexed, http://f1000r.es/T4CAVP

    Carl F Ware

    2012-08-01

    Full Text Available B lymphocytes promote the initial innate interferon response to viral pathogens without the need for antigen receptor activation. B cell dependent IFN production requires the cytokine, lymphotoxin-β. The LTβ pathway is well known to regulate lymphoid organogenesis and homeostasis by differentiating stromal cells and macrophages. However, in response to viral pathogens these same B cell-regulated populations rapidly produce type 1 interferons. Thus, B cells act as innate effector cells via LTβ homeostatic pathways, which serve as innate host barriers to viral pathogens.

  13. DIRAC RESTful API

    Casajus Ramo, A; Graciani Diaz, R; Tsaregorodtsev, A

    2012-01-01

    The DIRAC framework for distributed computing has been designed as a flexible and modular solution that can be adapted to the requirements of any community. Users interact with DIRAC via command line, using the web portal or accessing resources via the DIRAC python API. The current DIRAC API requires users to use a python version valid for DIRAC. Some communities have developed their own software solutions for handling their specific workload, and would like to use DIRAC as their back-end to access distributed computing resources easily. Many of these solutions are not coded in python or depend on a specific python version. To solve this gap DIRAC provides a new language agnostic API that any software solution can use. This new API has been designed following the RESTful principles. Any language with libraries to issue standard HTTP queries may use it. GSI proxies can still be used to authenticate against the API services. However GSI proxies are not a widely adopted standard. The new DIRAC API also allows clients to use OAuth for delegating the user credentials to a third party solution. These delegated credentials allow the third party software to query to DIRAC on behalf of the users. This new API will further expand the possibilities communities have to integrate DIRAC into their distributed computing models.

  14. Controlling active cabin suspensions in commercial vehicles

    Evers, W.J.E.; Besselink, I.J.M.; Teerhuis, A.P.; Knaap, van der A.C.M.; Nijmeijer, H.

    2009-01-01

    The field of automotive suspensions is changing. Semi-active and active suspensions are starting to become viable options for vehicle designers. Suspension design for commercial vehicles is especially interesting given its potential. An active cabin suspension for a heavy-duty truck is considered,

  15. Maraviroc is associated with latent HIV-1 reactivation through NF-κB activation in resting CD4+ T cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy.

    Madrid-Elena, Nadia; García-Bermejo, María Laura; Serrano-Villar, Sergio; Díaz-de Santiago, Alberto; Sastre, Beatriz; Gutiérrez, Carolina; Dronda, Fernando; Coronel Díaz, María; Domínguez, Ester; López-Huertas, María Rosa; Hernández-Novoa, Beatriz; Moreno, Santiago

    2018-02-14

    Maraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency reversing potential of maraviroc and the mechanisms involved, we performed a phase-II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (Eudra CT: 2012-003215-66). All patients completed full maraviroc dosing and follow up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signalling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4 + T-cells, estimated by HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-κB transcription factor was observed in these cells. In contrast, AP-1 and NFAT activity was not detected. To elucidate the mechanism of NF-κB activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, if maraviroc could be acting as a partial CCR5-agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-κB activity and NF-κB target genes expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-κB activation and functionality. Taken together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-κB as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients. IMPORTANCE HIV-1 persistence in a small pool of long-lived latently infected resting CD4 + T-cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency reversing agents to eliminate the reservoirs established in resting CD4 + T-cells. As no drug has been shown to be completely

  16. "Point de suspension"

    2004-01-01

    CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This wondrous show in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local population. There will be three perfo...

  17. "Point de suspension"

    2004-01-01

    http://www.cern.ch/cern50/ CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This wondrous show in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local pop...

  18. "Point de suspension"

    2004-01-01

    CERN - Globe of Science and Innovation 20 and 21 October Acrobatics, mime, a cappella singing, projections of images, a magical setting... a host of different tools of a grandeur matching that of the Universe they relate. A camera makes a massive zoom out to reveal the multiple dimensions of Nature. Freeze the frame: half way between the infinitesimally small and the infinitesimally large, a man suspends his everyday life (hence the title "Point de Suspension", which refers to the three dots at the end of an uncompleted sentence) to take a glimpse of the place he occupies in the great history of the Universe. An unusual perspective on what it means to be a human being... This spectacle in the Globe of Science and Innovation, specially created by the Miméscope* company for the official ceremony marking CERN's fiftieth anniversary, is a gift from the Government of the Republic and Canton of Geneva, which also wishes to share this moment of wonder with the local population. There will be three performances for...

  19. Physiology Of Prolonged Bed Rest

    Greenleaf, John E.

    1991-01-01

    Report describes physiological effects of prolonged bed rest. Rest for periods of 24 hours or longer deconditions body to some extent; healing proceeds simultaneously with deconditioning. Report provides details on shifts in fluid electrolytes and loss of lean body mass, which comprises everything in body besides fat - that is, water, muscle, and bone. Based on published research.

  20. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

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

    Marcia Ometto de Mello

    2001-09-01

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

  2. Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

    Matic, S.; Geisler, D.A.; Møller, I.M.

    2005-01-01

    remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes...... concentrations. Possible uses and limitations of this method for plant cell research are discussed.......The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage...

  3. EMF-REST: Generation of RESTful APIs from Models

    Hamza , Ed-Douibi; Cánovas Izquierdo , Javier Luis; Gómez , Abel; Tisi , Massimo; Cabot , Jordi

    2016-01-01

    In the last years, RESTful Web services have become more and more popular as a lightweight solution to connect remote systems in distributed and Cloud-based architectures. However, being an architectural style rather than a specification or standard, the proper design of RESTful Web services is not trivial since developers have to deal with a plethora of recommendations and best practices. Model-Driven Engineering (MDE) emphasizes the use of models and model transformations to raise the level...

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

    Xinhua Zhou; Rakesh Minocha; Subhash C. Minocha

    1995-01-01

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

  5. Inter-relationships between single carbon units' metabolism and resting energy expenditure in weight-losing patients with small cell lung cancer. Effects of methionine supply and chemotherapy

    Sengeløv, H; Hansen, O P; Simonsen, L

    1994-01-01

    The one-carbon unit metabolism was investigated in 8 weight-losing patients with small cell carcinoma of the lung (SCLC). At diagnosis, 6 of the 8 patients had elevated formiminoglutamic acid (FIGLU) excretion after a histidine load, suggesting a lack of one-carbon units. In accordance...

  6. Inter-relationships between single carbon units' metabolism and resting energy expenditure in weight-losing patients with small cell lung cancer. Effects of methionine supply and chemotherapy

    Sengeløv, H; Hansen, O P; Simonsen, L

    1994-01-01

    The one-carbon unit metabolism was investigated in 8 weight-losing patients with small cell carcinoma of the lung (SCLC). At diagnosis, 6 of the 8 patients had elevated formiminoglutamic acid (FIGLU) excretion after a histidine load, suggesting a lack of one-carbon units. In accordance, a signifi...

  7. Processing of strontium-doped lanthanum manganite suspensions for cathode production of the solid oxide fuel cell; Processamento das suspensoes de manganito de lantanio dopado com estroncio para fabricacao do catodo da celula a combustivel de oxido solido

    Chiba, R.; Vargas, R.A.; Andreoli, M.; Seo, E.S.M. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Ciencia e Tecnologia de Materiais. Lab. de SOFC - Insumos e Componentes

    2008-07-01

    The ceramic material, strontium-doped lanthanum manganite (La{sub 0,85}Sr{sub 0,15}MnO{sub 3} - LSM), has been used as cathode in Solid Oxide Fuel Cells (SOFCs). The cathode attainment as component of the SOFCs has been studied for diverse routes of synthesis and thin films forming in Yttria-stabilized zirconia (ZrO{sub 2}/Y{sub 2}O{sub 3} - YSZ) electrolyte. In this work, the LSM was synthesized by the citrate technique and deposited in YSZ substrate using the forming technique wet powder spraying. Rheological studies of suspensions and chemical, physical and microstructural characterizations of LSM powders were made, aiming at the deposition for thin films formation until 50 mum. The half unit cells LSM/YSZ sintered were characterized by scanning electron microscopy, for verification of porosity and adherence. In this sense, this work is a contribution for production of porous cathode using the forming technique wet powder spraying in the SOFCs. (author)

  8. Low-level HIV-1 replication and the dynamics of the resting CD4+ T cell reservoir for HIV-1 in the setting of HAART

    Wilke Claus O

    2008-01-01

    Full Text Available Abstract Background In the setting of highly active antiretroviral therapy (HAART, plasma levels of human immunodeficiency type-1 (HIV-1 rapidly decay to below the limit of detection of standard clinical assays. However, reactivation of remaining latently infected memory CD4+ T cells is a source of continued virus production, forcing patients to remain on HAART despite clinically undetectable viral loads. Unfortunately, the latent reservoir decays slowly, with a half-life of up to 44 months, making it the major known obstacle to the eradication of HIV-1 infection. However, the mechanism underlying the long half-life of the latent reservoir is unknown. The most likely potential mechanisms are low-level viral replication and the intrinsic stability of latently infected cells. Methods Here we use a mathematical model of T cell dynamics in the setting of HIV-1 infection to probe the decay characteristics of the latent reservoir upon initiation of HAART. We compare the behavior of this model to patient derived data in order to gain insight into the role of low-level viral replication in the setting of HAART. Results By comparing the behavior of our model to patient derived data, we find that the viral dynamics observed in patients on HAART could be consistent with low-level viral replication but that this replication would not significantly affect the decay rate of the latent reservoir. Rather than low-level replication, the intrinsic stability of latently infected cells and the rate at which they are reactivated primarily determine the observed reservoir decay rate according to the predictions of our model. Conclusion The intrinsic stability of the latent reservoir has important implications for efforts to eradicate HIV-1 infection and suggests that intensified HAART would not accelerate the decay of the latent reservoir.

  9. Low-level HIV-1 replication and the dynamics of the resting CD4+ T cell reservoir for HIV-1 in the setting of HAART

    Sedaghat, Ahmad R; Siliciano, Robert F; Wilke, Claus O

    2008-01-01

    Background In the setting of highly active antiretroviral therapy (HAART), plasma levels of human immunodeficiency type-1 (HIV-1) rapidly decay to below the limit of detection of standard clinical assays. However, reactivation of remaining latently infected memory CD4+ T cells is a source of continued virus production, forcing patients to remain on HAART despite clinically undetectable viral loads. Unfortunately, the latent reservoir decays slowly, with a half-life of up to 44 months, making it the major known obstacle to the eradication of HIV-1 infection. However, the mechanism underlying the long half-life of the latent reservoir is unknown. The most likely potential mechanisms are low-level viral replication and the intrinsic stability of latently infected cells. Methods Here we use a mathematical model of T cell dynamics in the setting of HIV-1 infection to probe the decay characteristics of the latent reservoir upon initiation of HAART. We compare the behavior of this model to patient derived data in order to gain insight into the role of low-level viral replication in the setting of HAART. Results By comparing the behavior of our model to patient derived data, we find that the viral dynamics observed in patients on HAART could be consistent with low-level viral replication but that this replication would not significantly affect the decay rate of the latent reservoir. Rather than low-level replication, the intrinsic stability of latently infected cells and the rate at which they are reactivated primarily determine the observed reservoir decay rate according to the predictions of our model. Conclusion The intrinsic stability of the latent reservoir has important implications for efforts to eradicate HIV-1 infection and suggests that intensified HAART would not accelerate the decay of the latent reservoir. PMID:18171475

  10. Clinical physiology of bed rest

    Greenleaf, John E.

    1993-01-01

    Maintenance of optimal health in humans requires the proper balance between exercise, rest, and sleep as well as time in the upright position. About one-third of a lifetime is spent sleeping; and it is no coincidence that sleeping is performed in the horizontal position, the position in which gravitational influence on the body is minimal. Although enforced bed rest is necessary for the treatment of some ailments, in some cases it has probably been used unwisely. In addition to the lower hydrostatic pressure with the normally dependent regions of the cardiovascular system, body fuid compartments during bed rest in the horizontal body position, and virtual elimination of compression on the long bones of the skeletal system during bed rest (hypogravia), there is often reduction in energy metabolism due to the relative confinement (hypodynamia) and alteration of ambulatory circadian variations in metabolism, body temperature, and many hormonal systems. If patients are also moved to unfamiliar surroundings, they probably experience some feelings of anxiety and some sociopsychological problems. Adaptive physiological responses during bed rest are normal for that environment. They are attempts by the body to reduce unnecessary energy expenditure, to optimize its function, and to enhance its survival potential. Many of the deconditioning responses begin within the first day or two of bed rest; these early responses have prompted physicians to insist upon early resumption of the upright posture and ambulation of bedridden patients.

  11. Clustering of resting state networks.

    Megan H Lee

    Full Text Available The goal of the study was to demonstrate a hierarchical structure of resting state activity in the healthy brain using a data-driven clustering algorithm.The fuzzy-c-means clustering algorithm was applied to resting state fMRI data in cortical and subcortical gray matter from two groups acquired separately, one of 17 healthy individuals and the second of 21 healthy individuals. Different numbers of clusters and different starting conditions were used. A cluster dispersion measure determined the optimal numbers of clusters. An inner product metric provided a measure of similarity between different clusters. The two cluster result found the task-negative and task-positive systems. The cluster dispersion measure was minimized with seven and eleven clusters. Each of the clusters in the seven and eleven cluster result was associated with either the task-negative or task-positive system. Applying the algorithm to find seven clusters recovered previously described resting state networks, including the default mode network, frontoparietal control network, ventral and dorsal attention networks, somatomotor, visual, and language networks. The language and ventral attention networks had significant subcortical involvement. This parcellation was consistently found in a large majority of algorithm runs under different conditions and was robust to different methods of initialization.The clustering of resting state activity using different optimal numbers of clusters identified resting state networks comparable to previously obtained results. This work reinforces the observation that resting state networks are hierarchically organized.

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

    GREGORY

    2011-12-16

    Dec 16, 2011 ... Antioxidant activity and phenolic compound was found in Justicia gendarussa via total phenolic content (TPC) and α,α-diphenyl-β-pycrilhydrazil hydrate (DPPH) radical scavenging assays. The assays were applied on aqueous and methanolic extracts of leaves, callus culture and cell suspension culture.

  13. Active Control of Suspension Bridges

    Thoft-Christensen, Palle

    In this paper some recent research on active control of very long suspension bridges, is presented. The presentation is based on research work at Aalborg University, Denmark. The active control system is based on movable flaps attached to the bridge girder. Wind load on bridges with or without...... flaps attached to the girder is briefly presented. A simple active control system is discussed. Results from wind tunnel experiments with a bridge section show that flaps can be used effectively to control bridge girder vibrations. Flutter conditions for suspension bridges with and without flaps...

  14. Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

    Dahlmann, Julia; Awad, George; Dolny, Carsten; Weinert, Sönke; Richter, Karin; Fischer, Klaus-Dieter; Munsch, Thomas; Leßmann, Volkmar; Volleth, Marianne; Zenker, Martin; Chen, Yaoyao; Merkl, Claudia; Schnieke, Angelika; Baraki, Hassina; Kutschka, Ingo; Kensah, George

    2018-01-01

    The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

  15. Modulation of mesenchymal stem cell behavior by nano- and micro-sized β-tricalcium phosphate particles in suspension and composite structures

    Smoak, Mollie; Hogan, Katie; Kriegh, Lisa; Chen, Cong; Terrell, LeKeith B.; Qureshi, Ammar T.; Todd Monroe, W.; Gimble, Jeffrey M.; Hayes, Daniel J.

    2015-04-01

    Interest has grown in the use of microparticles and nanoparticles for modifying the mechanical and biological properties of synthetic bone composite structures. Micro- and nano-sized calcium phosphates are of interest for their osteoinductive behavior. Engineered composites incorporating polymers and ceramics, such as poly-l-lactic acid (PLLA) and beta-tricalcium phosphate (β-TCP), for bone tissue regeneration have been well investigated for their proliferative and osteoinductive abilities. Only limited research has been done to investigate the effects of different sizes of β-TCP particles on human mesenchymal stromal cell behavior. As such, the aim of this study was to investigate the modulations of human adipose-derived stem cell (hASCs) behavior within cell/particle and cell/composite systems as functions of particle size, concentration, and exposure time. The incorporation of nanoscale calcium phosphate resulted in improved mechanical properties and osteogenic behavior within the scaffold compared to the microscale calcium phosphate additives. Particle exposure results indicate that cytotoxicity on hASCs correlates inversely with particle size and increases with the increasing exposure time and particle concentration. Composites with increasing β-TCP content, whether microparticles or nanoparticles, were less toxic than colloidal micro- and nano-sized β-TCP particles directly supplied to hASCs. The difference in viability observed as a result of varying exposure route is likely related to the increased cell-particle interactions in the direct exposure compared to the particles becoming trapped within the scaffold/polymer matrix.

  16. Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

    Dong, C.-Z.; Montillet, J.-L.; Triantaphylides, C.

    1994-01-01

    The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells (Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H + -ATPase activity. Plasma membrane-enriched vesicles were prepared and the H + -ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H + -ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2mM dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H + -ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH. This rapid irreversible inhibition of the plasma membrane H + -ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material. (author)

  17. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  18. Morphology of the Interstitial Tissue of Active and Resting Testis of the Guinea Fowl

    Dharani, Palanisamy; Kumary, S. Usha; Sundaram, Venkatesan; Joseph, Cecilia; Ramesh, Geetha

    2017-01-01

    SUMMARY: The morphology of the interstitial tissue of sexually active and resting testis of the guinea fowl were studied. Six adult health birds of active and resting phases of reproductive cycle were used for this study. The interstitial tissue consisted of loose connective tissue, interstitial cells (Leydig cells), few connective cells, blood vessels and adrenergic nerve fibres in the present study in both active and resting testes. The interstitial tissue was compact in sexually active tes...

  19. Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings

    Leeuwen, van W.; Vermeer, J.E.M.; Gadella, T.W.J.; Munnik, T.

    2007-01-01

    Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the

  20. Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose, pentose- and hexose-phosphates

    Krook, J.; Vreugdenhil, D.; Dijkema, C.; Plas, van der L.H.W.

    2000-01-01

    After a lag phase of 2 days, batch-grown cells of carrot (Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding 13C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO2 production from

  1. Four-Wheel Vehicle Suspension System

    Bickler, Donald B.

    1990-01-01

    Four-wheel suspension system uses simple system of levers with no compliant components to provide three-point suspension of chassis of vehicle while maintaining four-point contact with uneven terrain. Provides stability against tipping of four-point rectangular base, without rocking contact to which rigid four-wheel frame susceptible. Similar to six-wheel suspension system described in "Articulated Suspension Without Springs" (NPO-17354).

  2. Heavy vehicle pitch dynamics and suspension tuning

    Cao, Dongpu; Rakheja, Subhash; Su, Chun-Yi

    2008-01-01

    The influence of suspension tuning of passenger cars on bounce and pitch ride performance has been explored in a number of studies, while only minimal efforts have been made for establishing similar rules for heavy vehicles. This study aims to explore pitch dynamics and suspension tunings of a two-axle heavy vehicle with unconnected suspension, which could also provide valuable information for heavy vehicles with coupled suspensions. Based on a generalised pitch-plane model of a two-axle heav...

  3. Suspension-Firing of Biomass

    Shafique Bashir, Muhammad; Jensen, Peter Arendt; Frandsen, Flemming

    2012-01-01

    This paper is the second of two papers, describing probe measurements of deposit buildup and removal (shedding), conducted in a 350 MWth suspension-fired boiler, firing straw and wood. Investigations of deposit buildup and shedding have been made by use of an advanced online deposit probe and a s...

  4. Suspension-Firing of Biomass

    Bashir, Muhammad Shafique; Jensen, Peter Arendt; Frandsen, Flemming

    2012-01-01

    This paper is Part 1 in a series of two describing probe measurements of deposit build-up and removal (shedding) in a 350 MWth suspension boiler, firing straw and wood. The influence of fuel type (straw share in wood), probe exposure time, probe surface temperature (500, 550, and 600 °C), and flu...

  5. Nonlinear models of suspension bridges

    Malík, Josef

    2006-01-01

    Roč. 321, č. 2 (2006), s. 828-850 ISSN 0022-247X Institutional research plan: CEZ:AV0Z30860518 Keywords : suspension bridges * principle of minimum energy Subject RIV: BA - General Mathematics Impact factor: 0.758, year: 2006

  6. 49 CFR 570.8 - Suspension systems.

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Suspension systems. 570.8 Section 570.8 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY... Pounds or Less § 570.8 Suspension systems. (a) Suspension condition. Ball joint seals shall not be cut or...

  7. 49 CFR 570.61 - Suspension system.

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Suspension system. 570.61 Section 570.61... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VEHICLE IN USE INSPECTION STANDARDS Vehicles With GVWR of More Than 10,000 Pounds § 570.61 Suspension system. (a) Suspension condition. Ball joint seals shall not be cut...

  8. Modulation of mesenchymal stem cell behavior by nano- and micro-sized β-tricalcium phosphate particles in suspension and composite structures

    Smoak, Mollie; Hogan, Katie; Kriegh, Lisa; Chen, Cong, E-mail: cchen19@tigers.lsu.edu; Terrell, LeKeith B.; Qureshi, Ammar T.; Todd Monroe, W. [Louisiana State University and LSU AgCenter, Department of Biological and Agricultural Engineering (United States); Gimble, Jeffrey M., E-mail: Jeffrey.Gimble@pbrc.edu [Tulane University School of Medicine, Center for Stem Cell Research & Regenerative Medicine (United States); Hayes, Daniel J., E-mail: danielhayes@lsu.edu [Louisiana State University and LSU AgCenter, Department of Biological and Agricultural Engineering (United States)

    2015-04-15

    Interest has grown in the use of microparticles and nanoparticles for modifying the mechanical and biological properties of synthetic bone composite structures. Micro- and nano-sized calcium phosphates are of interest for their osteoinductive behavior. Engineered composites incorporating polymers and ceramics, such as poly-l-lactic acid (PLLA) and beta-tricalcium phosphate (β-TCP), for bone tissue regeneration have been well investigated for their proliferative and osteoinductive abilities. Only limited research has been done to investigate the effects of different sizes of β-TCP particles on human mesenchymal stromal cell behavior. As such, the aim of this study was to investigate the modulations of human adipose-derived stem cell (hASCs) behavior within cell/particle and cell/composite systems as functions of particle size, concentration, and exposure time. The incorporation of nanoscale calcium phosphate resulted in improved mechanical properties and osteogenic behavior within the scaffold compared to the microscale calcium phosphate additives. Particle exposure results indicate that cytotoxicity on hASCs correlates inversely with particle size and increases with the increasing exposure time and particle concentration. Composites with increasing β-TCP content, whether microparticles or nanoparticles, were less toxic than colloidal micro- and nano-sized β-TCP particles directly supplied to hASCs. The difference in viability observed as a result of varying exposure route is likely related to the increased cell–particle interactions in the direct exposure compared to the particles becoming trapped within the scaffold/polymer matrix.

  9. The influence of fast neutron irradiation through micropropagation, calli induction and cell aggregate suspension culture of tapak dara cultivate vinca rosea linn

    Wahid, Rosmiarty A.

    2000-01-01

    Study on the influence of fast neutron irradiation toward tissue induction of apical shoot, calli of leaf and corolla as well as development of bud micropropagation using variety of MS and Gamborg (B5) which were supplemented with growth hormone 2,4-D NAA, BAP and kinetin has been carried out. Cell aggregates were obtained from modified liquid media by mixing MS macro element and Gamborg vitamin. Influence of the iow level irradiation (0,5-10 Gy) was investigated for auxiliary bud micropropagation , middle (5 - 20 Gy) for calli induction, while for call aggregates higher doses (until 30 Gy) were used. Optimum growth of bud micropropagation was stimulate at dose range between 0,5-1Gy and grown on MS supplemented whit BAP and NAAN, while for leaf and corolla calli was at 5 Gy, on MS media which was supplemented whit 1 mg/L kinetin, 10mg/L BAP and 0,5 mg/L NAA. However, neutron dose of 10 Gy decreased the induction of leaf and corolla calli. The highest radioresistance was shown by cell aggregates of leaf calli that grew prosperously up to 20Gy. Key words : fast neutron, micropropagation, tissue culture, cell culture, vinca rosea L

  10. RESTful Java web services security

    Enríquez, René

    2014-01-01

    A sequential and easy-to-follow guide which allows you to understand the concepts related to securing web apps/services quickly and efficiently, since each topic is explained and described with the help of an example and in a step-by-step manner, helping you to easily implement the examples in your own projects. This book is intended for web application developers who use RESTful web services to power their websites. Prior knowledge of RESTful is not mandatory, but would be advisable.

  11. Identification of Alternaria alternata Mycotoxins by LC-SPE-NMR and Their Cytotoxic Effects to Soybean (Glycine max Cell Suspension Culture

    Edson Rodrigues-Filho

    2013-02-01

    Full Text Available This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1, alternariol (2, 3'-hydroxyalternariol monomethyl ether (3, and alternariol monomethyl ether (4, were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max cell cultures as a model. EC50 values which range from 0.11 (±0.02 to 4.69 (±0.47 μM showed the high cytotoxicity of these compounds.

  12. Clumping of labeled leukocyte suspension. A simple measure for avoiding it

    Goedemans, W.T.; Hardeman, M.R.; State Univ., Amsterdam

    1988-01-01

    Leukocytes in mixed suspensions can clump together, resulting in cell clusters which are responsible for false positive hot spots in lungs of patients, in the case of abscess localization studies using 111 In labeled leukocytes. Addition of extra ACD (acid-citrate-dextrose) in those labeled leukocyte suspensions prevented cell clumping and avoided occurrence of focal radioactivity accumulation in lungs. The acidification did not interfere in leukocyte migration under agar. (author)

  13. Suspensions with reduced violin string modes

    Lee, B H; Ju, L; Blair, D G

    2006-01-01

    We discuss the possibility of significantly reducing the number and Q-factor of violin string modes in the mirror suspension. Simulations of a bar-flexure suspension and an orthogonal ribbon have shown a reduction in the number of violin string modes when compared to a normal ribbon suspension. By calculating the expected suspension thermal noise, we find that the orthogonal ribbon provides a promising suspension alternative. A lower number of violin modes oscillating in the direction of the laser and a reduction in violin mode peak values of at least 23dB can be achieved with a slight increase in thermal noise above 40Hz

  14. Suspensions with reduced violin string modes

    Lee, B H; Ju, L; Blair, D G [School of Physics, University of Western Australia, Crawley 6009, WA (Australia)

    2006-03-02

    We discuss the possibility of significantly reducing the number and Q-factor of violin string modes in the mirror suspension. Simulations of a bar-flexure suspension and an orthogonal ribbon have shown a reduction in the number of violin string modes when compared to a normal ribbon suspension. By calculating the expected suspension thermal noise, we find that the orthogonal ribbon provides a promising suspension alternative. A lower number of violin modes oscillating in the direction of the laser and a reduction in violin mode peak values of at least 23dB can be achieved with a slight increase in thermal noise above 40Hz.

  15. Study on therapy of 188Re labelled stannic sulfur suspension in nude mice bearing human colon tumor

    Li Huiyuan; Wu Yuanfang; Dong Mo

    2003-01-01

    The effect of therapy, tissue distribution and stability are studied in nude mice bearing human colon tumor after injections of 188 Re labelled stannic sulfur suspension. The tissues are observed with electric microscope. The results show that 188 Re labelled stannic sulfur suspension is stabilized in the tumor and its inhibitive effects on human colon tumor cells are obvious. 188 Re labelled stannic sulfur suspension is a potential radiopharmaceuticals for therapy of human tumor

  16. The NASA Bed Rest Project

    Rhodes, Bradley; Meck, Janice

    2005-01-01

    NASA s National Vision for Space Exploration includes human travel beyond low earth orbit and the ultimate safe return of the crews. Crucial to fulfilling the vision is the successful and timely development of countermeasures for the adverse physiological effects on human systems caused by long term exposure to the microgravity environment. Limited access to in-flight resources for the foreseeable future increases NASA s reliance on ground-based analogs to simulate these effects of microgravity. The primary analog for human based research will be head-down bed rest. By this approach NASA will be able to evaluate countermeasures in large sample sizes, perform preliminary evaluations of proposed in-flight protocols and assess the utility of individual or combined strategies before flight resources are requested. In response to this critical need, NASA has created the Bed Rest Project at the Johnson Space Center. The Project establishes the infrastructure and processes to provide a long term capability for standardized domestic bed rest studies and countermeasure development. The Bed Rest Project design takes a comprehensive, interdisciplinary, integrated approach that reduces the resource overhead of one investigator for one campaign. In addition to integrating studies operationally relevant for exploration, the Project addresses other new Vision objectives, namely: 1) interagency cooperation with the NIH allows for Clinical Research Center (CRC) facility sharing to the benefit of both agencies, 2) collaboration with our International Partners expands countermeasure development opportunities for foreign and domestic investigators as well as promotes consistency in approach and results, 3) to the greatest degree possible, the Project also advances research by clinicians and academia alike to encourage return to earth benefits. This paper will describe the Project s top level goals, organization and relationship to other Exploration Vision Projects, implementation

  17. Source of cytotoxicity in a colloidal silver nanoparticle suspension.

    Hatipoglu, Manolya Kukut; Keleştemur, Seda; Altunbek, Mine; Culha, Mustafa

    2015-05-15

    Silver nanoparticles (AgNPs) are increasingly used in a variety of applications because of their potential antimicrobial activity and their plasmonic and conductivity properties. In this study, we investigated the source of cytotoxicity, genotoxicity, and reactive oxygen species (ROS) production on human dermal fibroblast and human lung cancer (A549) cell lines upon exposure to AgNP colloidal suspensions prepared with the simplest and most commonly used Lee–Meisel method with a variety of reaction times and the concentrations of the reducing agent. The AgNPs synthesized with shorter reaction times were more cytotoxic and genotoxic due to the presence of a few nanometer-sized AgNP seeds. The suspensions prepared with an increased citrate concentration were not cytotoxic, but they induced more ROS generation on A549 cells due to the high citrate concentration. The genotoxicity of the suspension decreased significantly at the higher citrate concentrations. The analysis of both transmission electron microscopy images from the dried droplet areas of the colloidal suspensions and toxicity data indicated that the AgNP seeds were the major source of toxicity. The completion of the nucleation step and the formation of larger AgNPs effectively decreased the toxicity.

  18. Interaction and rheology of vesicle suspensions in confined shear flow

    Shen, Zaiyi; Farutin, Alexander; Thiébaud, Marine; Misbah, Chaouqi

    2017-10-01

    Dynamics and rheology of a confined suspension of vesicles (a model for red blood cells) are studied numerically in two dimensions by using an immersed boundary lattice Boltzmann method. We pay particular attention to the link between the spatiotemporal organization and the rheology of the suspension. Besides confinement, we analyze the effect of concentration of the suspension, ϕ (defined as the area fraction occupied by the vesicles in the simulation domain), as well as the viscosity contrast λ (defined as the ratio between the viscosity of the fluid inside the vesicles, ηint, and that of the suspending fluid, ηext). The hydrodynamic interaction between two vesicles is shown to play a key role in determining the spatial organization. For λ =1 , the pair of vesicles settles into an equilibrium state with constant interdistance, which is regulated by the confinement. The equilibrium interdistance increases with the gap between walls, following a linear relationship. However, no stable equilibrium interdistance between two tumbling vesicles is observed for λ =10 . A quite ordered suspension is observed concomitant with the existence of an equilibrium interdistance between a vesicle pair. However, a disordered suspension prevails when no pair equilibrium interdistance exists, as occurs for tumbling vesicles. We then analyze the rheology, focusing on the effective viscosity, denoted as η , as well as on normalized viscosity, defined as [η ] =(η -ηext) /(ηextϕ ) . Ordering of the suspension is accompanied by a nonmonotonic behavior of [η ] with ϕ , while η exhibits plateaus. The nonmonotonic behavior of [η ] is suppressed when a disordered pattern prevails.

  19. Chain Dynamics in Magnetorheological Suspensions

    Gast, A. P.; Furst, E. M.

    1999-01-01

    Magnetorheological (MR) suspensions are composed of colloidal particles which acquire dipole moments when subjected to an external magnetic field. At sufficient field strengths and concentrations, the dipolar particles rapidly aggregate to form long chains. Subsequent lateral cross-linking of the dipolar chains is responsible for a rapid liquid-to-solid-like rheological transition. The unique, magnetically-activated rheological properties of MR suspensions make them ideal for interfacing mechanical systems to electronic controls. Additionally, the ability to experimentally probe colloidal suspensions interacting through tunable anisotropic potentials is of fundamental interest. Our current experimental work has focused on understanding the fluctuations of dipolar chains. It has been proposed by Halsey and Toor (HT) that the strong Landau-Peierls thermal fluctuations of dipolar chains could be responsible for long-range attractions between chains. Such interactions will govern the long-time relaxation of MR suspensions. We have synthesized monodisperse neutrally buoyant MR suspensions by density matching stabilized ferrofluid emulsion droplets with D2O. This allows us to probe the dynamics of the dipolar chains using light scattering without gravitational, interfacial, and polydispersity effects to resolve the short-wavelength dynamics of the dipolar chains. We used diffusing wave spectroscopy to measure these dynamics. The particle displacements at short times that show an independence to the field strength, but at long times exhibit a constrained, sub-diffusive motion that slows as the dipole strength is increased. The experiments are in good qualitative agreement with Brownian dynamics simulations of dipolar chains. Although there have been several important and detailed studies of the structure and interactions in MR suspensions, there has not been conclusive evidence that supports or contradicts the HT model prediction that long-range interactions exist between

  20. Robust Tensioned Kevlar Suspension Design

    Young, Joseph B.; Naylor, Bret J.; Holmes, Warren A.

    2012-01-01

    One common but challenging problem in cryogenic engineering is to produce a mount that has excellent thermal isolation but is also rigid. Such mounts can be achieved by suspending the load from a network of fibers or strings held in tension. Kevlar fibers are often used for this purpose owing to their high strength and low thermal conductivity. A suite of compact design elements has been developed to improve the reliability of suspension systems made of Kevlar.

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

    Tiago Santana Balbuena

    2009-03-01

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

  2. Polymorphism in Bacterial Flagella Suspensions

    Schwenger, Walter J.

    Bacterial flagella are a type of biological polymer studied for its role in bacterial motility and the polymorphic transitions undertaken to facilitate the run and tumble behavior. The naturally rigid, helical shape of flagella gives rise to novel colloidal dynamics and material properties. This thesis studies methods in which the shape of bacterial flagella can be controlled using in vitro methods and the changes the shape of the flagella have on both single particle dynamics and bulk material properties. We observe individual flagellum in both the dilute and semidilute regimes to observe the effects of solvent condition on the shape of the filament as well as the effect the filament morphology has on reptation through a network of flagella. In addition, we present rheological measurements showing how the shape of filaments effects the bulk material properties of flagellar suspensions. We find that the individual particle dynamics in suspensions of flagella can vary with geometry from needing to reptate linearly via rotation for helical filaments to the prevention of long range diffusion for block copolymer filaments. Similarly, for bulk material properties of flagella suspensions, helical geometries show a dramatic enhancement in elasticity over straight filaments while block copolymers form an elastic gel without the aid of crosslinking agents.

  3. RESTful web services with Dropwizard

    Dallas, Alexandros

    2014-01-01

    A hands-on focused step-by-step tutorial to help you create Web Service applications using Dropwizard. If you are a software engineer or a web developer and want to learn more about building your own Web Service application, then this is the book for you. Basic knowledge of Java and RESTful Web Service concepts is assumed and familiarity with SQL/MySQL and command-line scripting would be helpful.

  4. WEB-SERVICE. RESTFUL ARCHITECTURE

    M. Melnichuk

    2018-04-01

    Full Text Available Network technology for interaction between two applications via the HTTP protocol was considered in article.When client works with REST API - it means it works with "resources", and in SOAP work is performed with operations. To build REST web services, you must follow certain principles: explicit use of HTTP methods, access to resources by URI, stateless, HATEAOS, caching, transfer of objects in JSON or XML representation. But sometimes some principles are ignored to ensure a higher speed of work and to reduce development time.The pros and cons of using JSON and XML representations were considered, and it can be said that using the JSON format reduces the amount of data transfer, and with the use of XML, the readability of data increases.Also, two main ways of data transfer in REST web services were considered: converting the file to Base64 and transferring it as an object field or transferring the file using the usual HTTP multipart. The Base64 standard approach gives a higher speed for multiple files in a single request, because only one HTTP connection is created, but these files are stored in RAM during request processing, which increases chance of the application crashing.In the conclusion, the advantages of using web services and their wide use in other architectural approaches were considered, which increases the popularity of web services.

  5. Rest requirements and rest management of personnel in shift work

    Hammell, B.D. [PDG Environmental, Melbourne, FL (United States); Scheuerle, A. [Univ. of Texas, Houston, TX (United States)

    1995-12-31

    A difficulty-weighted shift assignment scheme is proposed for use in prolonged and strenuous field operations such as emergency response, site testing, and short term hazardous waste remediation projects. The purpose of the work rotation plan is to increase productivity, safety, and moral of workers. Job weighting is accomplished by assigning adjustments to the mental and physical intensity of the task, the protective equipment worn, and the climatic conditions. The plan is based on medical studies of sleep deprivation, the effects of rest adjustments, and programs to reduce sleep deprivation and normalize shift schedules.

  6. Adjustment of a rapid method for quantification of Fusarium spp. spore suspensions in plant pathology.

    Caligiore-Gei, Pablo F; Valdez, Jorge G

    2015-01-01

    The use of a Neubauer chamber is a broadly employed method when cell suspensions need to be quantified. However, this technique may take a long time and needs trained personnel. Spectrophotometry has proved to be a rapid, simple and accurate method to estimate the concentration of spore suspensions of isolates of the genus Fusarium. In this work we present a linear formula to relate absorbance measurements at 530nm with the number of microconidia/ml in a suspension. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Electrodialytic Remediation of Different Heavy Metal-Polluted Soils in Suspension

    Ottosen, Lisbeth M.; Jensen, Pernille Erland; Kirkelund, Gunvor Marie

    2013-01-01

    Electrokinetic remediation of heavy metal-polluted soil faces different challenges in relation to implementation. One challenge is to cope with the nonlinear and transient geochemical changes in the soil and another is to increase the remediation rate. Both these challenges are met when treating...... the soil in a suspension in an electrodialytic cell. The soil suspension is stirred and uniform during treatment. Previously, it has been shown that a faster remediation can be obtained when remediating a stirred soil suspension compared to a stationary water saturated soil (all other parameters the same...

  8. Geometrical Aspects During Formation of Compact Aggregates of Red Blood Cells

    Cardoso A.V.

    2002-01-01

    Full Text Available In the past forty years considerable progress has been achieved on the knowledge of human blood as a non-Newtonian shear-thinning suspension, whose initial state, that is at rest (stasis or at very low shear rates, has a gel-like internal structure which is destroyed as shear stress increases. The main goal of this communication is to describe the role of geometrical aspects during RBC (red blood cell aggregate formation, growth and compaction on naturally aggregate (porcine blood and non-aggregate (bovine blood samples. We consider how these aspects coupled with tension equilibrium are decisive to transform red cell linear roleaux to three-dimensional aggregates or clusters. Geometrical aspects are also crucial on the compaction of red blood cell aggregates. These densely packed aggregates could precipitate out of blood- either as dangerous deposits on arterial walls, or as clots which travel in suspension until they block some crucial capillary.

  9. Attractive and repulsive magnetic suspension systems overview

    Cope, David B.; Fontana, Richard R.

    1992-01-01

    Magnetic suspension systems can be used in a wide variety of applications. The decision of whether to use an attractive or repulsive suspension system for a particular application is a fundamental one which must be made during the design process. As an aid to the designer, we compare and contrast attractive and repulsive magnetic suspension systems and indicate whether and under what conditions one or the other system is preferred.

  10. Introducing Dual Suspension System in Road Vehicles

    Imtiaz Hussain; Jawaid Daudpoto; Ali Asghar Memon

    2013-01-01

    The main objective of suspension system is to reduce the motions of the vehicle body with respect to road disturbances. The conventional suspension systems in road vehicles use passive elements such as springs and dampers to suppress the vibrations induced by the irregularities in the road. But these conventional suspension systems can suppress vibrations to a certain limit. This paper presents a novel idea to improve the ride quality of roads vehicles without compromising vehicle?s stability...

  11. Time Varying Behavior of the Loudspeaker Suspension

    Pedersen, Bo Rohde; Agerkvist, Finn T.

    2007-01-01

    The suspension part of the electrodynamic loudspeaker is often modelled as a simple linear spring with viscous damping, however the dynamic behaviour of the suspension is much more complicated than predicted by such a simple model. At higher levels the compliance becomes non-linear and often chan...... changes during excitation at high levels. This paper investigates how the compliance of the suspension depends on the excitation, i.e. level and frequency content. The measurements are compared with other known measurement methods of the suspension....

  12. Introducing Dual Suspension System in Road Vehicles

    Imtiaz Hussain

    2013-04-01

    Full Text Available The main objective of suspension system is to reduce the motions of the vehicle body with respect to road disturbances. The conventional suspension systems in road vehicles use passive elements such as springs and dampers to suppress the vibrations induced by the irregularities in the road. But these conventional suspension systems can suppress vibrations to a certain limit. This paper presents a novel idea to improve the ride quality of roads vehicles without compromising vehicle?s stability. The paper proposes the use of primary and secondary suspension to suppress the vibrations more effectively.

  13. Sustainable Rest Area Design and Operations

    2017-10-01

    One way in which State Departments of Transportation (DOTs) can modernize their rest areas while reducing operations and maintenance costs is by incorporating sustainable practices into rest area design and operations. Sustainability practices that D...

  14. Suspension culture process for H9N2 avian influenza virus (strain Re-2).

    Wang, Honglin; Guo, Suying; Li, Zhenguang; Xu, Xiaoqin; Shao, Zexiang; Song, Guicai

    2017-10-01

    H9N2 avian influenza virus has caused huge economic loss for the Chinese poultry industry since it was first identified. Vaccination is frequently used as a control method for the disease. Meanwhile suspension culture has become an important tool for the development of influenza vaccines. To optimize the suspension culture conditions for the avian influenza H9N2 virus (Re-2 strain) in Madin-Darby Canine Kidney (MDCK) cells, we studied the culture conditions for cell growth and proliferation parameters for H9N2 virus replication. MDCK cells were successfully cultured in suspension, from a small scale to industrial levels of production, with passage time and initial cell density being optimized. The influence of pH on the culture process in the reactor has been discussed and the process parameters for industrial production were explored via amplification of the 650L reactor. Subsequently, we cultivated cells at high cell density and harvested high amounts of virus, reaching 10log2 (1:1024). Furthermore an animal experiment was conducted to detect antibody. Compared to the chicken embryo virus vaccine, virus cultured from MDCK suspension cells can produce a higher amount of antibodies. The suspension culture process is simple and cost efficient, thus providing a solid foundation for the realization of large-scale avian influenza vaccine production.

  15. Callus and cell suspension cultures of carnation

    Engvild, Kjeld Christensen

    1972-01-01

    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations...

  16. Restful API Architecture Based on Laravel Framework

    Chen, Xianjun; Ji, Zhoupeng; Fan, Yu; Zhan, Yongsong

    2017-10-01

    Web service has been an industry standard tech for message communication and integration between heterogeneous systems. RESTFUL API has become mainstream web service development paradigm after SOAP, how to effectively construct RESTFUL API remains a research hotspots. This paper presents a development model of RESTFUL API construction based on PHP language and LARAVEL framework. The key technical problems that need to be solved during the construction of RESTFUL API are discussed, and implementation details based on LARAVEL are given.

  17. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Priscila Cardoso Cristovam

    2008-10-01

    Full Text Available OBJETIVO: Avaliar a importância da presença de células 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo obtido de rimas córneo-esclerais. MÉTODOS: Rimas de diferentes doadores tiveram seus estroma posterior e endotélio removidos (n=6. Cada rima foi dividida em três segmentos iguais, que foram colocados em cultura em três diferentes condições: um segmento foi colocado na placa de cultura com o lado epitelial para cima (Grupo A. Os dois segmentos restantes foram tripsinizados e a suspensão de células obtida foi cultivada com (Grupo B ou sem (Grupo C células 3T3 irradiadas. As células foram mantidas em meio de cultura "supplemental hormonal epithelial médium" (SHEM, a migração epitelial e a formação de clones nos grupos A, B e C foram avaliadas pela microscopia de contraste de fase e por coloração pela rodamina B. Os resultados foram comparados estatisticamente. RESULTADOS: O crescimento de células epiteliais foi observado em 4/6 rimas (Grupo A. Todas as suspensões de células epiteliais que foram cultivadas com células 3T3 (Grupo B formaram clones. Nenhuma adesão ou formação de clones verdadeiros (holo ou meroclones foi observada na cultura de células que foi cultivada sem 3T3 (Grupo C (p=0,009. CONCLUSÕES: Suspensão de células epiteliais límbicas obtidas de rimas córneo-esclerais no modelo utilizado precisa ser cultivada com células 3T3 para formar clones e estabelecer colônias epiteliais com perspectivas para uso terapêutico na reconstrução da superfície ocular.PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR. METHODS: Corneo-scleral rims from different donors (n=6 had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the

  18. Changes of glucose utilization by erythrocytes, lactic acid concentration in the serum and blood cells, and haematocrit value during one hour rest after maximal effort in individuals differing in physical efficiency.

    Tomasik, M

    1982-01-01

    Glucose utilization by the erythrocytes, lactic acid concentration in the blood and erythrocytes, and haematocrit value were determined before exercise and during one hour rest following maximal exercise in 97 individuals of either sex differing in physical efficiency. In the investigations reported by the author individuals with strikingly high physical fitness performed maximal work one-third greater than that performed by individuals with medium fitness. The serum concentration of lactic acid was in all individuals above the resting value still after 60 minutes of rest. On the other hand, this concentration returned to the normal level in the erythrocytes but only in individuals with strikingly high efficiency. Glucose utilization by the erythrocytes during the restitution period was highest immediately after the exercise in all studied individuals and showed a tendency for more rapid return to resting values again in individuals with highest efficiency. The investigation of very efficient individuals repeated twice demonstrated greater utilization of glucose by the erythrocytes at the time of greater maximal exercise. This was associated with greater lactic acid concentration in the serum and erythrocytes throughout the whole one-hour rest period. The observed facts suggest an active participation of erythrocytes in the process of adaptation of the organism to exercise.

  19. Antioxidant isoenzyme responses to nickel-induced stress in tobacco cell suspension culture Resposta de isoenzimas antioxidantes ao estresse induzido por níquel em cultura de células em suspensão de fumo

    Georgia Bertoni Pompeu

    2008-01-01

    Full Text Available Exposure to nickel (Ni at high concentrations can lead to production of reactive oxygen species (ROS resulting in oxidative damage at the cellular level. We investigated the antioxidative responses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2 over a 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase (SOD, catalase (CAT and glutathione reductase (GR. Two main SOD isoenzymes were observed, a Mn-SOD (band I and a Fe-SOD (band II, as well as one CAT isoenzyme and four GR isoenzymes. Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GR isoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentration was used to induce the stress for the same period of treatment. These results illustrate the importance and advantages of determining individual isoenzyme activities.A exposição ao níquel (Ni, em altas concentrações, pode levar à produção de espécies reativas de oxigênio (EAOs, resultando em danos oxidativos em nível celular. Foram investigadas as respostas antioxidativas de células em suspensão do cultivar BY-2 de Nicotiana tabacum submetidas ao estresse por Ni (0.075 e 0.75 mM de NiCl2 por 72 h, com atenção especial às alterações potencias em isoenzimas de superóxido dismutase (SOD, catalase (CAT e glutatione redutase (GR. Duas principais isoenzimas de SOD foram observadas, uma Mn-SOD (banda I e outra Fe-SOD (banda II, bem como uma isoenzima CAT e quatro isoenzimas de GR. As análises revelaram que a atividade de CAT tem papel principal no momento inicial de resposta ao estresse oxidativo induzido por Ni, particularmente, quando sua concentração foi mais baixa, enquanto uma isoenzima específica de GR parece responder a este estresse na concentração mais alta de Ni

  20. Non-linear dielectric spectroscopy of microbiological suspensions

    Treo, Ernesto F; Felice, Carmelo J

    2009-01-01

    Background Non-linear dielectric spectroscopy (NLDS) of microorganism was characterized by the generation of harmonics in the polarization current when a microorganism suspension was exposed to a sinusoidal electric field. The biological nonlinear response initially described was not well verified by other authors and the results were susceptible to ambiguous interpretation. In this paper NLDS was performed to yeast suspension in tripolar and tetrapolar configuration with a recently developed analyzer. Methods Tripolar analysis was carried out by applying sinusoidal voltages up to 1 V at the electrode interface. Tetrapolar analysis was carried on with sinusoidal field strengths from 0.1 V cm-1 to 70 V cm-1. Both analyses were performed within a frequency range from 1 Hz through 100 Hz. The harmonic amplitudes were Fourier-analyzed and expressed in dB. The third harmonic, as reported previously, was investigated. Statistical analysis (ANOVA) was used to test the effect of inhibitor an activator of the plasma membrane enzyme in the measured response. Results No significant non-linearities were observed in tetrapolar analysis, and no observable changes occurred when inhibitor and activator were added to the suspension. Statistical analysis confirmed these results. When a pure sinus voltage was applied to an electrode-yeast suspension interface, variations higher than 25 dB for the 3rd harmonic were observed. Variation higher than 20 dB in the 3rd harmonics has also been found when adding an inhibitor or activator of the membrane-bounded enzymes. These variations did not occur when the suspension was boiled. Discussion The lack of result in tetrapolar cells suggest that there is no, if any, harmonic generation in microbiological bulk suspension. The non-linear response observed was originated in the electrode-electrolyte interface. The frequency and voltage windows observed in previous tetrapolar analysis were repeated in the tripolar measurements, but maximum were not

  1. Non-linear dielectric spectroscopy of microbiological suspensions

    Felice Carmelo J

    2009-09-01

    Full Text Available Abstract Background Non-linear dielectric spectroscopy (NLDS of microorganism was characterized by the generation of harmonics in the polarization current when a microorganism suspension was exposed to a sinusoidal electric field. The biological nonlinear response initially described was not well verified by other authors and the results were susceptible to ambiguous interpretation. In this paper NLDS was performed to yeast suspension in tripolar and tetrapolar configuration with a recently developed analyzer. Methods Tripolar analysis was carried out by applying sinusoidal voltages up to 1 V at the electrode interface. Tetrapolar analysis was carried on with sinusoidal field strengths from 0.1 V cm-1 to 70 V cm-1. Both analyses were performed within a frequency range from 1 Hz through 100 Hz. The harmonic amplitudes were Fourier-analyzed and expressed in dB. The third harmonic, as reported previously, was investigated. Statistical analysis (ANOVA was used to test the effect of inhibitor an activator of the plasma membrane enzyme in the measured response. Results No significant non-linearities were observed in tetrapolar analysis, and no observable changes occurred when inhibitor and activator were added to the suspension. Statistical analysis confirmed these results. When a pure sinus voltage was applied to an electrode-yeast suspension interface, variations higher than 25 dB for the 3rd harmonic were observed. Variation higher than 20 dB in the 3rd harmonics has also been found when adding an inhibitor or activator of the membrane-bounded enzymes. These variations did not occur when the suspension was boiled. Discussion The lack of result in tetrapolar cells suggest that there is no, if any, harmonic generation in microbiological bulk suspension. The non-linear response observed was originated in the electrode-electrolyte interface. The frequency and voltage windows observed in previous tetrapolar analysis were repeated in the tripolar

  2. 36 CFR 296.10 - Suspension and revocation of permits.

    2010-07-01

    ... AGRICULTURE PROTECTION OF ARCHAEOLOGICAL RESOURCES: UNIFORM REGULATIONS § 296.10 Suspension and revocation of... correct the situation which led to suspension of the permit. (b) Suspension or revocation for management...

  3. Design analysis of formula student race car suspension system

    Wirawan, Julian Wisnu; Ubaidillah, Aditra, Rama; Alnursyah, Rafli; Rahman, Rizki Abdul; Cahyono, Sukmaji Indro

    2018-02-01

    Design analysis of suspension especially for racecar suspension is very crucial to achieve maximum performance and handling. Suspension design may vary depending on the road terrain and the vehicle purpose itself, such as high speed or off-road vehicle. This paper focused on the suspension which used for racecar vehicle. The suspension type used was unequal double wishbone. This model is used because of its stability for high-speed usage compared to another kind of suspension. The suspension parameter was calculated to achieve desired performance. The result is the motion ratio of the designed suspension geometry. The obtained value of motion ratio was 1:2 for front suspension and 1:1 for the rear suspension. These calculation result the front suspension is still too soft, which the optimal motion ratio should be kept around 1:1 for better handling. This problem caused by the lack of space for suspension linkage.

  4. 39 CFR 320.9 - Revocation or amendment of suspensions.

    2010-07-01

    ... SUSPENSION OF THE PRIVATE EXPRESS STATUTES § 320.9 Revocation or amendment of suspensions. These suspensions... of operations (in dollar or volume terms, whichever is larger) lower than that antedating the...

  5. Linear viscoelastic properties of aging suspensions

    Purnomo, E.H.; Purnomo, E.H; van den Ende, Henricus T.M.; Mellema, J.; Mugele, Friedrich Gunther

    2006-01-01

    We have examined the linear viscoelastic behavior of poly-N-isopropylacrylamide (PNIPAM) microgel suspensions in order to obtain insight in the aging processes in these densely packed suspensions at various temperatures below the volume transition temperature. The system is found to display a strong

  6. 49 CFR 393.207 - Suspension systems.

    2010-10-01

    ... braking system. The vehicle shall be level (not tilting to the left or right). Air leakage shall not be... 49 Transportation 5 2010-10-01 2010-10-01 false Suspension systems. 393.207 Section 393.207... NECESSARY FOR SAFE OPERATION Frames, Cab and Body Components, Wheels, Steering, and Suspension Systems § 393...

  7. 41 CFR 105-74.670 - Suspension.

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Suspension. 105-74.670 Section 105-74.670 Public Contracts and Property Management Federal Property Management Regulations System...-GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 105-74.670 Suspension...

  8. 36 CFR 25.3 - Supervision; suspensions.

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Supervision; suspensions. 25.3 Section 25.3 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR NATIONAL MILITARY PARKS; LICENSED GUIDE SERVICE REGULATIONS § 25.3 Supervision; suspensions. (a) The guide service will operate under the direction...

  9. Cements and concentrated model suspensions. Flow, jamming and flocculation; Ciments et suspensions concentrees modeles. Ecoulement, encombrement et floculation

    Lootens, D

    2004-10-15

    Here we report about the rheological behavior of very concentrated dispersions -or pastes- of granular or colloidal particles of cement or of monodisperse spherical silica particles. The model system of silica particles is used in order to control the physical chemistry properties of the suspension. Our research project is centered on two points which are focusing on the slurries properties: (i) the first one is the study of the structuring process by the coagulation at rest and its relation with setting. We use special tools (ultrasonic, confocal) to follow and understand the evolution from coagulation to the setting of cement (ii) the second is the study of the dynamic structuring phenomena, under shear, typical of the concentrated suspensions and named jamming. The flow can enter into a shear-thickening and, possibly jamming regime. The onset of this regime is shown to involve giant fluctuations of stress, assigned to the formation and breakup of direct frictional contact chains. The surface state of the particles is a determining factor in this transition. The studies of the normal stress and microscopic observation give good reasons to believe that solid friction is involved in the jamming transition. (author)

  10. Osmotic consolidation of suspensions and gels

    Miller, K.T.; Zukoski, C.F.

    1994-01-01

    An osmotic method for the consolidation of suspensions of ceramic particles is demonstrated. Concentrated solutions of poly(ethylene oxide) are separated from a suspension of ceramic particles by a semipermeable membrane, creating a gradient in solvent chemical potential. Solvent passes from the suspension into the polymer solution, lowering its free energy and consolidating the suspension. Dispersions of stable 8-nm hydrous zirconia particles were consolidated to over 47% by volume. Suspensions of α-alumina in three states of aggregation (dispersed, weakly flocculated, and strongly flocculated) were consolidated to densities greater than or equal to those produced in conventional pressure filtration. Moreover, the as-consolidated alumina bodies were partially drained of fluid during the osmotic consolidation process, producing cohesive partially dried bodies with improved handling characteristics

  11. Stable, easily sintered BaCe0.5Zr0.3Y0.16Zn0.04O3-δ electrolyte-based proton-conducting solid oxide fuel cells by gel-casting and suspension spray

    Lin Bin; Dong Yingchao; Wang Songlin; Fang Daru; Ding Hanping; Zhang Xiaozhen; Liu Xingqin; Meng Guangyao

    2009-01-01

    Protonic ceramic membrane fuel cells (PCMFCs) based on oxide proton conductors exhibit more advantages than traditional solid oxide fuel cells (SOFCs) based on oxygen-ion conducting electrolytes, such as low activation energy and high energy efficiency. In order to develop a simple and cost-effective route to fabricate PCMFCs with SrCo 0.9 Sb 0.1 O 3-δ (SCS) cubic perovskite cathode, a dense BaCe 0.5 Zr 0.3 Y 0.16 Zn 0.04 O 3-δ (BCZYZn) electrolyte was fabricated in situ metal oxide on a porous anode support by gel-casting and suspension spray, which is cost-effective, easy to realize, and suitable for mass-production. The key part of this process is to directly spray well-mixed suspension of BaCO 3 , CeO 2 , ZrO 2 , Y 2 O 3 and ZnO instead of pre-synthesized BCZYZn ceramic powder on the anode substrate. With SCS cubic perovskite cathode synthesized by gel-casting on the bi-layer, single cells were assembled and tested with H 2 as fuel and the static air as oxidant. An open-circuit potential of 0.987 V, a maximum power density of 364 mW cm -2 , and a low polarization resistance of the electrodes of 0.07 Ω cm 2 was achieved at 700 deg. C.

  12. Intrasellar Symptomatic Salivary Gland Rest

    Chih-Hao Chen

    2007-05-01

    Full Text Available Ectopic salivary gland tissue in sellar turcica is frequently observed in microscopic examination at autopsy. This tissue is considered clinically silent. Only 2 symptomatic cases have been previously reported. Here we report a 28-year-old woman presenting with galactorrhea and hyperprolactinemia. Magnetic resonance imaging revealed a 6×5-mm nodule in the posterior aspect of the pituitary gland. This nodule showed isointensity on T1- and T2-weighted images and less enhancement on post-contrast T1-weighted images. Transsphenoidal exploration revealed a cystic lesion within the pituitary gland, which consisted of a grayish gelatinous content. The pathologic examination confirmed the diagnosis of salivary gland rest.

  13. Clogging in constricted suspension flows

    Marin, Alvaro; Lhuissier, Henri; Rossi, Massimiliano; Kähler, Christian J.

    2018-02-01

    The flow of a charged-stabilized suspension through a single constricted channel is studied experimentally by tracking the particles individually. Surprisingly, the behavior is found to be qualitatively similar to that of inertial dry granular systems: For small values of the neck-to-particle size ratio (D /d reported for granular systems and agree for moderate particle volume fraction (ϕ ≈20 % ) with a simple stochastic model for the number of particles at the neck. For larger neck sizes (D /d >3 ), even at the largest ϕ (≈60 %) achievable in the experiments, an uninterrupted particle flow is observed, which resembles that of an hourglass. This particularly small value of D /d (≃3 ) at the transition to a practically uninterrupted flow is attributed to the low effective friction between the particles, achieved by the particle's functionalization and lubrication.

  14. Next Generation Suspension Dynamics Algorithms

    Schunk, Peter Randall [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Higdon, Jonathon [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Chen, Steven [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-12-01

    This research project has the objective to extend the range of application, improve the efficiency and conduct simulations with the Fast Lubrication Dynamics (FLD) algorithm for concentrated particle suspensions in a Newtonian fluid solvent. The research involves a combination of mathematical development, new computational algorithms, and application to processing flows of relevance in materials processing. The mathematical developments clarify the underlying theory, facilitate verification against classic monographs in the field and provide the framework for a novel parallel implementation optimized for an OpenMP shared memory environment. The project considered application to consolidation flows of major interest in high throughput materials processing and identified hitherto unforeseen challenges in the use of FLD in these applications. Extensions to the algorithm have been developed to improve its accuracy in these applications.

  15. Graphite suspension in carbon dioxide

    Roche, R.

    1965-01-01

    Since 1963 the Atomic Division of SNECMA has been conducting, under a contract with the CEA, an experimental work with a two-component fluid comprised of carbon dioxide and small graphite particles. The primary purpose was the determination of basic engineering information pertaining to the stability and the flowability of the suspension. The final form of the experimental loop consists mainly of the following items: a light-phase compressor, a heavy-phase pump, an electrical-resistance type heater section, a cooling heat exchanger, a hairpin loop, a transparent test section and a separator. During the course of the testing, it was observed that the fluid could be circulated quite easily in a broad range of variation of the suspension density and velocity - density from 30 to 170 kg/m 3 and velocity from 2 to 24 m/s. The system could be restarted and circulation maintained without any difficulty, even with the heavy-phase pump alone. The graphite did not have a tendency to pack or agglomerate during operation. No graphite deposition was observed on the wall of the tubing. A long period run (250 hours) has shown the evolution of the particle dimensions. Starting with graphite of surface area around 20 m 2 /g (graphite particles about 1 μ), the powder surface area reaches an asymptotic value of 300 m 2 /g (all the particles less than 0.3 μ). Moisture effect on flow stability, flow distribution between two parallel channels, pressure drop in straight tubes, recompression ratio in diffusers were also investigated. (author) [fr

  16. Suspension chemistry and electrophoretic deposition of zirconia electrolyte on conducting and non-conducting substrates

    Das, Debasish; Basu, Rajendra N.

    2013-01-01

    Graphical abstract: - Highlights: • Stable suspension of yttria stabilized zirconia (YSZ) obtained in isopropanol medium. • Suspension chemistry and process parameters for electrophoretic deposition optimized. • Deposited film quality changed with iodine and water (dispersants) concentration. • Dense YSZ film (∼5 μm) fabricated onto non-conducting porous NiO-YSZ anode substrate. - Abstract: Suspensions of 8 mol% yttria stabilized zirconia (YSZ) particulates in isopropanol medium are prepared using acetylacetone, iodine and water as dispersants. The effect of dispersants concentration on suspension stability, particle size distribution, electrical conductivity and pH of the suspensions are studied in detail to optimize the suspension chemistry. Electrophoretic deposition (EPD) has been conducted to produce thin and dense YSZ electrolyte films. Deposition kinetics have been studied in depth and good quality films on conducting substrate are obtained at an applied voltage of 15 V for 3 min. YSZ films are also fabricated on non-conducting NiO-YSZ anode substrate using a steel plate on the reverse side of the substrate. Upon co-firing at 1400 °C for 6 h a dense YSZ film of thickness ∼5 μm is obtained. Such a half cell (anode + electrolyte) can be used to fabricate a solid oxide fuel cell on applying a suitable cathode layer

  17. Naratriptan hydrochloride in extemporaneosly compounded oral suspensions.

    Zhang, Y P; Trissel, L A; Fox, J L

    2000-01-01

    The purpose of this study was to determine the pharmaceutical acceptability and chemical stability of naratriptan hydrochloride in three extemporaneously compounded suspension formulations. The naratriptan-hydrochloride oral suspensions were prepared from 2.5-mg commercial tablets yielding a nominal naratriptan concentration of 0.5 mg/mL. The suspension vehicles selected for testing were Syrpalta, an equal-parts mixture of Ora-Plus and Ora-Sweet, and an equal-parts mixture of Ora-Plus and Ora-Sweet SF. The tablets were crushed and thoroughly triturated to a fine powder using a porcelain mortar and pestle. The powder was incorporated into a portion of the Syrpalta or Ora-Plus suspension vehicle and mixed until homogeneous. The mixtures were then brought to volume with Syrpalta, Ora-Sweet or Ora-Sweet SF, as appropriate. The suspensions were packaged in amber, plastic, screw-cap prescription bottles and stored at 23 deg C for seven days and 4 deg C for 90 days. An adequate suspension was never achieved in Syrpalta. The crushed-tablet powder did not produce a uniformly dispersed mixture and exhibited clumping and a high rate of sedimentation. A distinct layer of the solid tablet material settled immediately after shaking. Over the next four hours, a densely packed, yellow, caked layer formed at the bottom of the containers, making resuspension difficult. During storage, the caking became worse. Chemical analysis was not performed. The Ora-Plus and Ora-Sweet or Ora-Sweet SF suspensions had a slight greenish cast and were resuspended without difficulty by shaking for approximately ten seconds, yielding easily poured and homogeneous mixtures throughout the study. Visible settling and layering did not begin for four hours with the Ora-Sweet suspension and 24 hours for the Ora-Sweet SF suspension. High pressure liquid chromatographic analysis found that the naratriptan concentration in both suspension-vehicle combinations exhibited little or no loss for seven days at 23

  18. Physico-chemical and viscoelastic properties of high pressure homogenized lemon peel fiber fraction suspensions obtained after sequential pectin extraction

    Willemsen, K.L.D.D.; Panozzo, A.; Moelants, K.; Debon, S.J.J.; Desmet, C.; Cardinaels, R.M.; Moldenaers, P.; Wallecan, J.; Hendrickx, M.E.G.

    2017-01-01

    The viscoelastic properties of high pressure homogenized lemon peel cell wall fiber suspensions, obtained after sequential selective pectin extraction, were investigated in the current study. For comparison, a general pectin extraction was additionally performed on lemon peel under acid thermal

  19. Self-powered suspension criterion and energy regeneration implementation scheme of motor-driven active suspension

    Yan, Shuai; Sun, Weichao

    2017-09-01

    Active suspension systems have advantages on mitigating the effects of vehicle vibration caused by road roughness, which are one of the most important component parts in influencing the performances of vehicles. However, high amount of energy consumption restricts the application of active suspension systems. From the point of energy saving, this paper presents a self-powered criterion of the active suspension system to judge whether a motor-driven suspension can be self-powered or not, and then a motor parameter condition is developed as a reference to design a self-powered suspension. An energy regeneration implementation scheme is subsequently proposed to make the active suspension which has the potential to be self-powered achieve energy-saving target in the real application. In this implementation scheme, operating electric circuits are designed based on different working status of the actuator and power source and it is realizable to accumulate energy from road vibration and supply energy to the actuator by switching corresponding electric circuits. To apply the self-powered suspension criterion and energy regeneration implementation scheme, an active suspension system is designed with a constrained H∞ controller and calculation results indicate that it has the capability to be self-powered. Simulation results show that the performances of the self-powered active suspension are nearly the same as those of the active suspension with an external energy source and can achieve energy regeneration at the same time.

  20. Formulation development and rheological studies of palatable cefetamet pivoxil hydrochloride dry powder suspension

    G Divakar

    2011-05-01

    Full Text Available Background and the purpose of the study: Because of its intense bitter taste and susceptibility to moisture Cefetamet Pivoxil (CPH is presently available only in the form of tablet. The aim of this study was to develop taste masked CPH dry powder suspension. Methods: Methods employed for formulations were: a Film coating of CPH using Eudragit E100 and subsequent adsorption on different carriers such as spray-dried lactose, sodium starch glycolate and spry-dried mannitol and b Complexation of CPH with three different ion exchange resins viz; indion 234, amberlite IRP64 and amberlite IRP69. Results: Taste evaluation as recognized by volunteers revealed that coating with eudragit E100 and subsequent adsorption on different carriers do not mask the bitter taste of the drug. Suspensions prepared using amberlite IRP64 and amberlite IRP69 were extremely palatable with no bitter after taste. They showed pseudoplastic flow behavior and were too viscous even after shearing for sufficient duration of time and exhibited poor pourability. The suspension made with indion 234 was palatable with slight or no bitter after taste. It demonstrated plastic flow with negligible thixotropy. It had moderate viscosity at rest and could be poured after a reasonable amount of shaking. CPH dry powder suspensions were very unstable under different conditions except under refrigeration. A 5% degradation of drug was occurred in reconstituted suspension in 4 days period when stored at room temperature. Conclusion: Dry powder suspension prepared with indion 234 with 5% overages was stable even after 4th day of reconstitution and palatable with slight or no bitter after taste

  1. Vehicle lateral dynamics stabilization using active suspension

    Drobný V.

    2008-12-01

    Full Text Available The paper deals with the investigation of active nonlinear suspension control in order to stabilize the lateral vehicle motion in similar way as systems like ESP do. The lateral stabilization of vehicle based on braking forces can be alternatively provided by the different setting of suspension forces. The basis of this control is the nonlinear property of the tyres. The vehicle has at least four wheels and it gives one or more redundant vertical forces that can be used for the different distribution of vertical suspension forces in such a way that resulting lateral and/or longitudinal forces create the required correction moment for lateral dynamic vehicle stabilization.

  2. Suspension for the low frequency facility

    Cella, G; Di Virgilio, A; Gaddi, A; Viceré, A

    2000-01-01

    We introduce the working principles of the VIRGO Low Frequency Facility (LFF), whose main aim is the measurement of the thermal noise in the VIRGO suspension system. We evaluate the displacement thermal noise of a mirror, which is an intermediate element of a double pendulum suspension system. This double pendulum will be suspended to the last stage of a VIRGO Super-Attenuator (SA), the prototype VIRGO suspension system being tested at the Pisa section of INFN. In the proposed configuration, we evaluate the spectrum of the thermal noise for different choices of the parameters: based on this study, we comment on the future directions to be undertaken in the LFF experiment.

  3. Pro REST API development with nodejs

    Doglio, Fernando

    2015-01-01

    Pro REST API Development with Node.js is your guide to managing and understanding the full capabilities of successful REST development. API design is a hot topic in the programming world, but not many resources exist for developers to really understand how you can leverage the advantages. This book will provide a brief background on REST and the tools it provides (well known and not so well known). Understand how there is more to REST than just JSON and URLs. You will then cover and compare the maintained modules currently available in the npm community, including Express, Restify, Vatican,

  4. Feeding and resting postures of wild northern muriquis (Brachyteles hypoxanthus).

    Iurck, Maria F; Nowak, Matthew G; Costa, Leny C M; Mendes, Sérgio L; Ford, Susan M; Strier, Karen B

    2013-01-01

    Increased body size in Brachyteles has been regarded as an important evolutionary adaptation that allowed a greater reliance on leaves compared to other more frugivorous Atelidae, but its association with muriqui positional behavior and substrate use is still unknown. Here, we present original data on the feeding and resting postures of the northern muriqui (Brachyteles hypoxanthus) and evaluate predictions about the relationships between body size, postural behavior, and substrate use derived from previously published data for other atelids (e.g. Alouatta, Ateles, and Lagothrix). The study was undertaken from August 2002 to July 2003 on a large group of well-habituated muriquis inhabiting the Reserva Particular do Patrimônio Natural - Felíciano Miguel Abdala in Minas Gerais, Brazil. Consistent with our predictions, we found that B. hypoxanthus was highly suspensory during postural feeding (60.9%) and commonly used tail-hind limb suspension/horizontal tripod (38.0%) or tail-forelimb/hind limb suspension (21.4%). However, although tail-suspensory postures permitted the muriquis to use the terminal canopy and small-sized substrates, these areas were also accessed via tail-assisted above-branch postural behaviors involving multiple substrates. Unexpectedly, tail-suspensory postures were found to be frequently associated with large substrates, tree trunks, and the understory. We suggest that Brachyteles' ability to access food resources from all areas of a feeding tree and from tree crowns at different canopy levels may account for their ability to efficiently exploit food resources in seasonal disturbed forest fragments of the Brazilian Atlantic Forest today. © 2012 Wiley Periodicals, Inc.

  5. Investigating high-concentration monoclonal antibody powder suspension in nonaqueous suspension vehicles for subcutaneous injection.

    Bowen, Mayumi; Armstrong, Nick; Maa, Yuh-Fun

    2012-12-01

    Developing high-concentration monoclonal antibody (mAb) liquid formulations for subcutaneous (s.c.) administration is challenging because increased viscosity makes injection difficult. To overcome this obstacle, we investigated a nonaqueous powder suspension approach. Three IgG1 mAbs were spray dried and suspended at different concentrations in Miglyol® 840, benzyl benzoate, or ethyl lactate. Suspensions were characterized for viscosity, particle size, and syringeability; physical stability was visually inspected. Suspensions generally outperformed liquid solutions for injectability despite higher viscosity at the same mAb concentrations. Powder formulations and properties had little effect on viscosity or injectability. Ethyl lactate suspensions had lowest viscosity (Miglyol® 840 improved overall performance in high mAb concentration suspensions. This study demonstrated the viability of high mAb concentration (>300 mg/mL) in suspension formulations for s.c. administration. Copyright © 2012 Wiley Periodicals, Inc.

  6. Resting state fMRI: A review on methods in resting state connectivity analysis and resting state networks.

    Smitha, K A; Akhil Raja, K; Arun, K M; Rajesh, P G; Thomas, Bejoy; Kapilamoorthy, T R; Kesavadas, Chandrasekharan

    2017-08-01

    The inquisitiveness about what happens in the brain has been there since the beginning of humankind. Functional magnetic resonance imaging is a prominent tool which helps in the non-invasive examination, localisation as well as lateralisation of brain functions such as language, memory, etc. In recent years, there is an apparent shift in the focus of neuroscience research to studies dealing with a brain at 'resting state'. Here the spotlight is on the intrinsic activity within the brain, in the absence of any sensory or cognitive stimulus. The analyses of functional brain connectivity in the state of rest have revealed different resting state networks, which depict specific functions and varied spatial topology. However, different statistical methods have been introduced to study resting state functional magnetic resonance imaging connectivity, yet producing consistent results. In this article, we introduce the concept of resting state functional magnetic resonance imaging in detail, then discuss three most widely used methods for analysis, describe a few of the resting state networks featuring the brain regions, associated cognitive functions and clinical applications of resting state functional magnetic resonance imaging. This review aims to highlight the utility and importance of studying resting state functional magnetic resonance imaging connectivity, underlining its complementary nature to the task-based functional magnetic resonance imaging.

  7. Startup of electrophoresis in a suspension of colloidal spheres.

    Chiang, Chia C; Keh, Huan J

    2015-12-01

    The transient electrophoretic response of a homogeneous suspension of spherical particles to the step application of an electric field is analyzed. The electric double layer encompassing each particle is assumed to be thin but finite, and the effect of dynamic electroosmosis within it is incorporated. The momentum equation for the fluid outside the double layers is solved through the use of a unit cell model. Closed-form formulas for the time-evolving electrophoretic and settling velocities of the particles in the Laplace transform are obtained in terms of the electrokinetic radius, relative mass density, and volume fraction of the particles. The time scale for the development of electrophoresis and sedimentation is significantly smaller for a suspension with a higher particle volume fraction or a smaller particle-to-fluid density ratio, and the electrophoretic mobility at any instant increases with an increase in the electrokinetic particle radius. The transient electrophoretic mobility is a decreasing function of the particle volume fraction if the particle-to-fluid density ratio is relatively small, but it may increase with an increase in the particle volume fraction if this density ratio is relatively large. The particle interaction effect in a suspension on the transient electrophoresis is much weaker than that on the transient sedimentation of the particles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A Course in Fluid Mechanics of Suspensions.

    Davis, Robert H.

    1989-01-01

    Discusses a course focusing on fluid mechanics and physical chemistry of suspensions. Describes the main themes of the lectures and includes a list of course outlines. Possible textbooks and many journal articles are listed. (YP)

  9. Heat and mass transfer in particulate suspensions

    Michaelides, Efstathios E (Stathis)

    2013-01-01

    Heat and Mass Transfer in Particulate Suspensions is a critical review of the subject of heat and mass transfer related to particulate Suspensions, which include both fluid-particles and fluid-droplet Suspensions. Fundamentals, recent advances and industrial applications are examined. The subject of particulate heat and mass transfer is currently driven by two significant applications: energy transformations –primarily combustion – and heat transfer equipment. The first includes particle and droplet combustion processes in engineering Suspensions as diverse as the Fluidized Bed Reactors (FBR’s) and Internal Combustion Engines (ICE’s). On the heat transfer side, cooling with nanofluids, which include nanoparticles, has attracted a great deal of attention in the last decade both from the fundamental and the applied side and has produced several scientific publications. A monograph that combines the fundamentals of heat transfer with particulates as well as the modern applications of the subject would be...

  10. Experimental Evaluation of Mountain Bike Suspension Systems

    J. Titlestad

    2003-01-01

    Full Text Available A significant distinction between competitive mountain bikes is whether they have a suspension system. Research studies indicate that a suspension system gives advantages, but it is difficult to quantify the benefits because they depend on so many variables, including the physiology and psychology of the cyclist, the roughness of the track and the design of the suspension system. A laboratory based test rig has been built that allows the number of variables in the system to be reduced and test conditions to be controlled. The test rig simulates regular impacts of the rear wheel with bumps in a rolling road. The physiological variables of oxygen consumption and heart rate were measured, together with speeds and forces at various points in the system. Physiological and mechanical test results both confirm a significant benefit in using a suspension system on the simulated rough track, with oxygen consumption reduced by around 30 % and power transmitted through the pedals reduced by 30 % to 60 %.

  11. Crust formation in drying colloidal suspensions

    Style, R. W.; Peppin, S. S. L.

    2010-01-01

    and the equations of poroelasticity, while the equations of colloid physics govern processes in the suspension. We derive new equations describing this process, including unique boundary conditions coupling the two regions, yielding a moving-boundary model

  12. Increasing Possibilities of Nano suspension

    Sutradhar, K.B.; Khatun, S.; Luna, I.P.

    2013-01-01

    Nowadays, a very large proportion of new drug candidates emerging from drug discovery programmes are water insoluble and thus poorly bioavailable. To avoid this problem, nano technology for drug delivery has gained much interest as a way to improve the solubility problems. Nano refers to particles size range of 1-1000 nm. The reduction of drug particles into the submicron range leads to a significant increase in the dissolution rate and therefore enhances bioavailability. Nanosuspensions are part of nano technology. This interacts with the body at subcellular (i.e., molecular) scales with a high degree of specificity and can be potentially translated into targeted cellular and tissue-specific clinical applications designed to achieve maximal therapeutic efficacy with minimal side effects. Production of drugs as nanosuspensions can be developed for drug delivery systems as an oral formulation and no noral administration. Here, this review describes the methods of pharmaceutical nano suspension production including advantages and disadvantages, potential benefits, characterization tests, and pharmaceutical applications in drug delivery

  13. Suspension scheme for fuel pin

    Butts, C.E.; Gray, H.C.

    1975-01-01

    A description is presented of a nuclear fuel pin suspension arrangement comprising, in combination, a rod; a first beam member connected to said rod at one end; a plurality of parallel-spaced slidable fuel support plates attached to said first beam member, the longitudinal axis of first beam member being perpendicular to the longitudinal axis of each of said fuel support plates, a first coupling means disposed along the length of the first beam member for permitting slidable fuel support plates parallel movement with respect to the longitudinal axis of said first beam member, a second coupling means located at one end of each of slidable fuel plates for slidably engaging first coupling means of first beam member, a second beam member connected to the other end of each of parallel-spaced slidable fuel support plates and providing an extension, second beam member being provided with a third coupling means disposed along the length of second beam member at one end thereof; and a plurality of fuel pins provided with a fourth coupling means located at one end of each fuel pin for slidably engaging third coupling means of second beam member to permit each fuel pin parallel movement with respect to the longitudinal axis of second beam member. (U.S.)

  14. Characterization of Complex Colloidal Suspensions

    Seaman, J. C.; Guerin, M.; Jackson, B. P.; Ranville, J. M.

    2003-04-01

    Surface chemical reactions play a major role in controlling contaminant fate and transport in the subsurface environment. Recent field and laboratory evidence suggests that mobile soil and groundwater colloids may facilitate the migration of sparingly soluble groundwater contaminants. Colloidal suspensions collected in the field or generated in laboratory column experiments tend to be fairly dilute in nature and comprised of relatively small particulates (reserved for studying ideal systems to the characterization of mobile colloids. However, many of these analytical techniques, including total/selective dissolution methods, dynamic light scattering, micro-electrophoresis, streaming potential, and even scanning electron microscopy (SEM), can be biased in of larger size fractions, and therefore, extremely sensitive to sampling, storage, and fractionation artifacts. In addition, surface modifiers such as sorbed oxides or organics can alter particulate appearance, composition, and behavior when compared to synthetic analogues or mineral standards. The current presentation will discuss the limitations and inherent biases associated with a number of analytical characterization techniques that are commonly applied to the study of mobile soil and groundwater colloids, including field flow fractionation (FFF) and acoustic based methods that have only recently become available.

  15. Experimental data and theoretical predictions for the rate of electrophoretic clarification of colloidal suspensions

    Johnson, T.J.; Davis, E.J.

    2000-05-01

    An experimental and theoretical investigation of the electrophoretic clarification rate of colloidal suspensions was conducted. The suspensions included a coal-washing effluent and a model system of TiO{sub 2} particles. A parametric study of TiO{sub 2} suspensions was performed to validate and analysis of the electrophoretic motion of the clarification front formed between a clear zone and the suspension. To measure the electric field strength needed in the prediction of the location of the front, a moveable probe and salt bridge were connected to a reference electrode. Using the measured electric field strengths, it was found that the numerical solution to the unit cell electrophoresis model agrees with the measured clarification rates. For suspensions with moderately thick electric double layers and high particle volume fractions the deviations from classical Smoluchowski theory are substantial, and the numerical analysis is in somewhat better agreement with the data than a prior solution of the problem. The numerical model reduces to the predictions of previous theories as the thickness of the electric double layer decreases, and it is in good agreement with the clarification rate measured for a coal-washing effluent suspension with thin electric double layers.

  16. Experimental data and theoretical predictions of the rate of electrophoretic clarification of colloidal suspensions

    Johnson, T.J.; Davis, E.J. [University of Washington, Seattle, WA (USA). Dept. of Chemical Engineering

    2000-05-01

    An experimental and theoretical investigation of the electrophoretic clarification rate of colloidal suspensions was conducted. The suspensions included a coal-washing effluent and a model system of TiO{sub 2} particles. A parametric study of TiO{sub 2} suspensions was performed to validate an analysis of the electrophoretic motion of the clarification front formed between a clear zone and the suspension. To measure the electric field strength needed in the prediction of the location of the front, a moveable probe and salt bridge were connected to a reference electrode. Using the measured electric field strength, it was found that the numerical solution to the unit cell electrophoresis model agrees with the measured clarification rates. For suspensions with moderately thick electric double layers and high particle volume fractions the deviations from classical Smoluchowski theory are substantial, and the numerical analysis is in somewhat better agreement with the data than a prior solution of the problem. The numerical model reduces to the predictions of previous theories as the thickness of the electric double layer decreases, and it is in good agreement with the clarification rate measured for a coal-washing effluent suspension with thin electric double layers. 21 refs., 8 figs., 4 tabs.

  17. Compressed Air Production Using Vehicle Suspension

    Ninad Arun Malpure; Sanket Nandlal Bhansali

    2015-01-01

    Abstract Generally compressed air is produced using different types of air compressors which consumes lot of electric energy and is noisy. In this paper an innovative idea is put forth for production of compressed air using movement of vehicle suspension which normal is wasted. The conversion of the force energy into the compressed air is carried out by the mechanism which consists of the vehicle suspension system hydraulic cylinder Non-return valve air compressor and air receiver. We are co...

  18. A New, General Strategy for Fabricating Highly Concentrated and Viscoplastic Suspensions Based on a Structural Approach To Modulate Interparticle Interaction.

    Sakurai, Shunsuke; Kamada, Fuminori; Kobashi, Kazufumi; Futaba, Don N; Hata, Kenji

    2018-01-24

    We report a general strategy to fabricate highly concentrated, viscoplastic and stable suspensions by designing the particle surface structure to control the interparticle attractive forces. Unlike conventional methods, where the choice of solvent is critical in balancing interparticle interactions, suspensions showing excellent stability and viscoplastic properties were made using various solvents. We demonstrated this approach using highly sparse agglomerates of carbon nanotubes (CNTs) as the particles. Our results revealed that the essential feature of the CNT agglomerate to fabricate these suspensions was high porosity with a spacing size much smaller than the overall size, which was only possible using long single-walled carbon nanotubes (SWNTs). In this way, the agglomerate surface was characterized by fine network of CNT bundles. These suspensions exhibited solid-like behavior at rest (characterized by a high yield stress of c.a. 100 Pa) and a liquid-like behavior when subjected to a stress (characterized by a significant drop of an apparent viscosity to 1 Pa·s at a shear rate of 1000 s -1 ). Furthermore, in contrast to conventionally fabricated suspensions, these "CNT pastes" exhibited exceptional stability at rest, under flow, and at extremely high concentrations during the drying process, with only a weakly observable dependence on solvent type. As a result, highly uniform micrometer-thick SWNT films were successfully fabricated by dried blade-coated films of these pastes. Finally, we developed a simple, semiempirical model and clarified the importance of the CNT agglomerate microstructure (the ratio of spacing size/particle size and porosity) on tailoring the cohesive forces between particles to fabricate stable viscoplastic suspensions.

  19. Flow-induced structure in colloidal suspensions

    Vermant, J [Department of Chemical Engineering, K U Leuven, W de Croylaan 46, B-3001 Leuven (Belgium); Solomon, M J [Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109-2136 (United States)

    2005-02-02

    We review the sequences of structural states that can be induced in colloidal suspensions by the application of flow. Structure formation during flow is strongly affected by the delicate balance among interparticle forces, Brownian motion and hydrodynamic interactions. The resulting non-equilibrium microstructure is in turn a principal determinant of the suspension rheology. Colloidal suspensions with near hard-sphere interactions develop an anisotropic, amorphous structure at low dimensionless shear rates. At high rates, clustering due to strong hydrodynamic forces leads to shear thickening rheology. Application of steady-shear flow to suspensions with repulsive interactions induces a rich sequence of transitions to one-, two-and three-dimensional order. Oscillatory-shear flow generates metastable ordering in suspensions with equilibrium liquid structure. On the other hand, short-range attractive interactions can lead to a fluid-to-gel transition under quiescent suspensions. Application of flow leads to orientation, breakup, densification and spatial reorganization of aggregates. Using a non-Newtonian suspending medium leads to additional possibilities for organization. We examine the extent to which theory and simulation have yielded mechanistic understanding of the microstructural transitions that have been observed. (topical review)

  20. Cognitive Rest: An Integrated Literature Review

    Schneider, Kathleen H.

    2016-01-01

    Cognitive rest has been suggested as a treatment for school athletes who have sustained a concussion, but the concept has rarely been defined. The purpose of this article is to provide a comprehensive definition of cognitive rest, based on an integrative literature review. The method of synthesis was guided by Avant and Walker's concept analysis…